Protein kinase C and its substrates

e

r-‘arand ellular Endwinology

ELSEVIER

Molecular and Cellular Endocrinology

116 (1996) I-29

Review

Protein kinase C and its substrates Jun-Ping Liu’ Department of Medical Oncology. Newcastle Mater Misericordiae Hospital, locked Bag I, Hunter Region Mail Center, New South Wales 2310, Australia

Received I August 1995; accepted 10 October 1995

Keywords:

substrate:

Eukaryotic cell; Protein Molecular activation

kinase

C; Domain

structure;

1. Introduction The eukaryotic cell is a highly organised entity that is exquisitely responsive to changes in the extracellular and intracellular environment. The mechanisms by which the cell responds to extracellular stimuli (first messengers) initially involve a series of signal transductions across the cell membrane. The signal transduction is mostly mediated by the production of bioactive substances (second messengers) or by the ability of second messengers to cause conformational changes in regulatory proteins. Many of these proteins mediating this process have been identified to be enzymes that are activated through allosteric and covalent mechanisms. Protein kinase C, one of these proteins, is believed to be a key regulatory element in signal transduction and exerts its effects by catalysing specific substrate phosphorylation, the most common covalent chemical modification of proteins in eukaryotic cells. Since its discovery in 1977 as a proteolytically activated protein

Abbreviations: DAG, diacylglycerol; FA, fatty acid; IP,, myo-inosi, tol-D-1,4,%trisphosphate; MARCKS, myristoylated alanine-rich C kinase substrate; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanol; PI, phosphatidylinositol; PlP2, phosphatidylinositol 4,5-bisphosphate; PIP,, phosphatidylinositol 3,4,5trisphosphate; PLC, phospholipase C; PLD, phospholipase D; PKC, protein kinase C (Ca’ + - and phospholipid-dependent protein kinase); PKD. protein kinase D; PKM, active catalytic domain of PKC: PMA, phorbol 12-myristate 13-acetate; PS, phosphatidylserine. ’ Current and correspondence address: Dr. Jun-Ping Liu, Baker Medical Research Institute. P.O. Box 348, Prahran, Victoria 3181, Australia.

0303-7207/96/$15.00 0 1996 SSDI

0303-7207(95)03706-D

Intramolecular

regulation;

Intermolecular

regulation;

Protein

kinase (Inoue et al., 1977; Takai et al., 1977), protein kinase C (the Ca*+- and phospholipid-dependent protein kinase or PKC) has attracted enormous attention and a wealth of information has accumulated which is summarised in several reviews [Nishizuka, 1986, 1988; 1992; Berridge and Irvine, 1989; Parker et al., 1989; Rana and Hokin, 1990; Bell and Burns,,1991; Azzi et al., 1992; Beli et al., 1992; Scott and Soderling, 1992; Huang and Huang, 1993; Hug and Sarre, 1993). It is generally recognised that PKC is involved in diverse processes including growth, differentiation, neural development, synaptic transmission; axonal regeneration, smooth muscle contraction and relaxation, endocrine and exocrine secretion, tumor promotion, and aging. However, a complete understanding of the mechanisms -of activation and regulation of PKC, and the biological role of its substrates has yet to be attained. Thus, the purpose of the article is to ‘briefly review our current understanding on the mechanisms of PKC activation, its intracellular regulation, and its interactions with its major substrates. 2. Domain structure? PKC has been purified to homogeneity from several tissues including the brain, heart, spleen, adrenal and pituitary (Kikkawa et -al., 1982; Wise et al., 1982; Schatzman et al., 1983; Turgeon’et al., ,198$ Uchida and Filburn, 1984). The purified enzyme from brain is a single polypeptide chain with a molecular weight (Mw) of 77-87 kDa, an isoelectric point of 5.6, a pH optimum of 7.5-8.0, and a Michaelis-Menten constant

Elsevier Science Ireland Ltd. All rights reserved

J.-P. Liu

2

)IMoleu_hr cd C’ellulurEtdocrirzoloe,~ 116 (1996) I 29 Regulatory Domain

VI

PKCp

Cl

v2

c2

Catalytic Domain v3 c3v4

C4

v5

I

Fig. I, Domain structure of PKC family. Eleven PKC isoforms of the PKC family are depicted in four groups cPKC. nPKC. aPKC The conserved (Cl -C4) and variable (VI -V5) regions of PKC are indicated in the regulatory and catalytic domains. The cysteine-zinc ATP-binding site are pointed by arrows. (K,) for ATP of 6.6 ,uM (Kikkawa et al., 1982). Limited proteolysis of PKC generates a 50-kDa fully active catalytic domain (PKM) and a 30-kDa regulatory domain that binds the activators and cofactors, diacylglycerol (DAG), Ca*+ and phospholipid (Kikkawa et al., 1982). The catalytic domain is active, thereby suggesting that the regulatory domain basically inhibits the enzyme when it is not activated. The primary amino acid sequence of PKC can be divided into four conserved functional domains (Cl -C4) which are individually separated by variable regions (Vl -V5) (Fig. 1). Now there are at least eleven PKC subspecies (see below), all of which contain the catalytic domain (C3, V4, C4, V5) located in the highly conserved C-terminal third of the polypeptide that is homologous in amino acid sequence to other protein kinases (Nishizuka, 1988; Parker et al., 1989). The catalytic domain contains an acidic sequence in C4 section that functions as the substrate recognition site (House and Kemp, 1987). In addition, a consensus sequence of the ATP binding site (-G-X-G-X,-G-X,-GX,,-K- ) is also found within a 40-residue sequence in the C3 region (Coussens et al., 1986; Parker et al.. 1986; Ohno et al., 1987; Hanks et al., 1988). Deletion or point mutation of the ATP binding site abolishes the kinase activity (Kaibuchi et al., 1989; Ohno et al., 1990). Molecular cloning has led to the identification of various PKC isoforms and established that PKC is a multigene family. To date, at least eleven PKC isoforms (a, PI, PIL ‘r’,6, E, i, II. 0, r/i and p) have been cloned and characterised (Knopf et al., 1986; Nishizuka, 1992; Selbie et al., 1993). Structural differences in the regulatory domain and different activation conditions have allowed division of the PKC isoforms into four major categories: the Ca* + -dependent or conventional PKC

and PKC//. motifs and

(cPKC: x, PI, ,$‘I1 and y), Ca’+ -independent PKC or novel PKC (nPKC: ci, i:, q and H), atypical PKC (aPKC: [ and r//i), and PKCp (which takes an intermediate position between the subgroups of nPKC and aPKC). The structural differences, biochemical properties and tissue distribution of these proteins are illustrated in Fig. 1 and Table 1. The aPKC /i isoform from the mouse is 98% homologous to human aPKC I isoforrn suggesting that the aPKC E. is the mouse homologue of human aPKC/ (Selbie et al., 1993; Akimoto et al., 1994). PKCp is a recently cloned isoform from human, which has an extended N-terminus containing a signal peptide and two unique hydrophobic domains and is independent of Cal+ - and phorbol esters (Johannes et al., 1994). However, PKCp has an overall 92”/0 homology to the recently cloned mouse protein kinase D (PKD) that binds phorbol esters with high affinity, and is believed to be a distinct protein kinase based on its distinct structure including a putative transmembrane domain and a pleckstrin homology (PH) domain without sequence homology to the PKC pseudosubstrate motif (see below) or to the catalytic domains of PKC family (Valverde et al.. 1994; Van Lint et al., 1995). It is possible that PKD is a close murine variant of human PKC,U and various isoforms of PKC may possess similar or dissimilar properties depending on their respective structures and be activated by various bioactive molecules in various tissues. The structure of the regulatory domain in cPKC (x, /j’I, pII, and 7 subspecies) includes Vl, C 1, V2, C2 and V3 regions in the N-terminal half. The Cl domain contains a tandem repeat of cysteine-rich sequences which have been detected in all PKC isoforms except the atypical PKCs, I and l/i, which have only one

J.-P. Liu / Molecular and Cellular Endocrinology I I6 (1996) I --29 Table Protein

1 kinase

PKC isoforms

nPKC (5 ‘I 0 aPKC

r/i

C isoforms Numbers of amino acids

Activators

76.8 76.8 76.9 77.5

672 671 673 697

PS, PS, PS, PS.

Ca’+, Ca”. Ca’+. Ca’+

77.5 83.5 78.0 81.6

674 737 683 707

PS, PS, PI, PI,

DAG. DAG, PMA PMA

67.7 67.2

592 586

PS, FA, PIP3. cerdmide, unknown

115.0

912

unknown

Mw (kDa)

Tissue distribution

.

DAG, DAG. DAG. DAG.

FA, FA, FA. FA,

1.25-D,. PMA PMA 1.25-D,.

PMA

ubiquitous most tissues most tissues neural

PMA

FA, PI, PMA PI. 1,25-D,. PMA

ubiquitous neural, immune. epithelium. heart neural. epithelium ovary. skeletal muscle, platelets

PA

most tissues ubiquitous

PKC /’

cysteine-rich sequence of the tandem. The cysteine-rich sequence that resembles the consensus sequence of a ‘cysteine-zinc finger’ found in many DNA-binding proteins is essential for DAG and phorbol ester binding and activation of the kinase (Ono et al., 1989a; Hubbard et al., 1991). Recent evidence suggests that two Zn2+ ions bind each zinc finger, and each Zn’+ atom coordinates with one histidine nitrogen and three cysteine sulfur atoms, to stabilise a particular conformation (Hubbard et al., 1991). Phorbol esters bind to this region in two cysteine-rich sites (Burns and Bell, 1991). The binding to the first cysteine-rich domain would be sufficient for lipid-dependent interaction with DAG and to confer translocation of the protein to plasma membranes (Quest and Bell, 1994; Quest et al., 1994). However, the recently cloned PKCC( that lacks the C2 domain and possesses a long spacing of 74 amino acids between the two cysteine-rich motifs shows no efficient phorbol ester binding (Johannes et al., 1994) suggesting that there may be cooperativity between the two cysteine-rich domains in phorbol ester binding to the Cl domain. Preceding the Cl region, there is also a pseudosubstrate sequence of 13-30 residues, which contains more than six basic residues. This region resembles a natural substrate recognition sequence for the kinase and maintains the enzyme in the inactive form in the absence of activators by binding to the active site in C4 region and blocking substrate access (House and Kemp, 1987). An antibody against this pseudosubstrate sequence or mutation of it increases the basal kinase activity (Makowske and Rosen, 1989; Pears et al., 1990; Cazaubon et al., 1994; Orr and Newton, 1994; Zhang et al., 1994). The regulatory domain of nPKC and aPKC does not contain the C2 region (Nishizuka, 1988; Ohno et al., 1988, 1989b). These isozymes, when expressed in COS-7 cells, do not possess an absolute requirement for Ca2 + , suggesting that the C2 region is required for the binding

unknown

of Ca’+ although it has not an EF hand or calelectrinlike sequence (Nishizuka, 1988; Ono et al., 1989b; Schaap et al., 1989; Akita et al., 1990a; Luo and Weinstein, 1993). The role of the C2 domain in Ca” binding is further supported by the studies in which recombinant C2 domain interacts with Ca’+ in the presence of PS or Cl domain (Luo and Weinstein, 1993; Luo et al., 1993). It is interesting to note that Ca’+ has been shown to bind mainly to the Cl domain with the C2 domain required to confer specificity for Ca’+ and therefore Cl and C2 may form a pocket for Ca’ + binding (Luo and Weinstein, 1993). Deletions in the C2 region appear to influence the affinity of PKC for Ca’ + , whereas deletions within the Cl region result in the loss of phorbol ester binding (Ono et al., 1989b). The Cl cysteine-rich sequence has also been found in several other proteins including the sn-1,2-diacylglycerol kinase (Sakane et al., 1990) the steroid receptors (Carson-Jurica et al., 1990). the neuronal protein chimaerin (Hall et al., 1990; Ahmed et al., 1993). the proto-oncogene product raf (Ishikawa et al., 1986) and PKD (Valverde et al., 1994) whereas a homologous region to the C2 domain has been found in proteins including phospholipase C, phospholipase Al, synaptotagmin. and the annexins (Klee, 1988; Perin et al., 1990; Clark et al., 1991; Sharp et al., 1991; Shirataki et al., 1993). Therefore, the Cl domain is an important region in mediating interactions with lipids and a possible recognition motif for protein-protein interaction, whereas the C2 domain is important for Ca’+ interaction. 3. Molecular activation 3.1. Intrructions

with endogenous

uctiautors

Activation of cPKC, the most studied of the PKCs, appears to involve complex interactions of the kinase

Agonist

(5)

(1) (2) (3) (4) (5)

-

(2)(4)

Receptor activation Translocation Down-regulation Membrane insertion Substrate phosphorylation

Fig. 2. Schematic representation of proposed mechanisms of PKC activation in eukaryotic cells. Two pathways to activate intracellular PKC from extracellular environment: receptor-mediated production of DAG through G-protein-coupled phospholipase C and phorbol ester-induced direct pharmacological activation, Both DAG and phorbol esters (such as PMA. phorbol l2-myristate 13-acetate) bind to PKC Cl region of the regulatory subunit to cause a physical association of PKC with the plasma membrane (translocation). whereas DAG promotes a complex formation of PKC, phosphatidylserine (PS) and Ca’ +- but phorbol esters induce a plasma membrane insertion of PKC and then down-regulation. Both DAG and phorbol esters induce a conformational change of PKC structure so that the inhibitory pseudosubstrate sequence moves away from the substrate binding site to allow binding and phosphorylation of PKC substrates to occur.

with its activators (Fig. 2). First, it requires association of the enzyme with plasma membrane phospholipids. in particular phosphatidylserine (PS). PKC binds to PS in the absence of metal cations through electrostatic interaction of both the regulatory and catalytic domains (Huang and Huang, 1993). The binding is highly cooperative and is greatly enhanced in the presence of Ca’ + in a concentration-dependent manner (Hannun and Bell, 1986; Hannun et al., 1986; Newton and Koshland, 1989; Bazzi and Nelsestuen, 1990; Mosior and Epand, 1993; Liu et al., 1994b). It is postulated that Ca’+ may participate in phospholipid clustering (Bazzi and Nelsestuen, 1991) and form a bridge between PKC and the acidic lipid (Hannun et al., 1985, 1986). In the absence of phospholipids, PKC binds to Ca’+ one molecule per molecule; in the presence of PS, however, PKC binds at least eight Ca’ + ions per protein (Bazzi and Nelsestuen, 1990). The binding of Ca”+ ions may require four carboxyl groups of four serine residues on the cytoplasmic surface of the plasma membrane. Ca’ + binding sites may thus locate at the interface between PKC and plasma membrane. PKC associates into this phospholipid-Ca’ + complex but becomes active only after associating with DAG. This interaction with DAG occurs through a hydrogen bond provided by the m-3 hydroxyl group of DAG to an acceptor atom on Cl region of the kinase, after interaction with the carbonyl

oxygens in positions sn-1 and m-2 of DAG (Brockerhoff, 1986; Ganong et al., 1986). When fully active, PKC is thought to be a quaternary complex consisting of anionic phospholipid, Ca2 + , DAG and the enzyme. The stoichiometry for PKC activation has suggested that one molecule of PKC requires four molecules of PS. one molecule of Ca’+ and one molecule of DAG for complete activation (Hannun and Bell, 1986). Unsaturated DAG renders PKC fully active by increasing PKC apparent affinity for PS and by lowering the Ca’+ requirement into the physiological range ( < 1 ,uM). Without DAG, PKC is only active when intracellular Ca’+ concentrations are increased lOO-fold (Kishimoto et al., 1980; Nishizuka, 1986). In addition, kinetic analysis shows that PS and Ca’+ decrease the Km of the enzyme for a protein substrate whereas DAG increases both the Km and I’,,,, suggesting that the binding of PKC to PS and Ca2 + increases PKC affinity for substrate, and that DAG interacts predominantly with bound PKC resulting in activation of the enzyme (Hannun and Bell, 1990). It is currently believed that the molecular mechanism of PKC activation by DAG involves the lipid-induced allosterism of PKC. In the presence of Ca’ + , the binding of PS and DAG to the N-terminal regulatory domain of PKC causes conformational changes including an exposure of arginine-19 (the first amino acid residue) of the pseudosubstrate

J.-P. Liu I Moiwular and Cellular Endmrinoiogy

prototope (see below) to proteolysis (Orr and Newton, 1994). Under resting condition, the arginine-19 is shielded by a cluster of acidic residues when the pseudosubstrate occupies the substrate-binding site. Proteolysis of the arginine-19 is accompanied by a dissociation of the N-terminal pseudosubstrate inhibitory region from the C-terminal catalytic domain substrate binding site, by that allowing substrate to access the enzyme for phosphorylation (Fig. 2) (House and Kemp, 1987; Pears et al., 1990; Orr et al., 1992). DAG is transiently produced by the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP,) when receptors are stimulated and degraded rapidly (within one minute in some cells). This reaction is catalysed by phosphatidylinositol hydrolysing-phospholipase C (PIPLC) and results in the concomitant release of an equimolar amount of n~yo-inositol-D-1,4,5triphosphate (IP,). The IP3 binds to a specific receptor located on an intracellular particle termed the ‘calciosome’ and causes the release of Ca’+ from this organelle (Worley et al., 1987; Ferris et al.. 1989; Ross et al., 1989). Increasing evidence suggests that DAG may be also generated from sources other than PI hydrolysis. These include the hydrolysis of phosphatidylcholine (PC), phosphatidylethanol (PE) and inositol-containing glycolipid. These reactions are catalysed by other forms of PLC and phospholipase D (PLD) and produce phosphatidic acid (PA) (Bocckino et al., 1985; Besterman et al., 1986; Bocckino et al., 1987; Irving and Exton, 1987; Pai et al., 1988; Rosoff et al., 1988; Slivka et al., 1988; Agwu et al., 1989: Martin and Michaelis, 1989; Billah et al., 1989; Chan et al., 1989; Cook and Wakelam, 1989; Diaz-Meco et al., 1989; Liscovitch and Amsterdam, 1989; Larrodera et al., 1990); PA can be then degraded to DAG by phosphohydrolase. Following receptor stimulation, a rapid net increase in the mass of cellular DAG occurs in a number of tissues - for instance, in mouse pancreas stimulated with various pancreatic secretagogues (cholinergic stimulation, cholecystokinin, bombesin) (Banschbach et al., 1981; Pandol and Schoeffield, 1986), in platelets stimulated with thrombin (Rittenhouse-Simmons, 1979; Preiss et al., 1986). in hepatocytes stimulated with arginine vasopressin, epinephrine and angiotensin II (Bocckino et al., 1985), and in pituitary GH, cells stimulated with thyrotropin releasing hormone (Macphee and Drummond, 1984; Martin et al., 1990). In addition, it has been reported that arginine vasopressin can activate PI-PLC, phosphatidylcholine-catalysing (PC)-PLC and phospholipase D (PLD) in some cells leading to the direct and indirect formation of DAG. The amount of DAG formed by PI-independent hydrolysis is much greater than that formed from PI and the duration is prolonged, providing additional sources of DAG capable of activating PKC (Bocckino et al., 1987; Grillone et al., 1988: Hepler et al., 1990; Huang and Cabot, 1990).

116 (1996) l-29

5

PKC can also be activated by the phosphoinositides phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), PIP,, and phosphatidylinositol 3,4,5trisphosphate (PIP,) in the presence of CaZ+ and phosphatidylserine (Lee and Bell, 1991; Kochs et al., 1993; Nakanishi et al., 1993; Singh et al., 1993). PIP, PIP, and PIP, are formed by the sequential phosphorylation of PI, PIP and PIP* by PI-4 kinase, PI-5 kinase and PI-3 kinase, respectively. The affinity and potency of these phosphoinositides in activating PKC increase with the incremental phosphorylation and is PIP, > PIP2 > PIP > PI (Singh et al., 1993). However, in the absence of divalent cations, these phosphoinositides cause irreversible inactivation of PKC with a similar order of potency (PIP, > PIP, > PIP) (Huang and Huang, 1991). It has been proposed that in the presence of Ca* + , PIP, may bind to the Cl domain of PKC with hydrogen bonds through its carbonyl groups and interact electrostatically with the C2 domain and Ca*+ through its phosphate groups; while without Ca*+ the phosphate groups may bind to the basic groups of PKC to cause inactivation (Singh et al., 1993). More recently, it has been shown that the phosphoinositides PI(3,4)PZ and PIP, activate only nPKC, but not cPKC and PKC[ (Toker et al., 1994). The activation by PI(3,4)P, and PIP, of nPKC is particularly interesting if one considers the fact that these lipids are the physiologically important products of PI catalysed by PI-3 kinase and potentially act as second messengers specifically for nPKC (Downes and Carter, 1991; Stephens et al., 1991; Jackson et al., 1992; Nakanishi et al., 1993; Toker et al., 1994). PI-3 kinase consists of an 85-kDa regulatory domain and a 1IO-kDa catalytic domain; the 85-kDa regulatory domain, like PLCy 1 and p21’““GAP, associates with specific phosphorylated tyrosine-containing domains of growth factor receptors through SH2 (Src homology-2) domain (Fantl et al., 1992; Kashishian et al., 1992; Reedijk et al., 1992). When they are activated. the growth factor receptors undergo autophosphorylation of their tyrosine residues in their cytoplasmic domains that then facilitates their binding to PI-3 kinase. PI-3 kinase could also be activated by certain oncogene products through protein-protein interaction (Kucera and Rittenhouse, 1990; Varticovski et al., 1991). Thus, activation of PI-3 kinase can catalyse the production of PI(3,4)P2 and PIP, that in turn activate nPKC, providing a mechanism by which PKC may mediate intracellular signalling for growth factors and oncogenes that increase PI-3 kinase activity. In addition, all cPKC can be activated by cis-unsaturated fatty acids including arachidonic, oleic and linoleic acids derived from the cell membrane catalysed by phospholipase A2 (Murakami et al., 1987; Bronfman et al., 1988; Verkest et al., 1988; Shinomura et al., 1991; Chen and Murakami, 1992). These fatty acids activate cPKC in the presence of Ca2 + less than 1 p M or absence

and this increase can be further enhanced by of Ca”, Zn’+ in presence of < 5 ,LLMCa’+ (Murakami et al., 1987). The activation of PKC by these fatty acids is independent of PS, suggesting that soluble fatty acids activate soluble PKC to phosphorylate a different set of substrates (Khan et al., 1992). The fatty acids also potentiate DAG-stimulated PKC activity in intact platelets (Yoshida et al., 1992). Micromolar amounts of long chain saturated and unsaturated fatty acyl-CoAs also increase cPKC activity in the presence of DAG, PS, and Ca’+ but inhibit activity at high. non-physio( > 0.5 mM), suggesting logical Ca’ * concentrations that acyl-CoAs are important in the modulation of PKC activation induced by the PLC/PI pathway (Bronfman et al.. 1988). It is noteworthy that the key metabolite of steroid hormone vitamin D,, 1x.25-dihyconcentrations actidroxyvitamin D,, at physiological vates PKC, including x. ;’ and I: isoforms in a manner similar to that by DAG (Slater et al.. 1995). Although several lines of evidence suggest that besides acting on its nuclear receptor to regulate genomic-dependent Ca’ + homeostasis the vitamin D, metabolite stimulates a rapid mobilization of Ca’+ , the turnover of phosphoinositides and phosphatidylcholines leading to increases in the levels of IP, and DAG, and translocation of PKC, the direct effect of this compound on PKC activity is proposed to result from an interaction at a distinct site of PKC and to be responsible for its various functions in non-genomic cellular responses to the hormone (Slater et al., 1995). Whether PKC is a plasma membrane receptor for 1x,25-dihydroxyvitamin D, is an interesting issue and requires further study. Furthermore. ceramide, the immediate product of sphingomyelinase hydrolysis of sphingomyelin, has been reported to activate PKCC both in vitro and in NIH 3T3 fibroblasts (Begum, 1995), although other effects of this compound on the signalling pathway of PKC have also been observed (see below).

Tumor-promoting phorbol esters. such as phorbol 12-myristate 13-acetate (PMA), possess a molecular structure that is similar to that of DAG. These agents can therefore substitute for DAG and activate PKC directly in vitro and in vivo (Castagna et al., 1982; Sano et al., 1983; Yamanishi et al., 1983). Like DAG, phorbol esters dramatically increase the affinity of PKC for Ca’+, resulting in its complete activation at physiological Ca’+ concentrations. Since the phorbol esters are slowly metabolised. however, they persist in tissues for much longer times than DAG and cause a prolonged activation of PKC. In addition, phorbol esters induce irreversible insertion of PKC into phospholipid vesicles (Kazanietz et al., 1992), which possibly causes additional perturbation of the cell membrane (Kikkawa and

Nishizuka, 1986); in contrast. DAG is inefficient in promoting PKC insertion, suggesting that the ability of the compounds to promote PKC insertion could contribute marked differences in the biological behaviour of the known PKC activators (Kazanietz et al., 1992). Phorbol ester-induced activation of PKC causes ditferential substrate phosphorylation from that stimulated by Ca’ + and phosphatidylserine, suggesting that different activators may also alter PKC substrate specificity (Robinson, 1992). The idea that PKC is the phorbol ester ‘receptor’ is based on studies of phorbol ester binding. A cytosolic phorbol ester binding site was shown to co-purify with PKC activity, to require PS, and to possess a similar order of affinity for phorbol ester analogues as that seen in intact cells (Ashendel et al., 1983; Kikkawa et al., 1983; Sando and Young, 1983; Leach and Blumberg, 1985). Kinetic analysis with purified PKC suggests that one molecule of PMA can activate one molecule of PKC (Kikkawa et al., 1983). Although there is evidence to suggest that PKC is not the only phorbol ester ‘receptor’ and to argue that the effects of the phorbol esters are not all mediated through the activation of PKC, it is generally agreed that PKC is the major cellular site of action for phorbol esters (Rana and Hokin, 1990). Four additional classes of phorbol ester receptors have recently been identified: the chimaerin family, UNC-13, Vav and PKD. The chimaerin family contains three members: /J-chimaerin in testis and two splicing variants of n-chimaerin in the brain; they possess an N-terminal domain homologous to the cysteine-rich zinc finger region of PKC (Hall et al., 1993; Areces et al., 1994). UNC-13 contains domains homologous to both the cysteine-rich zinc finger and C2 regions of PKC (Maruyama and Brenner. 199 1). Vav is a proto-oncogene specifically expressed in hematopoietic cells, the product of which contains SH2 and SH3 domains. a Dbl-homology domain, a PH domain and a single cysteine-rich domain (Gulbins et al.. 1994). PKD is a newly cloned protein kinase containing a putative transmembrane domain, two cysteine-rich zinc finger motifs and a PH domain in the N-terminus (Valverde et al.. 1994; Van Lint et al., 1995). All these proteins have been shown to bind phorbol esters with high affinity (Ahmed et al., 1993: Areces et al., 1994; Gulbins et al., 1994; Valverde et al., 1994; Van Lint et al., 1995). Interestingly, phorbol esters not only bind to n-chimaerin, Vav and PKD but also stimulate the rdc-GAP activity of chimaerin, the ras-guanine nucleotide exchange activity of Vav and the kinase activity of PKD (Ahmed et al., 1993; Gulbins et al.. 1994: Van Lint et al., 1995), suggesting that multiple potential sites of action for the phorbol esters exist in cells, and some protein phosphorylation and effects on mitogen-activated protein kinase pathways may be stimulated by phorbol esters independently of PKC.

J.-P. Liu / Molecular and Cehdar Endocrinology 116 (1996) l-29

3.3. Permissive

phosphorylation

Activation of PKC depends not only on activators such as phospholipids and Ca2+ but also on phosphorylation of the protein. Recent evidence suggests that PKC activation requires permissive post-translational phosphorylation of the protein catalytic subunit (Cazaubon and Parker, 1993). Site-directed mutagenesis of PKCa cDNA shows that a region including three threonine residues in the catalytic subunit is involved in controlling PKC activity and that substitution of these threonine residues, especially threonine-497, with alanine residues results in the retentions of phorbol ester binding but abolishes the kinase activity (Cazaubon and Parker, 1993; Cazaubon et al., 1994). It has been suggested that this region containing the threonine residues is equivalent to the ‘T-loop’ of cyclin-dependent kinase 2 (De Bondt et al., 1993) and the ‘lip’ of the L12 loop of MAP kinase ERK2 (Zhang et al., 1994) and is involved in phosphorylation-induced conformational activation of the protein kinases allowing substrate binding to the catalysis core. Site-specific mutation of PKCPI cDNA shows that threonine-642 is important in the processing for newly synthesized PKCgI to function (Zhang et al., 1994). In PKCBII, it has been shown that threonine-500 is the residue phosphorylated by another protein kinase (Orr and Newton, 1994). Dephosphorylation of PKC/?II by the catalytic subunit of protein phosphatase-I results in a loss in the kinase activity and the dephosphorylated enzyme cannot autophosphorylate or be phosphorylated by other PKCs, suggesting that phosphorylation at this site is carried out by a different protein kinase (Dutil et al., 1994). Molecular modelling further suggests that the residue threonine-500 is part of the ‘lip’ structure at the entrance of the catalytic site and phosphorylation on this lip (or an activation loop) is important to activation of protein kinases (Orr and Newton, 1994; Taylor and Radzio-Andzelm, 1994). Thus, a PKC kinase is required to phosphorylate PKC at its catalytic subunit before it can be activated by second messengers. The identity of the PKC kinase has not been fully elucidated. It has recently been suggested. however, that each PKC isoform is rendered catalytically competent by phosphorylation on its activation loop by another specific PKC isoform (Newton and Taylor, 1995). A trans-phosphorylation cascade within the PKC family has been interestingly described to operate almost following the reverse order of the PKC isoforms. namely PKCE phosphorylates PKCS, PKCS phosphorylates PKQ, PKCy phosphorylates PKC/?I and PKC/?I phosphorylates PKC/?II that in turn phosphorylates PKCcr (Newton and Taylor, 1995). It is also worthwhile noting that PKCor is tyrosine phosphorylated in Chinese hamster ovary cells in response to insulin stimulation, and dephosphorylation by a spe-

7

cific tyrosine phosphatase induces a 35% decrease in the kinase activity, suggesting that PKC is also subject to tyrosine phosphorylation under certain circumstances under which its activity is regulated (Liu and Roth, 1994). 3.4. Translocation One particularly important concomitant of PKC activation is the intracellular redistribution of the enzyme. The phenomenon of PKC redistribution was first observed when EL4 mouse thymoma cells were exposed to phorbol esters (Kraft and Anderson, 1983). It is now generally accepted that activation of PKC is associated with translocation of the enzyme from the cytosol to the cell membrane and that the enzyme translocation can therefore be used as an index of the enzyme activation (Kraft and Anderson, 1983; Vilgrain et al., 1984; Costa-Casnellie et al., 1985; Drust and Martin, 1985; Farrar and Anderson, 1985; Fearon and Tashjian, 1985; Hirota et al., 1985; May et al., 1985; Wolf et al., 1985; Hannun and Bell, 1986, 1989; McArdle and Conn, 1986; Murakami et al., 1987; Nishizuka, 1988; Seifert et al., 1988; Berridge and Irvine, 1989; McFadden et al., 1989; Newton and Koshland, 1989; O’Flaherty et al., 1990; Akita et al., 1990a). In resting anterior pituitary cells, for example, 60-80% of the total PKC is found in the cytosol; upon activation by phorbol esters, 70-90% of total PKC becomes tightly associated with the cell membrane within 30 min (Liu et al., 1990). Incubation of the cells with argjnine vasopressin also causes PKC translocation from cytosol to the plasma membrane (Liu et al., 1992). However, there is also evidence that the translocation of PKC does not temporally correlate with activation of the enzyme as measured by substrate phosphorylation. In cultured anterior pituitary cells, the translocation of PKC induced by arginine vasopressin occurred later than the arginine vasopressin induced PKC phosphorylation of the myristoylated alanine rich C-kinase substrate (MARCKS) protein, a major PKC substrate (see below) (Liu et al., 1992. 1994a). A dissociation between PKC translocation and substrate phosphorylation is also observed in the 132 1N 1 astrocytoma cell line in response to muscarinic receptor stimulation (Trilivas et al., 1991). In addition, in several cell lines such as renal mesangial cells, Swiss 3T3 cells and HL60, PKC isoform c does not translocate to the plasma membrane when stimulated with phorbol esters (Olivier and Parker, 1992; Ways et al., 1992; Olivier and Parker, 1994). These findings suggest that measurement of PKC substrate phosphorylation is a more accurate index of PKC activation. Selective translocation of PKC isozymes also occurs in response to specific activation. For example, thyro-

tropin releasing hormone causes translocation of PKC isozymes other than PKC-t:, while bryostatin 1 stimulates selective translocation of PKC-;7 in GH, rat pituitary cells (Kiley et al., 1991; Mackanos et al.. 1991). In addition. reverse translocation (from cell membrane to cytosol) has also been observed when bovine adrenocortical cells are stimulated with ACTH (Vilgrain et al., 1984) or when human monocytes are stimulated with concanavalin A (Costa-Casnellie et al., 1985). In this regard. a pool of membrane-associated activatable PKCs has been reported in a number of cells (Chakravarthy et al., 1994). The molecular mechanisms underlying the translocation of PKC are not yet clear, but several factors have been proposed to promote PKC adherence to vesicles and PKC activation. Such factors include the production of acidic phospholipids (Hannun and Bell. 1986, 1989: Newton and Koshland, 1989), long chain unsaturated fatty acids (Seifert et al., 1988). low pH (McFadden et al.. 1989), increases in intracellular Ca’+ concentrations (Wolf et al., 1985: May et al., 1985) and/or regional changes in Ca’+ concentration (O’Flaherty et al.. 1990). as well as effects of ATP (Wolf et al.. 1985: Chen and Tai, 1987) and such cations as Zn’ and Cd’+ (Murakami et al., 1987). The binding of PKC to cell membrane involves direct binding of the protein to phospholipids and is enhanced by Ca” in a concentration-dependent manner (Liu et al., 1994b). Besides associating with phospholipids, PKC may also interact with some plasma membrane anchoring proteins; such proteins include PKC inhibitor protein (PKCI), annexin 1 and RACKS (Mochly-Rosen et al.. 1991; Toker et al., 1992: Ron et al., 1994). It is interesting that these membrane anchoring proteins possess a conserved sequence of 16 amino acids which binds to PKCP 1 and may represent a PKC binding domain (Mochly-Rosen et al., 1991). Cloning of the cDNA encoding RACKS suggests that RACKI has a structural homology and functional analogy to the p subunit of G-proteins involved in membrane anchorage of the a-adrenergic receptor kinase (PARK) (Ron et al.. 1994). The plasma membrane PKC binding proteins may thus direct PKC binding to membrane specific sites. Several lines of evidence suggest that PKC, Ca’dependent and independent forms, also translocates to nuclei when cells are stimulated with phorbol esters or other mitogens (Cambier et al., 1987; Leach et al.. 1989: Fields et al., 1990; Eldar et al., 1992; Li et al.. 1992; Hocevar et al., 1993; Goss et al., 1994). Examination of PKC in nuclei shows the enzyme associated with the nuclear membranes belonging to a permanently active membrane-inserted form is responsible for long term action (Buchner et al., 1992). Whether the PKC is converted from cytosol into an integral nuclear membrane protein or is chemically covalently modified to

couple to the membrane remains unknown. It has been suggested that the,hinge and catalytic domains of PKC interact with a PKC ‘receptor’ present in the nuclear envelope and thus facilitate the translocation of PKC to the nucleus (James and Olson, 1992). Nuclear PKC causes phosphorylation of several nuclear substrates including nuclear lamins (Hornbeck et al.. 1988; AbdelGhany et al.. 1989; Fields et al., 1990; Hocevar et al., 1993; Goss et al., 1994), DNA topoisomerase II (Sahyoun et al., 1986) CCAAT-enhancer binding protein (Mahoney et al., 1992). and myogenin (Li et al., 1993) suggesting that the PKC is facing the interior of the nucleus by an unknown translocation mechanism (Buchner et al., 1992). PKC phosphorylation may play important roles in regulation of these protein functions. Phosphorylation of the nuclear lamin B by PKC pII has been implicated in the regulation of a nuclear envelope breakdown during mitosis (Goss et al.. 1994). whereas phosphorylation of DNA topoisomerase II by PKC stimulates the specific activity of the enzyme apparently via an enhancement in ATP hydrolysis (DeVore et al., 1992). In addition fibroblast growth factor stimulates PKCx translocation to the nuclear membrane to phosphorylate the muscle-specific transcription factor myogenin, resulting in an inhibition of myogenin interaction with DNA, gene transcription from musclespecific promoters and possibly the differentiation of myoblasts into mydfibres (Li et al., 1992). The treatment of isolated intact rat liver nuclei with phorbol esters also stimulates the phosphorylation of nuclear IP, receptors by native PKC, increasing the potency by which IP, may release Ca” from nuclei. These findings suggest that PKC phosphorylation of the IP, receptor may be a key mechanism in maintaining nuclear Ca’+ homeostasis (Matter et al., 1993). 4. Intra- and inter-molecular

regulation

The basal cellular activity of PKC is usually maintained at a low level and this is accomplished by auto-inhibitory domains such as the high affinity intramolecular pseudosubstrate site (House and Kemp, 1987). The pseudosubstrate prototope is the amino acid sequence that extends between residues 19 and 3 I of the Cl region. It resembles a substrate phosphorylation site in its distribution of basic residue recognition determinants (House and Kemp, 1990) and it maintains the enzyme in an inactive state by binding to the acidic sequence of the C4 domain blocking the substrate binding site (Coussens et al., 1986; Parker et al., 1986; House and Kemp. 1987; Kemp et al., 1989). Activation of the enzyme by the bindings of PS and DAG to the C I region causes a conformational change that removes the pseudosubstrate sequence from the active site and

J.-P. Liu / Molcwlur

and Ceilulur Endocrinology

allows the enzyme to reach protein substrates (House and Kemp, 1987; Hardie, 1988). A synthetic peptide analog that corresponds to this pseudosubstrate sequence has been shown to be a potent and specific substrate antagonist, inhibiting both PKC autophosphorylation and substrate protein phosphorylation. When a serine is substituted for the alanine residue at position 25, the inhibitory peptide is converted into an excellent substrate (House and Kemp, 1987). These studies have provided new insight into the intra-molecular regulation of PKC activation and a means of modifying PKC activity. 4.2. Autuphosphorylution As indicated above, the activation of PKC requires an interaction of the enzyme with the cell membrane. However, when the enzyme becomes activated, it causes phosphorylation of substrate proteins and undergoes autophosphorylation (Kikkawa et al., 1982; Huang et al., 1986; Mochly-Rosen and Koshland, 1987; Woodgett and Hunter, 1987; Mitchell et al., 1989). This autophosphorylation may require aggregates of PKC, Ca* + , PS and Mg* + in a highly ordered manner (Bazzi and Nelsestuen, 1992), and it occurs at six basic amino acid residues in three regions of rat PKC PII, with the maximal phosphate incorporation of - 1.5 molecule per molecule protein (Flint et al., 1990). The three regions are the amino-terminal peptide, the carboxylterminal tail, and the hinge region between the regulatory domain and catalytic domain (Flint et al., 1990). Several lines of evidence suggest that autophosphorylation of PKC increases the affinity of the enzyme for phorbol esters and calcium (Huang et al., 1986), and increases its ability to phosphorylate Hl histone (Mochly-Rosen and Koshland, 1987). Autophosphorylation of PKC at distinct sites may cause distinct types of enzyme regulation. Autophosphorylation of an Nterminal region suggests that this region may play a role in the activation of PKC. Many protein kinases are regulated by a pseudosubstrate inhibitor sequence that keeps the enzyme inactive before stimulation. Since the region of PKCDII between amino acids 19 and 3 1 serves such an inhibitory function (House and Kemp, 1987), it is possible that autophosphorylation of the amino-terminal peptide might result in a conformational change that removes, or dislodges, the inhibitory peptide from the active site to allow substrate binding. Autophosphorylation of the threonine residues in the carboxyl tail of the catalytic domain may, however, enhance the kinase catalytic activity like the similarly located autophosphorylation site in pp60’+” (TyrS2’) and other tyrosine kinases. Finally, autophosphorylation of the hinge region may alter the susceptibility of the kinase to proteolysis, thus affecting its down-regulation (Flint et al., 1990).

I16 (1996) I-29

4.3. Feedback

9

regulu tion

The feedback regulation of PKC could occur at various levels including the components such as receptors, G-proteins, phospholipases and PKC itself. Several lines of evidence suggest that PKC is also capable of negatively regulating the activity of PLC, the enzyme that catalyses the formation of DAG (Drummond, 1985; Woods et al., 1986; Orellana et al., 1987; Hepler et al., 1988; Stassen et al., 1989; Sanchez-Bueno et al., 1990; Park et al., 1992; Rhee and Choi, 1992). By inhibiting PLC activity, PKC may decrease the production of IP, and be responsible for the sinusoidal oscillations in intracellular Ca2+ (Bird et al., 1993). In anterior pituitary cells, down-regulation or inhibition of PKC also causes an increased basal secretion of ACTH (Liu et al., 1990, 1992). More recently, studies in rat basophilic RBL-2H3 cells have shown that different isozymes of PKC mediate feedback inhibition of PLC and stimulatory signals for exocytosis; PKCc( and E inhibit antigen-induced PLC activation at 0.1 PM Ca’+, whereas PKCp and S are sufficient to stimulate exocytosis in these cells (Ozawa et al., 1993). The mechanism of PKC inhibition of PLC is likely to be due to PKC mediated serine phosphorylation of PLC, which may reduce tyrosine phosphorylation that activates PLC (Ozawa et al., 1993). In contrast to the effect on PLC, PKC may exert a positive feedback control over the activity of PLD. In several types of cells (Nishizuka, 1992; Ha and Exton, 1993) and various plasma membrane preparations (Pai et al., 1988; Qian and Drewes, 1990; Siddiqui and Exton, 1992) phorbol esters activate PLD. This activation by phorbol esters may be independent of protein phosphorylation, since PKC activates fibroblast plasma membrane PLD in an ATP-independent manner suggesting the involvement of an allosteric protein-protein interaction (Conricode et al., 1992). Additionally, over-expression of PKC in fibroblasts up-regulates agonist-stimulated activation of PLD, suggesting a role for PKC in the regulation of agonist-induced activation of PLD (Pachter et al., 1992; Eldar et al., 1993). Recent studies have attempted to determine which isoform of PKC is responsible for the stimulation of PLD. This has been suggested to be the pl-isoform in Rat-6 fibroblasts (Pai et al., 1991) the cc-isoform in Swiss 3T3 fibroblasts (Eldar et al., 1993) and the e-isoform in rat renal mesangial cells (Pfeilschifter and Huwiler, 1993). Thus PKC activation may act as a switch that terminates inositol phospholipid hydrolysis and activates the hydrolysis of phosphatidylcholine (Bishop et al., 1992). 4.4. Down-regulation Another important mechanism is down-regulation of the enzyme in response to continuous stimulation with

phorbol esters. When intact cells are exposed to maximal or submaximal concentrations of phorbol esters for reasonably long periods ( IO-72 h), phorbol ester binding PKC activity, and PKC immunoreactivity gradually fall to very low levels. Distinct PKC isozymes different susceptibilities to proteolysis by possess trypsin or endogenous proteases. For example, thyrotropin-releasing hormone selectively down-regulates PKCr: without affecting x- and /J-PKCs in GH4Cl cells (Akita et al., 1990b: Kiley et al.. 1991). Bombesin induces selective down-regulation of PKC6 and PKCI: without affecting x- and [-isoforms in Swiss 3T3 cells (Olivier and Parker, 1994). The PKC isozyme /j and x are degraded at ditferent rates in response to phorbol ester pretreatment (Huang et al.. 1989). Phorbol esters induce slow down-regulation of PKCI: and no downregulation of PKC< (Liyanage et al.. 1992; Olivier and Parker, 1992. 1994; Ways et al., 1992). This differential regulation of PKC isozymes occurs at the protein and not the mRNA level and is thought to be related to the selective compartmentation of the isoforms (Isakov et al.. 1990; Kiley et al.. 1991). It has been reported that PKCI: is localized to the Golgi apparatus in NIH 3T3 cells (Lehel et al.. 199.5). whereas PKC,Y is associated with the mitotic apparatus in primary cell cultures of the shark rectal gland (Lehrich and Forrest, 1994). It therefore seems that the relative susceptibility of different PKC species to down-regulation varies among different cell types and is at least partly dependent on the properties of the isoforms. The mechanism for the down-regulation of PKC has not been delineated. The disappearance of PKC is not due to decreased PKC biosynthesis. since cycloheximide does not alter phorbol ester binding or affect the recovery of these receptors from down-regulation (Collins and Rozengurt, 1984); in addition, the phenomenon is not prevented by agents known to prevent clustering and/or internalisation of hormone-receptor complexes (Solanki and Slaga, 1982). Phorbol ester-induced PKC down-regulation is mainly due to an increased rate of degradation of the enzyme. In GH, cells, PMA accelerated the proteolytic loss of immunologically identified [?]Met-labelled PKC in a concentrationand time-dependent manner (Ballester and Rosen. 1985). A rat brain Ca’ + -dependent neutral protease catalyses the limited proteolysis of PKC to a phospholipidand Cal+ -independent 50-kDa form (PKM) (Kishimoto et al., 1983). Protease inhibitors are capable of inhibiting the down-regulation of PKC (Chida et al., 1986; Ito et al., 1988b; Olivier and Parker, 1992). In addition, several proteases have been proposed to be responsible for the proteolysis of PKC, including calpain (Eto et al., 1995; Melloni et al., 1986; Kishimoto et al., 1989) and serine proteases (Chida et al., 1986). Recently, a 190kDa calpain-PKC complex composed of one molecule /(-calpain and one molecule PKCr has been demon-

strated in skeletal muscle, and immunofluorescence staining reveals their co-localization on the muscle fibre plasma membranes (Savart et al., 1995). In rat pituitary GH4C I cells. thyrotropin-releasing factor causes translocation of /I-calpain onto the plasma membrane, and the calpain-specific inhibitor. inhibitor 1 or calpastatin. inhibits thyrotropin-releasing hormone induced PKCc down-regulation (Eto et al., 1995). These data strongly suggest that calpain, a Ca’+-requiring thiol proteinase. is involved in the proteolysis of PKC converting it into PKM and further degrading the enzyme for down-regulation. In addition. PKC down-regulation requires plasma membrane association; PKC{, which does not translocate onto plasma membrane. does not undergo downregulation (Liyanage et al., 1992; Olivier and Parker. 1992, 1994; Ways et al., 1992). In contrast, a chimeric molecule containing the regulatory domain of PKCb fused to the catalytic domain of PKC; undergoes down-regulation when expressed in S. pomhr (Goode and Parker, 1994). It has been proposed that the binding of PKC to plasma membrane induces a conformational change that exposes the cleavage sites for proteases rendering the protein sensitive to proteolysis (Orr and Newton, 1994). The plasma membrane association of PKC thus is an important prerequisite condition for down-regulation. Moreover, it has been suggested that autophosphorylation of PKC may prime down-regulation by targeting the protein for proteolytic degradation, since point mutation of the ATP binding site of PKCr abolishes the kinase activity and downregulation (Ohno et al., 1990). More recently, it has been shown that insufficient down-regulation of PKC can be up-regulated in fruns by the presence of other types of PKC catalytic activity, suggesting that downregulation requires the function of certain PKC isotypes (Goode et al., 1995). Thus, PKC activation, autophosphorylation, catalytic subunit function and down-regulation are coupled events and down-regulation may be physiologically significant in cellular signalling. 4.5. L?.sospkingolipi~ls In addition, PKC is also negatively regulated by sphingosine and lysosphingolipids (Hannun and Bell, 1989; Merrill and Stevens, 1989). Sphingolipids are a major class of plasma membrane lipids and composed of a long-chain sphingoid base. an amide-linked fatty acid, and a polar head group at the l-position. Sphingosine, the major breakdown product of the sphingolipids, is a potent inhibitor of PKC (Bazzi and Nelsestuen, 1987; Hannun and Bell, 1989), and causes competitive inhibition of the binding of DAG and phorbol esters and noncompetitive inhibition of the binding of Ca’ + (Bazzi and Nelsestuen, 1987; Hannun and Bell. 1989). In addition. sphingosine may also

J.-P. Liu

I Molecular and Cellular Endocrinology 116 (1996) l-29

inhibit the catalytic domain under certain conditions (Nakadate et al., 1988). The inhibition of PKC by sphingosine affects its positive charge and hydrophobic nature (Hannun and Bell, 1989) and may be related to simple charge neutralisation of the lipid (Bazzi and Nelsestuen, 1987; Rando, 1988). The inhibition of PKC by sphingosine has raised the possibility that PKC may be physiologically regulated by both positive (DAG) and negative (sphingosine and lysosphingolipids) messengers (Hannun and Bell, 1989). The notion that sphingosine may function as a second messenger is supported by the findings that sphingomyelin levels (Nelson and Murray, 1982) and activation of a neural sphingomyelinase (Merrill and Stevens, 1989) are associated with dexamethasone-induced differentiation of 3T3-Ll pre-adipocytes; that exogenous DAG activates a sphingomyelinase in GH, pituitary cells (Kolesnick, 1989); that sphingomyelinase inhibits phorbol ester-induced differentiation of HL-60 cells (Kolesnick, 1989) and that vitamin D, decreases sphingomyelin levels and increases sphingomyelinase activity and phosphatidylcholine levels (Okazaki et al., 1989). 4.6. Cerumide Ceramide is a metabolite of sphingomyelin breakdown catalyzed by sphingomyelinase. It is produced in response to stimulation by various agonists including dihydroxyvitamin D1, tumor necrosis factors, interleukins and interferons, suggesting that it is a second messenger (Hannun, 1994; Kolesnick and Golde, 1994). Although ceramide has been shown to have a variety of potential functions including activating a specific protein kinase, a protein phosphatase and the mitogenactivated protein kinase, it appears that this molecule is also involved in the regulation of protein kinase C activity. However, the action of ceramide on PKC is isoform dependent and includes both direct and indirect effects. It has been shown that ceramide activates PKCi in vitro and in NIH 3T3 cells (Lozano et al., 1994). In contrast, ceramide inhibits PKCa translocation from cytosol to plasma membrane in both mouse epidermal and human skin fibroblast cells (Jones and Murray, 1995). While the mechanisms underlying the actions of ceramide are not clear, it has also been well documented that ceramide inhibits DAG kinase activity and antagonizes the action of DAG causing apoptosis in leukemia cells (Jarvis et al., 1994; Jayadev et al., 1995).

11

lated as a dimeric zinc-binding heat-stable 125-amino acid peptide with an apparent molecular weight of 17-kDa. This protein inhibits PKC activity specifically with a half maximal inhibition at 2.2 PM (Pearson et al., 1990), but the physiological relevance and mechanism of the inhibition are currently unclear (Fraser and Walsh, 1991). It does not compete for the binding of Ca’+, DAG and phospholipid, and unlike the autoinhibitory domain of PKC, PKC inhibitor I does not contain a peudosubstrate site. Since this protein binds to Zn2+ through a specific sequence motif, it is proposed that it may sequester cytosolic free Zn2 + or bind to PKC to modulate the interaction of PKC with the cell membrane (Pearson et al., 1990). 4.8. 14-3-3 Proteins 14-3-3 proteins belong to a highly conserved and widespread family of eukaryotic proteins. They are acidic proteins with a molecular weight of 29-33 kDa and an isoelectric point of about 4.5 playing a role in a variety of cellular functions including the regulation of PKC activity (Aitken et al., 1995; Jones et al., 1995). Seven isoforms of 14-3-3 proteins have been described from cc-q, but the isoforms tl and S are the phosphorylated forms of j3 and [ (Aitken et al., 1995). The 14-3-3 proteins are potent PKC inhibitors and are thus called kinase C inhibitor proteins (KCIP) (Toker et al., 1990, 1992). Whereas the phosphorylated forms possess a potent inhibitory effect on PKC, the non-phosphorylated forms have recently been shown to interact with and activate the proto-oncogene product raf. The 14-33 proteins inhibit PKC by acting on the Cl domain and competing with the binding of the PKC substrates ATP 3 Ca’+ , DAG or phorbol dibutyrate (Toker et al., 1990; Robinson et al., 1994). Sequence analysis has revealed an N-terminal region at amino acids 60-63 that is similar to part of the pseudosubstrate prototope, suggesting a possible mechanism for PKC inhibition. In addition, the region at residues 134- 150 shows a close similarity to the conserved carboxyl terminus of the lipocortin family of phospholipid-dependent Ca’ + binding proteins (Klee, 1988). A synthetic peptide based on this region prevents PKC binding to RACKS (Mochly-Rosen et al., 1991). suggesting that this region of 14-3-3 may play a role in modulating the targeting of PKC to the cell membrane. 4.9. Other fuctors

4.7. PKC inhibitor I A number of proteins have been suggested to inhibit PKC activity by inter-molecular protein-protein interaction (Aitken et al., 1990; Pearson et al., 1990; Schlaepfer et al., 1992). PKC inhibitor I is one of the best characterised endogenous protein inhibitors, iso-

Besides membrane sphingolipids, many other cellular factors also suppress PKC activity. These factors include Ca2 + binding proteins such as calmodulin and troponin C (Saitoh and Dobkins, 1986; Funder and Sheppard, 1987; Pate1 and Kligman, 1987; Baudier et al., 1989; McIlroy et al., 1991) annexin V (Schlaepfer et

al., 1992) reactive oxygen species (Gopalakrishna and Anderson, 1987). polyamines (Qi et al., 1983). interferon /? (Ito et al., 1988a), CAMP (Narindrasorasak et al., 1987), and cGMP (Pandey, 1994ab; Barnett et al.. 1995). and RNA aptamers (Conrad et al., 1994). 5. Protein substrates Like other serine and threonine kinases, PKC catalyses the transfer of phosphate from ATP to the free hydroxyl group of serine or (less frequently) threonine residues of substrate proteins (Nishizuka, 1980; Edeland Kemp, 1991). The man et al., 1987; Pearson protein substrates contain motifs that determine kinase specificity and these sequences can be either S*IT*XK: R, K/RXXS*/T*, K/RXXS*/T*XK/R. K/RXS*/T* or K/RXS*,‘T*XK/R (single amino acid codes. X is any amino acid) (Pearson and Kemp, 1991). A large number of potential substrates for PKC have been reported (Kuo et al., 1984; Nishizuka. 1984; Nishizuka et al.. 1984; Nairn et al.. 1985; Niedel and Blackshear, 1986; Witters and Blackshear, 1987) and, up to 1986, at least seven receptors and 18 endogenous proteins or enzymes have been found phosphorylated by purified PKC in in vitro cell-free systems, or in intact cells in response to agents known to activate PKC (Niedel and Blackshear, 1986). Table 2 shows the 110 currently known PKC substrate proteins several of which have been extensively studied over the last few years. These proteins include neuromodulin, the MARCKS protein, catecholaminergic and acetylcholinergic receptors, receptors for growth factors, the large molecular mass GTP-binding protein dynamin I and some proto-oncogene products. The following section provides a detailed description of our current knowledge of the PKC substrates neuromodulin, the MARCKS protein. dynamin I and some proto-oncogene products, focussing on their interaction with PKC and their roles in signal transduction and cellular function. 5.1. Neuronlodulirl Neuromodulin, also known as B50, GAP43, GAP48. Fl, ~57, or pp46, is a nervous tissue-specific protein, which is highly expressed in neurons during development and nerve regeneration and has been implicated in neurite outgrowth. long-term potentiation. neurotransmitter release and signal transduction. Neuromodulin has a molecular weight ranging from 43 to 57-kDa in SDS-PAGE. In bovine brain, there is approximately60 pmol neuromodulin per mg membrane protein from cerebellum, pineal gland, internal capsule, thalamus and hypothalamus, striatum and hippocampus (Andreasen et al.. 1983; Cimler et al., 1985). As a major component of growth-cone membranes, neuromodulin accumulates near the plasma membrane associating

with the cortical cytoskeleton and membranes (Skene et al., 1986; Aigner and Caroni, 1995). The bovine neuromodulin is a single polypeptide of 239 amino acid residues with a calculated molecular weight of 24.721kDa (Cimler et al., 1987; Karns et al., 1987; Wakim et al., 1987; Ng et al.. 1988). At the N-terminus, the cysteine residues at positions 3 and 4 are palmitoylated (Skene and Virag, 1989) and a nine-amino acid sequence constitutes a putative calmodulin binding site (Alexander et al., 1988). Neuromodulin derived from the brain of the cat (Sheu et al., 1990a), rat (Akers and Routtenberg, 1987; Nelson et al., 1989; Deloulme et al.. 1990) and bovine (Alexander et al., 1988; Baudier et al.. 1989) is phosphorylated by PKC. Studies with rat hippocampal synaptosomes show that this reaction is enhanced by the presence of Ca’+ (Akers and Routtenberg, 1987) and is mediated by the fr isozyme of PKC (Rosenthal et al., 1987; Sheu et al., 1990b). Although neuromodulin contains 14 serine and 14 threonine residues as potential phosphorylation sites, phosphoamino acid analysis indicates that the phosphorylation site of neuromodulin is at serine-41, which is adjacent to the single aromatic residue (Phe4’) contained within the 16 amino acid residues that binds to calmodulin (Coggins and Zwiers, 1989; Ape1 et al., 1990) and acidic phospholipids (Houbre et al., 1991). Mutagenesis of the serine-41 into threonine or alanine results in neuromodulin products with mobilities on two-dimensional electrophoresis that are similar to those of non-mutated recombinant neuromodulin and rat brain neuromodulin. However, the mutations prohibit PKC phosphorylation, suggesting that the serine-41 is the sole PKC phosphorylation site (Nielander et al., 1990). The consequence of phosphorylation of this site by PKC is a lowering of the affinity of the molecule for calmodulin, suggesting that phosphorylation of neuromodulin may increase the local concentration of free calmodulin in neuron (Liu and Storm, 1990). Neuromodulin is also subjected to dephosphorylation by calcineurin, a Ca2+and calmodulin-dependent protein phosphatase (protein phosphatase IIB) (Liu and Storm, 1989) and by protein phosphatase I and IIA (Han et al., 1992). Dephosphorylation of neuromodulin by calcineurin is a specific reaction with a rapid turnover of 70 nmol Pi/min/mg and a Km of 2.6 ,LIM (Liu and Storm, 1989). Thus, PKC phosphorylation and calcineurin dephosphorylation may serve as dual molecular switches to control calmodulin binding to and release from neuromodulin. In addition. the phosphatase calcineurin can be phosphorylated by both PKC and the Ca’ + /calmodulin-dependent protein kinase (PKII), and this reaction abolishes its ability to cause dephosphorylation (Hashimoto and Soderling, 1989). It is then possible that activation of PKC may increase the intracellular concentrations of tiee calmodulin by both direct phosphorylation and indirect inhibi-

J.-P. Liu 1 Moleculur and CeNulur Endocrinology I I6 (I 9961 I - 29 Table 2 Protein kinase

C substrate

proteins

Substrate

Reference

Receptors and G proteins (n = 20)

P-adrenergic receptor acetylcholine receptor serotonin receptor GABA receptor glutamate receptor CCK receptor vitamin D receptor insulin receptor insulin-like growth factor EGF receptor transferrin receptor interleukin II receptor ANP receptor T-cell antigen receptor thromboxane A2 receptor rhodopsin photoreceptor Gsr Gir GZ transducin (G, protein)

1 receptor

(Niedel and Blackshear, 1986) (Safran et al., 1990) (Raymond, 1991) (Kellenberger et al.. 1992) (Tan et al., 1994) (Gates et al., 1993) (Hsieh et al., 1993) (Lewis et al., 1990) (Niedel and Blackshear. 1986) (Niedel and Blackshear. 1986) (Niedel and Blackshear, 1986) (Niedel and Blackshear. 1986) (Sharma et al.. 1989) (Niedel and Blackshear. 1986) (Kinsella et al.. 1994) (Newton and Williams. 1993) (Pyne et al., 1992) (Yatomi et al., 1992) (Lounsbury et al., 1993) (Sagi-Eisenberg et al.. 1989)

Enzymes (n = 35)

adenylate cyclase guanylate cyclase cGMP phosphodiesterase tyrosine hydroxylase PI-phospholipase C phospholipase A2 tyrosine kinase protein kinase A protein tyrosine phosphatase 2C MAP kinase Ca’+ - and CaM-dependent kinase ribosomal protein S6 and S6 kinase creatine phosphokinase B pyruvate kinase /?ARK kinase phosphatase IIB (calcineurin) Na + /K + -ATPase Dynamin I GTPase exchanger Na+/H+ lipoxygenase cytochrome ~450 keratinocyte transglutaminase nitric oxide synthase AMP deaminase glycogen synthase glycogen synthase kinase-3 sepiapterin reductase NADPH oxidase poly(ADP-ribose) synthetase acetyl-CoA carboxylase RNA polymerase II eukaryotic initiation factors eukaryotic elongation factors deoxycytidine kinase S-adenosylmethionine synthetdse ~68 RNA helicase Cytoskeleton

(Yoshimasa et al.. 1987) (Louis et al., 1993) (Udovichenko et al.. 1994) (Niedel and Blackshear, 1986) (Bennett and Crooke, 1987) (Nemenoff et al.. 1993) (Gandino et al., 1994) (Newton and Taylor. 1995) (Zhao et al., 1994) (Kribben et al., 1993) (Waxham and Aronowski, 1993) (Lawen et al.. 1989: Meier et al., 1990) (Chida et al.. 1990) (Rosler and Schoner. 1990) (Pitcher et al., 1992) (Hashimoto and Soderling. 1989) (Lowndes et al., 1990) (Liu et al., 1994b) (Winkel et al., 1993) (Epstein et al.. 1989) (Chakravarty et al., 1990) (Bredt et al., 1992) (Thakkar et al., 1993) (Niedel and Blackshear. 1986) (Goode et al.. 1992) (Katoh et al.. 1994) (Niedel and Blackshear. 1986) (Tanaka et al., 1987) (Haystead and Hardie, 1988) (Chuang et al.. 1987) (Morley et al., 1991) (Kielbassa et al., 1995) (Wang and Kucera. 1994) (Pajares et al., 1994) (Buelt et al.. 1994)

proteins (R = 16)

myosin myelin basic protein adducin

(Niedel and Blackshear, 1986; Murakami (Niedel and Blackshear, 1986) (Bennett et al., 1988; Kaisr et al., 1989)

et al.. 1994)

I4 Table 2 (continued) Substrate

Reference

vinculin tahn cdldesmon vimentin desmin troponin calponin connexin fibronectin receptor integrins neurofilament protein microtubule-associated proteins cardiac sarcolemma protein dystrophin

(Werth and Pastan. 1984) (Litchfield and Bail. 1986) (Litchtield and Ball. 1987) (Lopez-Briones et al., 1990) (Kitamura et al.. 1989) (Noland et al.. 1989) (Mino et al.. 1995) (Saez et al.. 1990) (Freed et al., 1989) (Sihag et al.. 1988) (Hoshi et al.. 1988) (Niedel and Blackshear. 1986) (Senter et al.. 1995)

Proto-oncogene

products (n = 13)

(Jeng et al.. 1987) (Ghosh and Baltimore. 1990) (Kolch et al.. 1993) (Li et al.. 1992) (Hardy et al.. 1993) (Kapiloff et al.. 1991) (Goldberg et al.. 1988) (Abate et al.. 1991) (Baker et al.. 1992) (Luttrell et al.. 1989) (Gandino et al.. 1990) (Pendergast et al.. 1987) (Saksela et al., 1989)

P2 1r.‘* inhibitor-,B Raf-I myogenin MRF4 Pit-l p46c-erbA /OS ;lttl pp60c-src C-tNcI rrhl L-IllJ,1~ Nuclear

proteins (n = 9)

lamin B DNA topoisomerase I and II DNA methyl transferase IP, receptor CCAAT enhancer binding protein CREB Pit-l p53 PICKI

(Hocevar et al.. 1993) (Samuels et al., 1989) (Niedel and Blackshear. 1986) (Matter et al., 1993) (Mahoney et al.. 1991) (Yamamoto et al.. 1988) (Howard and Maurer. 1994) (Takenaka et al.. 1995) (Staudinger et al.. 1995)

Others (n = 17)

MARCKS neuromodulin neurogrdnin pleckstrin GTPase activating protein protamines glucose transporter lipocortins annexins sterol carrier protein amyloid precursor peptide NAP-22 PEA-15 phospholemman A-type K+ channel N-type Ca’+ channel Na-K-Cl cotransportet

(GAP)

(Niedel and Blackshear. 1986) (Niedel and Blackshear. 1986) (Baudier et al.. 1991) (Tyers et al.. 1988) (Gschwendt et al.. 1993) (Souvignet and Chambaz, 1990) (Niedel and Blackshear, 1986) (Stoehr et al., 1990) (Toker et al., 1992) (Steinschneider et al.. 1989) (Gandy et al., 1988) (Maekawa et al., 1994) (Danziger et al.. 1995) (Walaas et al., 1994) (Covarrubias et al.. 1994) (Hell et al.. 1994) (Torchia et al.. 1994)

tion of calcineurin dephosphorylation of neuromodulin, and after calmodulin is released calcineurin is then activated to dephosphorylate neuromodulin providing a

mechanism calmodulin subsequent

for feedback control of the increase in free concentrations. The resultant increase and decrease in free calmodulin might then reg-

J.-P. Liu / Molecular and Cellular Endocrinology

ulate the activities of the Ca2 + /calmodulin-dependent enzymes adenylate cyclase and PKII (Liu and Storm, 1990). Neuromodulin binds fluorescein-labelled calmodulin with the dissociation constants 0.2 and 1.0 PM in the presence or absence of excess Ca* + chelator, suggesting that it has a higher affinity for calmodulin in the absence than in the presence of Ca2+ and that most, if not all, calmodulin is bound to neuromodulin in unstimulated cells (Alexander et al., 1988). The binding of calmodulin to neuromodulin has been localized to residues 39-55 of neuromodulin (Alexander et al., 1988; Chapman et al., 1992). However, the findings that the binding calmodulin produces a structural conformation by stabilizing a basic, amphiphilic a-helix within a neuromodulin molecule while Ca2 + is absent at physiological salt concentrations suggest that calmodulin regulates the biological activities of neuromodulin through an allosteric, Ca*+ -sensitive mechanism that can be uncoupled by PKC-mediated phosphorylation (Gerendasy et al., 1995). Furthermore, neuromodulin inhibits Ca2 + -activated calmodulin-dependent nitric oxide synthase through the interaction with calmodulin that is prevented by PKC phosphorylation and can be regained by calcineurin dephosphorylation (Slemmon and Martzen, 1994). Thus, neuromodulin may serve as a local calmodulin store at the plasma membrane of growth cones in neurons, regulated by Ca* + , phosphorylation and dephosphorylation (Liu and Storm, 1990). Neuromodulin may play an important role in synaptic plasticity. It has been shown that regulation of neuronal PKC activity directly affects the persistence of long-term potentiation (LTP), a model of neuronal plasticity (Lovinger et al., 1986; Routtenberg et al., 1986; Linden et al., 1987) and the in vitro phosphorylation of neuromodulin by PKC correlates with persistence of LTP (Lovinger et al., 1986). Examination of PKC activity and endogenous neuromodulin phosphorylation in the cerebral cortex of the developing cat has also shown that an increased level of neuromodulin phosphorylation occurred during a critical period, after which time adult levels of the PKC substrate are maintained in the adult (Sheu et al., 1990a). In contrast, a 46% decline in neuromodulin phosphorylation has been noted in the hippocampus of the aging rat, suggesting a possible role for neuromodulin phosphorylation in the age-related decline in hippocampal synaptic plasticity (Barnes et al., 1988). In addition, immunocytochemical studies have shown that neuromodulin is present in the O-2A glial cell line, including O-2A bipotential glial precursor cells derived from newborn rat brain, oligodendrocytes and type 2 astrocytes, suggesting that neuromodulin may play a more general role in neuronal plasticity during development of the central nervous system (Deloulme et al., 1990). Furthermore, that neuromodulin may enhance a neurite outgrowth has been

I I6 (1996) l-29

15

suggested by the enhanced nerve growth factor responsiveness of PC-12 cells that overexpress neuromodulin (Yankner et al., 1990) and by the reduced neuritogenesis in the presence of intracellular anti-neuromodulin antibodies (Shea et al., 1991). However, it has been also shown that PC12 cells (Baetge and Hammang, 1991) or sensory neurons (Aigner and Caroni, 1995) that lack the protein can still extend neurites, but the growth cones of the sensory neuron become devoid of local f-actin and fail to produce spreading and branching in response to stimulation (Aigner and Caroni, 1995). These data suggest that neuromodulin may play roles in promoting evoked morphogenic activity and resistance to retraction, probably by promoting f-actin accumulation and modulating the transduction machinery of the growth cones. Besides its role in neuronal plasticity, neuromodulin may also be involved in neurotransmitter release from nerve terminals. Chemical depolarisation (30 mM K+) and activation of PKC with phorbol esters increase neuromodulin phosphorylation and stimulate neurotransmitter release from hippocampal slices, and both of these events are inhibited by the PKC inhibitor polymyxin B. Treatment of rat brain synaptosomes with high K+ or veratridine also causes a parallel increase in neuromodulin phosphorylation and noradrenaline release, and the phorbol ester phorbol 12,13dibutyrate enhances depolarisation-induced neuromodulin phosphorylation and noradrenaline release (Dekker et al., 1990). In addition, affinity-purified anti-neuromodulin antibodies which interfere with PKC phosphorylation of neuromodulin inhibit Ca2 + -induced noradrenaline and cholecystokinin-8 release from streptolysin O-permeated synaptosomes (Hens et al., 1993). A reduction of endogenous neuromodulin in PC12 cells transfected with a recombinant expression vector coding for antisense human neuromodulin cRNA blocks Ca2+ -mediated neurotransmitter dopamine release, although appreciable quantities of dopamine and secretory granules with a normal appearance have been observed (Ivins et al., 1993). These studies suggest that neuromodulin and PKC phosphorylation may be essential in the process of neurotransmitter release from nerve terminals. Although PKC phosphorylation and neuromodulin regulation of calmodulin are clearly involved in some of the functions of neuromodulin in neuronal plasticity and neurotransmitter release, detailed molecular mechanisms of the function of neuromodulin’s have not been fully elucidated. It is interesting to note that in addition to serving as a local calmodulin store, neuromodulin interacts with G, protein (Strittmatter et al., 1990; Strittmatter et al., 1991). The N-terminal motif of neuromodulin amino acid residues I-10 mimics the cytoplasmic tail of Go-linked receptors to activate G, by enhancing guanine nucleotide exchange, increasing the

dissociation of GDP from, and association of GTP with, the a-subunit (Sudo et al., 1992). In addition. neuromodulin increases the GTPase activity of G,,, independent of phospholipids and fiy subunits and not blocked by pertussis toxin (Strittmatter et al., 1990, 199 1). These suggest that neuromodulin may be a guanine nucleotide release protein potentially involved in the intracellular regulation of receptor-coupled G,,, a major component of growth cones of neurons. However, the precise relationship between G,, neuromodulin, calmodulin, and PKC remains to be determined. 5.2. MARCKS The MARCKS protein, another major PKC substrate from mammalian cells and tissues (Niedel and Blackshear, 1986; Blackshear. 1993). migrates in SDSPAGE with an apparent molecular mass of 80-87 kDa and was therefore initially called 80K or 87K protein. MARCKS purified from the brain of several species appears to be homologous and its amino acid sequence and the cDNA encoding its gene have been reported (Sakai et al., 1989; Stump0 et al., 1989). This protein contains alanine and glutamate in large proportion but has no tyrosine or tryptophan (Albert et al., 1987; Pate1 and Kligman, 1987; Morris and Rozengurt, 1988). In the N-terminal of MARCKS, there is a myristoylation consensus sequence where the glycine residue is myristoylated in intact cells (Aderem et al., 1988; Graff et al., 1989a; McIlhinney and McGlone, 1990). In the middle region of the protein, there is a 25residue basic domain with four serine residues which serve as PKC phosphorylation sites (Stump0 et al., 1989). The MARCKS cDNA in the bovine has an open reading frame of 1005 base pairs, punctuated by one 1794-bp intron, and predicts a protein of 335 amino acids with M, 31 949 (Sakai et al., 1989; Stump0 et al., 1989). When expressed in cells lacking MARCKS, this cDNA encodes a protein which migrates on SDS-PAGE with an apparet al.. 1989). This ent Mw of 80-87 kDa (Stump0 presumably results from its particular rod-like structure, acidic pl 4.4, and general hydrophilicity. The MARCKS protein is an extremely elongated monomer (frictional ratio 3.2 and axial ratio 60) (Albert et al., 1987), and its dimensions are 4.4 x 36 nm (Hartwig et al., 1992). Recently. a MARCKS relative called F52. MacMARCKS or MRP (MARCKS-related protein) has been cloned from mouse, rabbit and human cDNA libraries (Blackshear, 1993). With homology of sequences and analogy of biochemical properties. MRP is expressed with high levels in macrophages and reproductive tissues and its gene is located on a different chromosome from that encoding MARCKS (Blackshear, 1993). These suggest that there is a superfamily of MARCKS proteins with distinct properties and functions in eukaryotic cells.

MARCKS is an acidic and heat-stable protein present in all bovine and rat tissues examined. The mRNA of MARCKS is most highly expressed in the brain. spinal cord, spleen, and lung and these sites contain the highest concentration of immunoreactivity. MARCKS is found in the brain of the rat, mouse, rabbit, cow, monkey and in man (Albert et al., 1986; Blackshear et al., 1986; Ouimet et al., 1990). It is widely distributed throughout the rat brain including the pineal gland and pituitary and is concentrated in the piriform and entornhinal cortices, portions of the amygdaloid complex, the intralaminar thalamic nuclei, the hypothalamus, the nucleus of the solitary tract, nucleus ambiguus, and many catecholaminergic and serotoninergic nuclei (Ouimet et al., 1990). Electron microscopic analysis has revealed that MARCKS is present in both neuron and glial cells (Ouimet et al., 1990) and in the neuron MARCKS is concentrated in higher-order dendrites, axons, axon terminals, and is often associated with microtubules. However, the MARCKS protein is present throughout the cell membrane and cytosol of glial cells, suggesting that it could be involved in membrane-cytoskeleton interactions (Ouimet et al., 1990). In bovine cerebral cortex, 40% of the protein is contained within the synaptosomal membrane. 25% within the cytosolic fraction, 2O”X within the nuclear fraction, 10%) within the synaptosomal cytosol, and 5% within the microsomal fraction (Albert et al.. 1986). In human neutrophils, however, 80-90% of MARCKS is associated with the plasma membrane where it co-localises with PKC (Thelen et al.. 1991). MARCKS is phosphorylated on serine residues of the 25-residue basic domain by PKC, the stoichiometry being two to four molecules of “P per molecule MARCKS protein (Albert et al., 1987; Pate1 and Kligman, 1987; Morris and Rozengurt, 1988; Manenti et al.. 1992). Sequencing indicates that at least three serine residues (Ser15’, Ser”‘. Serlh’) are phosphorylated by PKC (Seki et al.. 1995). The phosphorylation of MARCKS appears to be mediated by different PKC isozymes, with the 80K-L and the 80K-H species phosphorylated by the type III, and type I and II isozymes, respectively (Sakai et al., 1989; Hirai and Shimizu, 1990). This may suggest the existence of several species of MARCKS proteins that may possess different affinity for different PKC isoforms. Recent studies indicate that the recombinant MARCKS protein is phosphorylated by PKCx, S and c isoforms with high affinity, but not by [ isoform. The K,,, values for the PKC isoforms 2. b and i; are 10.7, 20.7 and 29.8 nM, respectively (Fujise et al., 1994). The phosphorylation of MARCKS by PKC has been shown to be reversed by a combination of protein phosphatase 1, phosphatase 2A and phosphatase 2B (calcineurin) (Seki et al.. 1995). Phosphatase 2A and calcineurin appear to preferentially dephosphorylate serine’“’ and serine”“,

J.-P. Liu / Molecular and Cellular Endocrino1og.v 116 (1996) 1-29

respectively, but dephosphorylate serine15’ poorly (Seki et al., 1995). These data suggest that MARCKS phosphorylation is controlled by multiple factors including different PKC isoforms and protein phosphatases. In support of this notion, recent studies have shown that MARCKS is also phosphorylated in vivo on six serine residues in the N-terminal half of the molecule by proline-directed protein kinase (Taniguchi et al., 1994). In addition, MARCKS phosphorylation by PKC is inhibited by calmodulin and Ca*+ both in vitro and in intact cells (Chakravarthy et al., 1995; Sheu et al., 1995). The findings that calmodulin inhibition of MARCKS phosphorylation requires the presence of Ca”+ (Sheu et al., 1995; Chakravarthy et al., 1995) and is not significantly prevented by the calcineurin inhibitor cyclosporin A (Chakravarthy et al., 1995) suggest a direct interaction of MARCKS with the Ca2 + -bound calmodulin conformation, implicating both a possible feedback control mechanism for MARCKS phosphorylation and that MARCKS is also phosphorylated in vivo on six serine residues in the N-terminal half of the molecule by proline-directed protein kinase (Taniguchi et al., 1994). The phosphorylation of MARCKS by PKC may be important in diverse cellular functions, including growth receptor-dependent mitogenesis, macrophage activation and neurosecretion (Blackshear et al., 1986; Aderem et al., 1988; Thelen et al., 1991; Hartwig et al., 1992). For example, in adipocytes, soleus muscle and BCjH-1 myocytes, insulin is capable of stimulating MARCKS phosphorylation accompanied by PKC translocation (Arnold et al., 1993). In the macrophage, the MARCKS protein is distributed in a punctate fashion at the interface between pseudopodia and filopodia and is co-localised with vinculin and talin (Rosen et al., 1990). When PKC is activated by phorbol esters, rapid disappearance of punctate MARCKS staining follows, accompanied by cell spreading and loss of filopodia. The morphological changes in the macrophage and the disappearance of the punctate distribution of MARCKS follow a time course that closely approximates the PKC-dependent phosphorylation of MARCKS and its release from the plasma membrane (Rosen et al., 1990). In anterior pituitary cells, however, arginine vasopressin causes a marked, but temporary increase in MARCKS phosphorylation followed by dephosphorylation; the phosphorylation of the MARCKS protein appears to closely parallel hormone-induced ACTH release suggesting a possible involvement for MARCKS in the process of ACTH exocytosis (Liu et al., 1994a). It is possible that PKCdependent phosphorylation of MARCKS plays roles in the interplay of cellular signalling pathways in the regulation of the membrane cytoskeleton functions. Support for this notion also comes from the observations that murine MARCKS binds large unilamellar

17

phospholipid vesicles through electrostatic interaction of its basic domain with acidic lipids, an interaction inhibited by phosphorylation, calmodulin and high ionic strength (Kim et al., 1994). It is intriguing to speculate that MARCKS might bind intracellular transport vesicles, thereby regulating cell secretion and membrane trafficking. When it is phosphorylated by PKC in isolated nerve terminals or macrophages MARCKS protein undergoes a stimulation-dependent translocation from the plasma membrane to a cytosolic compartment (Wang et al., 1989; Rosen et al., 1990). The phenomenon of MARCKS translocation appears to be dependent on the cell type as it does not occur in BC,Hl myocytes (James and Olson, 1989a) or in Nl E-l 15 neuroblastoma cells, although phorbol ester-induced phosphorylation is apparent (Byers et al., 1993). The translocation of MARCKS in human neutrophils is reversible since the phorbol ester P-TPA and f-Met-Leu-Phe cause a transient phosphorylation and translocation of MARCKS followed by dephosphorylation and reassociation of the protein with the plasma membrane within a few minutes (Thelen et al., 1991). The ability of phosphorylation to regulate the subcellular localisation of MARCKS is confirmed by a recent analysis of the difference in the protein found in membrane-associated and cytosolic pools. This study has revealed that the cytoplasmic preparation contains more phosphorylated species than the membrane preparation, and that in vitro phosphorylation of MARCKS leads to the incorporation of more phosphate groups into the membraneassociated form than into the cytosolic form (4 vs. 2.9 molecules/molecule) (Manenti et al., 1992). It is also of interest that a similar phosphorylation-regulated binding of the protein to filamentous actin has been established (Hartwig et al., 1992). Dephosphorylated MARCKS, but not the phosphorylated form, crosslinks actin filaments with a periodical association of about 20 nm. This cross-linking is inhibited by Ca2+ and calmodulin, suggesting an involvement of the phosphorylation site domain in the actin interaction. These studies suggest that activation of PKC and increases in intracellular Ca’ + concentrations regulate the interaction of the MARCKS protein with both the plasma membrane and the actin cytoskeleton during murine macrophage chemotaxis (Hartwig et al., 1992). As a membrane-associated protein, myristoylation of MARCKS is thought to be necessary for targeting this protein to the cell membranes (Aderem et al., 1988; Graff et al., 1989a), although factors in addition to myristojrlation also appear to be required for this’to occur (James and Olson, 1989b; Kim et al., 1994). However, MARCKS undergoes translocation from plasma membrane to cytosol upon phosphorylation by PKC, though phosphorylation does not affect myristoylation of MARCKS (James and Olson, 1989,;b).Neither

myristoylation nor plasma membrane association is required for MARCKS to undergo phosphorylation, as a mutation that prevents myristoylation of MARCKS does not prevent the phosphorylation of the protein in intact cells stimulated by phorbol esters (Grdff et al., 1989a). Myristoylation of MARCKS is inhibited by cycloheximide in BC,Hl myocytes (James and Olson, 1989b) but not in the rat brain (McIlhinney and McGlone, 1990), suggesting that MARCKS myristoylation might be related to protein synthesis and thus probably determined by tissue-specific factors. Recent studies show that the MARCKS protein binds to the plasma manner, membrane ~~60’.“” in a myristate-dependent suggesting that the MARCKS protein may associate with cell membrane partly through binding to the plasma membrane cytoplasmic protein ‘receptor’ (Resh and Ling, 1990). In addition, MARCKS also binds to cell membranes which have been boiled or trypsin digested, suggesting that the protein binds to cell membrane lipids (George and Blackshear, 1992; Taniguchi and Manenti, 1993). This has also been shown by studies in which purified MARCKS protein binds pure phospholipid membranes in a phosphorylation-dependent fashion (Taniguchi and Manenti, 1993; Kim et al., 1994). These findings suggest that the MARCKS protein associates with plasma membrane through both the N-terminal myristoyl moiety and the middle 2% residue basic phosphorylation domain (Taniguchi and Manenti, 1993). PKC phosphorylation may cause MARCKS dissociation from the plasma membranes of nerve endings (Wang et al., 1989) and macrophages (Rosen et al., 1990) by making the molecule strongly negatively charged and decreasing electrostatic interactions between the basic phosphorylation domain and membrane phospholipids. Furthermore, the MARCKS protein may dissociate from plasma membrane by demyristoylation catalyzed by an unidentified cytoplasmic factor in an ATP-dependent manner regulated by calmodulin and Ca’+ (Manenti et al., 1994). Since MARCKS protein and neuromodulin shares several characteristics such as an anomalous migration on SDS-PAGE, an acidic isoelectric point, and an unusual amino acid composition, it has been proposed that the MARCKS protein may also bind to calmodulin. The MARCKS protein from bovine or avian sources can cross-link an equimolar amount of “‘I-labelled calmodulin with an apparent dissociation constant (&) of 0.65 PM (Tokumitsu et al., 1989; Graff et al., 1989b). Moreover, the binding is dependent on Ca’+ and is regulated by phosphorylation. The finding that phosphorylation of MARCKS by PKC prevented ‘251-calmodulin binding and cross-linking suggests that the calmodulin-binding domain of MARCKS may be located at, or near, the PKC phosphorylation sites (Graff et al., 1989b). Indeed, the 25-residue domain of basic amino acids in MARCKS protein is similar to

previously described calmodulin binding domains; further, it has been shown that a synthetic peptide correwith sponding to this domain could compete MARCKS for binding to calmodulin and could also be cross-linked to ‘~51-calmodulin in a calcium-dependent manner (Graff et al., 1989b). Phosphorylation of the synthetic 25-amino acid peptide by PKC inhibits the binding of this peptide to calmodulin and causes the dissociation of calmodulin with a time course that paralleled phosphorylation (McIlroy et al., 199 1). These studies therefore strongly suggest that in basal state the MARCKS protein binds and sequesters calmodulin. and that when activation of PKC occurs the PKC-induced phosphorylation of the MARCKS protein releases calmodulin which then stimulates other calmodulin-dependent enzymes such as adenylate cyclase, phosphodiesterase. PKII and calcineurin. Calmodulin also regulates both the polymerisation and cross-linking of cytoskeleton polymers including actin and spectrin, by interaction with a number of different cytoskeleton proteins (Stromqvist et al., 1988; Carraway and Carraway, 1989; Steiner et al., 1989) and is also essential for cell proliferation (Davis et al.. 1986; Rasmussen and Means, 1989). Studies in mice deficient in MARCKS have shown that the MARCKS protein plays a vital role in the normal development of neurulation, brain hemisphere fusion, forebrain commissure formation and formation of cortical and retinal laminations; deletion of the MARCKS gene from the mice results in death and severe cerebral abnormalities including agenesis of the corpus callosum and other forebrain commissures, and failure of fusion of the cerebral hemispheres (Stump0 et al., 1995). It is not yet known how the MARCKS protein is critically involved in normal mouse brain development and postnatal survival and whether posttranslational modifications such as phosphorylation play roles in these processes. In the brains of patients with Alzheimer’s disease (Cole et al.. 1988) and in NIH 3T3 cells transformed by a variety of oncogenes such as raf and ras (Oh-uchida et al., 1990) phosphorylation of the MARCKS protein is decreased suggesting that MARCKS phosphorylation may be involved in the pathogenesis of Alzheimer’s disease and inversely correlated with cellular transformation. The hypothesis that MARCKS phosphorylation may be involved in cellular transformation is also supported by studies with preneoplastic and neoplastic mouse JB6 cells, in which a progressive decline in the MARCKS protein and its mRNA was observed during progression from a preneoplastic to neoplastic phenotype. The loss of MARCKS protein expression in neoplastic cells cannot be attributed to a lack of PKC, since PKC activity is similar in cells of all phenotypes tested (Simek et al.. 1989). In contrast. synthesis of the MARCKS protein is increased in human neutrophils stimulated by tumor

J.-P. Liu / Molecuhr

und Cellulur Endocrinolog_v I16 (1996) I-29

necrosis factor-m (TNF-cr) and bacterial lipopolysaccharide (LPS). Although TNF-a and LPS do not cause MARCKS protein phosphorylation, they enhance MARCKS phosphorylation when neutrophils are exposed to PKC activators (Thelen et al., 1991). The MARCKS protein in quiescent Swiss 3T3 cells is rapidly phosphorylated by phorbol esters, bombesin, and bradykinin (Erusalimsky et al., 1988; Issandou and Rozengurt, 1990; Rozengurt, 1990); bombesin-induced MARCKS phosphorylation is accompanied by a chain of events including Ca2+ mobilisation, increased Na + and K’ fluxes, transmodulation of the EGF receptor, enhancement of CAMP accumulation, and expression of the proto-oncogenes c-fos and c-myc (Rozengurt, 1990). Bombesin-stimulated mitogenesis may also be partly mediated by activation of G-protein and PKC, and MARCKS phosphorylation (Rozengurt, 1990). Taken together, these findings indicate that MARCKS and its phosphorylation by PKC may be involved in the regulation of cell growth, differentiation and normal function. 5.3. Dynamin I Dynamin I is a large molecular weight GTP-binding protein with high intrinsic GTPase activity involved in controlling intracellular membrane trafficking (Liu and Robinson, 1995). It was initially observed as a phosphoprotein in nerve terminals (also called P96 or dephosphin) (Krueger et al., 1977; Robinson and Dunkley, 1983) then isolated from bovine brain as a microtubule-binding protein and later revealed to be a member of GTP-binding protein superfamily (Obar et al., 1990). It is a single polypeptide chain of 851-864 amino acids containing the well-conserved tripartite consensus sequence elements, characteristic of most GTP-binding proteins, within the N-terminal 300 amino acids (Obar et al., 1990; Chen et al., 1991; Robinson et al., 1993). Dynamin I also contains a middle unknown function domain, a PH domain and the C-terminal proline-rich tail containing an Src homology 3 (SH3) binding domain. It is present in four intracellular pools of rat brain - 10% cytosolic, 20% peripheral membrane-associated, 15% integral membrane-associated and 55% cytoskeleton-associated (Liu et al., 1994b). The major association with the particulate fractions suggests that dynamin I may interact with plasma membrane phospholipids. Tuma et al. have demonstrated that phospholipids stimulate dynamin I GTPase activity (Tuma et al., 1993) and in a phospholipid-binding assay purified dynamin I binds phospholipids (Liu et al., 1994b). However, dynamin I binding to plasma membranes or phospholipids is regulated by GTP, ATP and Ca2+; both GTP and ATP prevent dynamin I binding to the plasma membrane, but at physiological concentrations Ca’ + enhances the bind-

19

ings (Liu et al., 1994b). These findings suggest that dynamin I is a member of the group of Ca2 + -sensitive phospholipid-binding proteins such as PKC, phospholipase C-y, or annexins (Klee, 1988; Clark et al., 1991), although it possesses no sequence homology to these proteins. Analysis of interaction between dynamin I and PKC has shown that PKC phosphorylation of dynamin I has extraordinary kinetics, with an initial increase phase reaching to almost a plateau followed by inhibition of the phosphorylation (Liu et al., 1994b). The incremental phase of dynamin I phosphorylation shows a high affinity of the protein for PKC (S,,, 0.36 + 0.1 p M). It is thus possible that this high affinity interaction between dynamin I and PKC at least partly renders dynamin I phosphorylated by PKC in resting nerve terminals as previously reported (Robinson, 1992). But when higher than 0.5 PM dynamin I is used phosphorylation of dynamin I by PKC was markedly inhibited. These unusual kinetics may implicate a more complex interaction between dynamin I and PKC when the dynamin I concentrations are high, and it could be of physiological significance for dynamin I to serve as a PKC inhibitory factor when one considers the possible concentrations of dynamin I in nerve terminals to be 20-100 PM. In addition, dynamin I is stoichiometritally phosphorylated by PKC at more than one site (Liu et al., 1994b). Papain digestion of dynamin I to cleave off the C-terminal tail shows the phosphorylation sites are located in the tail region. Furthermore, PKC phosphorylation of dynamin I promotes the formation of a tetramer form of dynamin I (unpublished data), suggesting that PKC regulates the structure of dynamin I and may thus affect its function. Dynamin I also is a substrate for calcineurin both in vitro and in intact nerve endings (Liu et al., 1994~). Calcineurin at low concentrations causes a rapid dephosphorylation of purified PKC phosphorylated dynamin I. Analysis of the dephosphorylation suggests that dynamin I possesses high affinity for calcineurin (K,,, 0.5 + 0.01 PM), the highest affinity ever reported for a calcineurin protein substrate. The dephosphorylation is also specific, in that other protein phosphatases tested have no effect. In the intact nerve terminals, depolarisation induces a reversible dephosphorylation of dynamin I (Robinson et al., 1993) and a decrease of dynamin I GTPase activity (Liu et al., 1994~). The decrease of dynamin I GTPase activity is abolished by removal of extracellular Ca2 + or by the calcineurin inhibitor cyclosporin A (Liu et al., 1994~). Signal transduction and Ca* + -mediated regulation of dynamin I in nerve terminals are therefore established at least in part with PKC and calcineurin as upstream regulators of the GTPase, placing the protein phosphatase calcineurin squarely into the context of neurotransmission (Liu et al., 1994c).

What is the functional consequence of PKC phosphorylation and calcineurin dephosphorylation of dynamin I’? Purified dynamin I possesses a very high intrinsic GTPase activity (0.2 s ‘), much greater than the GTP hydrolysis rates ( b’,,i,,) of C, (224 min ‘), and higher than that of Ras proteins in the presence of GTPase activating protein (GAP) (0.4 min ‘) (Shpetner and Vallee, 1992). The GTPase activity of dynamin 1 has been shown to be stimulated several fold by microtubules (Maeda et al., 1992; Shpetner and Vallee, 1992), phospholipids and endogenous brain vesicles (Tuma et al.. 1993) and by some SH3 containing proteins (Gout et al., 1993; Herskovits et al., 1993b). Although none of these stimulators of dynamin 1 GTPase activity has yet been demonstrated to play a regulatory role for dynamin I in vivo, PKC phosphorylation stimulates dynamin I GTPase activity to a similar level to that by microtubules (more than ten-fold) (Robinson et al.. 1993). This suggests that in resting nerve terminals dynamin 1 is phosphorylated by PKC (Robinson et al.. 1993) and its GTPase activity is thus up-regulated (Liu et al.. 1994~). Upon depolarisation of the nerve terminals, the GTPase activity of dynamin I is decreased by calcineurin dephosphorylation (Liu et al.. 1994~). Thus, PKC phosphorylation and calcineurin dephosphorylation may serve as molecular switches to control dynamin I GTPase activity in nerve terminals. Dynamin I GTPase activity has been implicated in membrane endocytic trafhcking (Liu and Robinson. 1995). Sequence analysis of the dynamin I gene reveals that it is a homologue of a Dmsop/~ib protein expressed by a temperature-sensitive mutant shi6ir~ (Kessell et al., 1989; Chen et al.. 1991; Van der Bliek and Meyerowitz. 1991). The high degree of sequence conservation between rat dynamin I and the product of the D~~sophi/rr shihirr gene suggests closely related functions. Adult flies with this phenotype show a temperature-sensitive paralysis. The shibiw flies behave normally at permissive temperatures ( < 21°C) but undergo reversible paralysis within a minute at the non-permissive temperatures ( > 28°C). The paralysis is traced to an inability to reform synaptic vesicles after releasing their contents in the nerve terminals at the neuromuscular junction (Poodry and Edgar, 1979; Kosaka and Ikeda. 1983b). Subsequent studies have shown that it is a general defect in the formation of clathrin-coated and noncoated vesicles at the plasma membrane of neuron and other cells (Kosaka and Ikeda, 1983b; Kessell et al., 1989). The shihiw defect is thought to occur at an early stage of the pinching-OR mechanism of the vesicle (Kosaka and Ikeda. 1983a: Van der Bliek et al., 1993) and to be due to specific mutations in the GTP-binding domain (Chen et al., 1991; Van der Bliek and Meyerowitz. 1991). Studies transfecting a mutated dynamin I gene encoding the GTP binding domain into mammalian cells further demonstrate a blockade of

constitutive and receptor-mediated endocytosis (Herskovits et al., 1993a: Van der Bliek et al., 1993;). These studies indicate that dynamin I GTPase activity plays a general function in the endocytic membrane trafficking involved in endocytosis in both neuronal and non-neuronal tissues. Although it is still not clear how dynamin I is involved in the control mechanism of endocytosis, recent studies have provided interesting findings that dynamin I forms spring-like helical arrays around the invaginated pits in the synaptosomes in the presence of GTP;qS, suggesting that dynamin I may promote budding in the GTP-bound conformation and induce pinching off when the GTP is hydrolyzed (Take1 et al., 1995).

Proto-oncogenes are normal cellular genes and are counterparts of the viral oncogenes involved in neoplasia. They have been very highly conserved through evolution and their products are elements of a cellular signalling network which includes external hgands and growth factors, plasma membrane receptors, small GTP-binding proteins. cytoplasmic protein kinases. and nuclear transcription factors (Cantley et al., 1991; Hunter. 1991). Most proto-oncogene products are involved in the cascade of events by which growth factors stimulate normal cell division. During signal transduction, however, some proto-oncogene products interact with other signalling systems including the PKC pathway. In this regard, several proto-oncogene products have been reported to be PKC substrate proteins (Table 2). P21 I%“,a small GTP-binding protein, is the product of c-ras proto-oncogene family. It binds GTP to catalyse its hydrolysis to GDP in order to stimulate cellular growth (Field et al., 1987; Satoh et al.. 1988). P21’“‘ is required for the initiation of a cycle of DNA synthesis (S phase) in all normal cell types tested (Mulcahy et al., 1985). However, it has been shown that the Harvey (H)-ras p21 can be phosphorylated by PKC in vitro at a serine site (Jeng et al., 1987). In addition, studies with various ras proteins in both a mouse adrenocortical cell line (Y 1) and NIH 3T3 cells have shown that PKC also phosphorylates cellular Kirsten ras p2 1, suggesting that cKi-ras is a substrate for PKC (Ballester et al.. 1987). The function of the phosphorylation is not known, since it affected neither the binding nor the GTPase activity of the ras protein. Recently, other interesting connections have been proposed between PKC and ~21’~“‘“. Stimulation of the antigen receptor in T lymphocytes leads to the rapid activation of p2 1C-T:“,converting ~21’~‘“’ from the predominantly GDP-bound form to the GTP-bound state. The mechanism by which ~21’~‘“” is stimulated on T-cell activation is not fully understood, but upon activation of PKC the

J.-P. Liu / Molecular and Cellular Endocrinology 116 (1996) 1-29

activity of GTPase activating protein (GAP) is reduced several fold, suggesting that PKC may regulate ~21’~‘“” both directly and indirectly (Downward et al., 1990). The indirect action of PKC is further supported by the findings that PKC forms a complex with and phosphorylates GAP (Gschwendt et al., 1993). Furthermore, PKC activation of transcription of the human T cell receptor /3 gene requires the activity of ras (Wotton et al., 1993). Activated ~21’“” increased levels of DAG, choline, and phosphocholine (Price et al., 1989). Ras directly interacts PKC[ responding to platelet-derived growth factor stimulation (Diaz-Meco et al., 1994b), probably targeting PKC[ to plasma membrane thereby to be activated by PIP3, the product of PI 3-kinase (Nakanishi et al., 1993) and then to phosphorylate Inhibitor-,B (I,B) (Diaz-Meco et al., 1994a). PI 3 kinase has been shown to be a direct effector of ras (Rodriguez-Viciana et al.. 1994). These findings suggest that PKC activation is also modulated by ras and that PKC functions downstream of ras, like raf, to mediate the mitogenic potential of ras. I,B is a 68-kDa protein able to specifically inhibit the DNA binding of a nuclear factor (NF)-,B, a ubiquitous intracellular DNA-binding protein mediating inducible and tissue-specific gene expression (Lenardo and Baltimore, 1989). Activation of PKC causes phosphorylation of I,B in the inactive complex I,B/NF-,B, which releases NF-,B allowing its translocation into the nucleus to transactivate kB-dependent promoters (Ghosh and Baltimore, 1990). NF-kB is a heterodimer of SO-kDa (~50, NF-,Bl) and 65kDa (~65, relA: a member of the rel family of proteins) subunits. It was initially identified as a protein that could bind to a IO-bp site (GGGACTTTCC) in the k light chain enhancer of B cells (Kieran et al., 1990; Bours et al., 1990) and was subsequently found to be a gene regulator during development and involved in the inducible expression of a large number of genes in different cell types (Lenardo and Baltimore, 1989). These include the K light chain of immunoglobulin, cytokines, cytokine receptors, major histocompatibility antigens and associated proteins, serum amyloid A protein, and a variety of viruses including human immuno-deficiency virus, SV40, and cytomegalovirus. It has recently been suggested that PKCi is involved in the activation of NFkB by ras, PC-PLC and tumor necrosis factor-a (Diaz-Meco et al., 1994a,b). However overexpression of mouse PKC[ in NIH 3T3 cells shows no alteration in NF-,B activity stimulated by tumor necrosis factor-a, suggesting that further study is required (Montaner et al., 1995). Since NF-,B is present in the cytoplasm bound to I,B, phosphorylation of 1,B by PKC to release NF-,B provides an important mechanism for activation of transcription by PKC and a direct communication between extracellular stimuli and modulation of nuclear events.

21

Raf-1 is the 70-75-kDa phosphoprotein encoded by the c-raf-1 gene which is the cellular homolog of u-raf, the transforming gene of the murine sarcoma virus 36 11. Raf- 1 is present in all cell types tested and has an intrinsic kinase activity toward serine and threonine residues. Sequence analysis suggests that the amino terminus of the Raf-1 contains certain similarities to the PKC regulatory domain, whereas the kinase domains of Raf-1 family are most closely related to the tyrosine kinases (Li et al., 1991). Interestingly, PMA induces Raf-1 hyperphosphorylation and activation in all cell types, and Raf-1 molecules undergo a mobility shift from an apparent molecular mass of 70 kDa to a series of bands of sizes ranging from 72 to 74 kDa (Sturgill et al., 1988; Baccarini et al., 1990; Carroll et al., 1990; Siegel et al., 1990; Kanakura et al., 1991). Because phorbol esters do not directly interact with c-Raf (Kazanietz et al., 1994), these findings suggest that PKC regulates Raf- 1. It has now been established that Raf is an important physiological substrate of PKC. Purified PKC phosphorylates the residues serine497 and serine619 and stimulates the kinase activity of Raf-1 (Carroll and May, 1994). PKC isoform tl directly phosphorylates Raf-1 at several sites including a serine residue at position 499 which is necessary for maximal activation of the kinase (Kolch et al., 1993). The phosphorylation of c-raf by PKC stimulated by T cell receptor activation is distinct from that for tyrosine kinase growth factor receptors (Adrian et al., 1983). These studies suggest that PKC is a kinase kinase for Raf-1 playing an intermediate role in response to external biological stimuli. When activated, Raf-1 then functions as a kinase kinase to trigger a protein kinase cascade by direct phosphorylation of MAP kinase kinase, that in turn activates a number of signalling proteins, including cytosolic enzymes (like S6 kinase and phospholipase A,) and nuclear transcription factors (like c-Myc and Jun) in the signal processes required for cell growth and differentiation. p46c-erbA is a product of the proto-oncogene encoding the nuclear receptor for a thyroid hormone (T,). It stimulates transcription from specific target promoters upon binding to &-acting DNA sequence elements. Both PKC and PKA phosphorylate distinct serine residues on p46c-erbA, suggesting a possible role of phosphorylation in the regulation of the function of the erbA-encoded transcriptional factors (Goldberg et al., 1988). In addition to causing proto-oncogene product phosphorylation, PKC activation by PMA has also been linked to the induction of a variety of proto-oncogenes and other cellular genes. These PMA-inducible primary response genes possess unique 5’-upstream DNA sequences responsive to activation of PKC (Angel et al., 1991; Lee et al., 1991). The promoter regions of these genes possess specific binding sites for the gene regulatory factors which are activated by PMA and

22

J.-P. Liu

Mokular

and C’rllulor Endocrirlology 116 11996) l-29

which then regulate gene transcription through multiple cis- and trans-acting mechanisms (Chiu et al., 1987). Such PMA-response elements include AP-1 (Angel et al., 1991; Lee et al., 1991). AP-2, AP-3 (Mitchell et al., 1987) and NF-,B (Sen and Baltimore, 1986), and all have been demonstrated to regulate gene expression. PKC is also involved in the induction of the c-Jbs proto-oncogene and in mediating the actions of growth hormone (Doglio et al., 1989), platelet-derived growth factor (Hall and Stiles, 1987), bombesin (Rozengurt, 1990), arginine vasopressin (Nambi et al., 1989) angiotensin II (Taubman et al., 1989), ras (Gauthier-Rouviere et al., 1990), heparin (Wright et al., 1989) and ultraviolet irradiation (Buscher et al., 1988). 6. Prospectus PKC constitutes a structurally homologous family of enzymes that are activated by cell membrane lipids and that catalyze the rapid and reversible phosphorylation of serine or threonine residues in a wide variety of proteins. By this mechanism, PKC modulates the biological functions of these proteins. PKC is activated by the physiological second messenger DAG that is generated from the hydrolysis of plasma membrane inositol phospholipids by PLC and of phosphatidylcholine by PLD, resulting in the formation of a highly ordered complex consisting of the kinase, DAG, the cofactors Ca’ + and phosphatidylserine, and the substrate. Other cell membrane products such as arachidonic acid and PIP, also physiologically activate PKC and synergise with DAG to maximally activate PKC. Phorbol esters cause a pharmacological prolonged activation of PKC and down-regulate the enzyme by inducing enzyme insertion into the plasma membrane whereby it is gradually proteolysed. PKC activation requires a permissive post-translational phosphorylation of the catalytic subunit by an unknown protein kinase. Activated PKC becomes physically associated with the cell membrane or nuclear membrane by a process known as translocation. PKC is inserted into the nuclear membrane in a permanently active form and thereby causes long term effects. PKC is also subjected to positive or negative regulation by other factors and regulates other enzymes by protein phosphorylation or protein-protein interactions. Different members of the PKC family undergo differential activation, translocation and down-regulation and play distinct roles by causing differential phosphorylation of their substrates. The coupling between PKC activation and its biological cellular responses is mediated by various protein substrates which become physiologically phosphorylated by PKC in response to agonist stimulation. In addition to multiple isozymes of PKC (at least eleven members), the multiple substrates account for the multiple roles of PKC in eukaryotic cells. Although more

than 100 putative substrates have been currently considered, very few have the characteristics of being a primary target of PKC in cells. Neuromodulin, MARCKS and dynamin I are the ones among others that have been best studied, and they represent a model of how PKC regulates other enzymes and signalling pathways such as the Ca2+ - and calmodulin-dependent pathway, and of how PKC thus regulates cellular structure and function. The proto-oncogene products of PKC substrates provide further examples of how PKC may be involved in mitogenesis and oncogenesis. While the currently identified substrates require further study, it is likely that new PKC substrates will be recognised and identification of their individual functions will promote our understanding of the sophisticated roles of PKC in cellular function. Therefore. much work remains to be done to elucidate the mechanisms by which cellular responses are mediated by members of the PKC family via distinct substrate phosphorylation in eukaryotic cells. Acknowledgments The author wishes to thank John W. Funder, Dennis Engler, Phillip Robinson and He Li for critical reading of the manuscript and valuable discussion. This work was supported by a grant from the National Health and Medical Research Council of Australia. References Abate.

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