Protein kinase D (PKD): A novel target for diacylglycerol and phorbol esters

Fundamental and Molecular Mechanisms of Mutagenesis

ELSEVIER

Mutation Research 333

(1995)153- 160

Protein kinase D (PKD) : A novel target for diacylglycerol phorbol esters *, James

Enrique Rozengurt Im~rricrl

Cmcer

Sinnett-Smith,

Research Fund. P.O. Bo.r 123. 11

and

Johan Van Lint, Angela M. Valverde Lincolrc‘s Inn

Fields. London

WC’ZA 3PX. UK

Abstract A novel serine/threonine protein kinase regulated by phorbol esters and diacylglycerol (named PKD) has been identified. PKD contains a cysteine-rich repeat sequence homologous to that seen in the regulatory domain of protein kinase C (PKC). A bacterially expressed NH?-terminal domain of PKD exhibited high affinity phorbol ester binding activity (K, = 3.5 nM). Expression of PKD cDNA in COS cells conferred increased phorbol ester binding to intact cells. The catalytic domain of PKD contains all characteristic sequence motifs of serine protein kinases but shows only a low degree of sequence similarity to PKCs. The bacterially expressed catalytic domain of PKD efficiently phosphorylated the exogenous peptide substrate syntide-2 in serine but did not catalyse significant phosphorylation of a variety of other substrates utifised by PKCs and other major second messenger regulated kinases. PKD expressed in COS cells showed syntide-2 kinase activity that was stimulated by phorbol esters in the presence of phospholipids. We propose that PKD may be a novel component in the transduction of diacylglycerol and phorbol ester signals.

1. Introduction

A variety of hormones, neurotransmitters and growth factors elicit their characteristic effects on cellular processes upon binding to specific receptors on the surface of their target cells. A rapid increase in the synthesis of lipid-derived second messengers is an important mechanism for transducing these extracellular signals across the plasma membrane (Majerus,

1992; Nishizuka,

1992; Bet-ridge.

1993). A

in this process is the phospholipase C (PLC)-mediated hydrolysis of polyphosphoinositides to produce two second messengers: inositol I ,4.5-trisphosphate and diacylglycerol (DAG). Inositol key reaction

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I ,4,5_trisphosphate induces mobilisation of calcium from intracellular stores (Berridge. 1993). DAG activates protein kinase C (PKC) - originally described as a Ca’+-activated, phospholipid-dependent protein kinase (Nishizuka, 1988). The early findings that the potent tumour promoters of the phorbol ester family can substitute for DAG in PKC activation and that the phorbol ester receptor and PKC copurify supported the hypothesis that the cellular target of the phorbol esters i5 PKC (Nishizuka, 1989). A large number of cellular studies demonstrated that phorbol esters, presumably acting through PKC, induce a wide range of biological effects including changes in ion fluxes, gene expression, cell-cell communication, cell differentiation and proliferation (Kikkawa and Nishizuka, 1986; Rozengurt. 1986; Weinstein, 1988; Herschman, I99 1; Nishizuka, 1992).

Subsequent studies revealed the diversity of the individual components of the DAG-PKC signal transduction pathway. It has become apparent that PLC exists in several molecular forms that are regulated by G proteins and tyrosine phosphorylation (Rhee and Choi. 1992). Furthermore. DAG can be generated by several alternative routes including the hydrolysis of phosphatidylcholine (Exton. 19901. Molecular cloning has demonstrated the presence of multiple related PKC isoforms which are differentially expressed in cells and tissues (Azzi et al.. 1992; Nishizuka, 1992; Hug and Sarre, 19931. All members of the PKC family. i.e.. conventional PKCs (a, PI, PII, r>, novel PKCs (6. E. 7, 13) and atypical PKCs ({, A, L) possess a highly conserved catalytic domain. Most of the variation between the PKC subspecies occurs in the regulatory domain. The Cl region of this domain of both conventional and novel PKCs has a characteristic tandem repeat of zinc finger-like cysteine rich motifs which is indispensable to confer phospholipid-dependent phorbol ester and DAG binding to these PKC isoforms (One et al., 1989a; Bums and Bell, 1991; Hubbard et al., 1991; Quest et al., 1994). In contrast, atypical PKCs contain a single cysteine-rich motif and neither bind phorbol esters nor are regulated by DAG (One et al., 1989b; Ways et al.. 1992: Selbie et al.. 1993). These studies emphasised the complexity of the signalling pathways initiated by DAG but did not exclude the possibility that other protein kinases, unrelated to the PKC family in their catalytic domain. could also play a role in the mediation of the cellular effects of DAG and phorbol esters. Indeed, the identification of such novel phorbol ester and DAG targets could have important implications for our understanding of signal transduction and tumour promotion. 1. I. Protein kinase D Recently, we reported the molecular cloning. sequence and expression analysis of protein kinase D (PKD), a novel serine/threonine protein kinase with distinct structural features and enzymological properties (Valverde et al.. 1994). PKD. a protein of 918 amino acids with a predicted molecular mass of 102 kDa, may be a novel component in the transduction of DAG and phorbol ester signals. This paper reviews the salient structural and functional features of

PKD and provides additional evidence demonstrating that this enzyme can serve as a phorbol ester receptor in eukaryotic cells. PKD (accession No. 234524 in the EMBL/Gen Bank database) possesses a modular structure and consists of catalytic and regulatory domains. 1.1.1. Catalytic domairl The putative catalytic domain of PKD contains a protein kinase consensus region between residues 589 and 845 which is composed of distinct subdomains (Hanks and Quinn, 1991). This kinase region includes an ATP-binding motif (GXGXXGI in subdomain I at residues 596-601 (GSGQFG) and a lysine in subdomain II (residue 618) that is highly conserved in protein kinases. The motifs DFG in subdomain VII and APE in subdomain VIII of PKD are clearly indicative of a protein kinase. The presence of the sequences DLKPEN and GTPXYLAPE in the subdomains VIB and VIII, respectively, are indicative of specificity towards strongly serine/threonine substrates. An unusual feature of the sequence shown in Fig. 1 is that the highly conserved arginine residue in the HRDL motif of subdomain VIB of many protein kinases is substituted by a cysteine in PKD. This was observed previously only in two members of the protein kinase superfamily. the yeast ras suppressor, YAK-l (Garrett and Broach, 1989) and in a human putative protein-serine kinase (Hanks and Quinn, I99 1). The products of these genes have not been shown directly to possess catalytic activity. Searching of the protein databases with the sequence corresponding to the catalytic domain of PKD revealed the highest identity with the catalytic domains of myosin light chain kinase (MLCK) of Dictyostelium (41%) (Fig. 1A). The rank order of homologies within the catalytic domain was: MLCK (Dictyosteliuml > calcium/calmodulin dependent protein kinase (CaM kinase) type II > CaM kinase type IV > CAMP-dependent protein kinase > phosphorylase B kinase. In particular, the catalytic domain of PKD exhibits only a low degree of similarity to the highly conserved regions characteristic of all members of the PKC family. Specific examples of sequence differences between PKD and conserved regions of PKCs in subdomains I, II-III, VI and VII are shown in Fig. 1B. The important motif

E. Ro,-mgurt

et al. /Mutation

Research 333

C1995)

155

153- 160

A 678

nLclr

IFPDEVMSQQFGIVCGGKHRKTGRDVAIKIIDKLRFP'I?(QESQLRNEVAILQNLHHPGVVNLECMFETPERVFVVMEKL~GDMLE~ILS I III I I I I II/I II I I III IIIIIII I III I YEFKEEMRQAFSIWLGENKQTKQRYAIKVINKSELGKDYE~LK~VDILKK~HPNIIALKELFDTPEKLYLVMELVTGGELFDKIV

PKD

SEKGWLPEHITKFLITQILVALRHLHFKNIVHCDLllPEMTLLASADPFPQVKLCDFQFARIIGEKSFRRSWG"PAYLllPEVLRNKGYNR

768

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I

I

I

I

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IIIIII

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-EKGSYSEADAANLVKKIVSAVGYLHGLNIVHRDLXPElPLLLKSKENHLEVAIADFQLSKIIGQTLVMQTACGTPSYVAPEVLNATGYDK

PM

SLDEkiSVQVIIYVSLSGTFPFNEDEDIHD--QIQNAAF~PPNPWKEISHE4IDLINNLLQVKMRKRYSVDKTLSHPWL IllI

MLCK

III

I

I

I

II

I

I I

I

I

IIll

I

III

II

97

186

845 I/II

EVDMWSIQVITYILLCGFPPFYGDTVPEIFEQIMEVNYEFPEEYWGGISKEAKDFIGKLLWDVSKRLNATNALNHPWL

265

B

I

PXD

LGSGQFGIVCG

GRDVAIKIIDKLRFPTKQESQLRN

HCDLKPEN

GVKLCOFGFARI

mouse PKC alfa rat PKC beta I rat PKC gamma mouse PKC delta mouse PKC epsilon mouse PKC eta mouse PKC theta mouse PKC zeta

LGKGSFGKVML LGKGSFGKVML LGKGSFGKVML LGKGSFGKVLL LGKGSFGKVML LGKGSFGKVML LGKGSFGKVFL IGRGSYAKVLL

EELYAIKILKKDWIQOOOVECTH DELYAVKILKKDWIQDOOVECTkl DELYAIKILKKDVIVQODOVDCTL DKYFAIKCLKKDWLIDDDVECTH DEVYAVKVLKKOVILQDDDVDCTM GELYAVKVLKKDVILQDDDVECTM NQFFAIKALKKDWLMODDVECTM DQIYAMKVVKKELVHDDEDIDWVQ

YPJILKLDN YRDLKLDN YRDLKLDN YRDLKLDN YRDLKLDN YRDLKLDN YRDLKLDN YRDLKLDN

HIKIADFGMCKE HIKIADFGMCKE HIKITDFGMCKE HIKIAOFGMCKE HCKLADFGMCKE HIKIADFGMCKE HIKIADFGMCKE HIKLTOYGMCKE

II-III

VI

VII

C PKD

PWALFVHSYRAPAFCDHCGEMLWG-LVRQGLKCEGCGLNYHKRCAFKIPNNC

194

mouse

PKC alpha rat PKC beta-l PKC gamma mouse PKC delta mouse PKC epsilon mouse PKC eta mouse PKC theta rouse PKC zeta

DWKFIARFFKQPTFCSHCTDFIWG-FGKQGFQCQVCCFWHKRCHEFVTFSC NHKFTARFFKQPTFCSHCTDFI~-FGKQGFQCQVCCFWHKRCHEFVTSFC SHKFTARFFKQPTFCSHCTDFIWG-IGKQGLQCQVCSFWHRRCHEFV'IFEe NKEFIATFFGQPTFCSVCKEFVh'G-LNKQGYKCRQCNAAIHKKCIDKIIGRC GHKFMATYLRQPTYCSHCROFIWGVIGKQGYQCQVCTCWHKRCHELIITKC GWKFMATYLRQPTYCSHCREFIWGVFGKQGYQCQVETCWH CHEFTATFFPQPTFCSVCHEFVWG-LNKQGYQCRQCNAAIWKKCIOKVIAKC GHLFQAKRFNRGAYCGQCSERIWG-LSRQGYRCINCKLLVHKRCHVLVPLTC

86 86 85 208 220 222 209 180

PM

PHTFVIHSYTRPTVCQFCKKLLKG-LFRQGLQCKDCRFNCHKRCAPKVPNNC

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mouse PKC alfa rat PKC beta I rat PKC gamma mouse PKC delta mouse PKC epsilon mouse PKC eta mouse PKC theta

KHKFKIHTYGSPTFCDHCGSLLYG-LIHQGMKCDTCDTCDHNVHK@XJINDPSLC KHKFKIHTYSSPTFCDHCGSLLY$+LIHQGMXCDTCHMNVWKRCVMNVPSLC KHKFRLHSYSSPTFCDHCGSLLYG-LVHQGMKCSCCEMNVHRRCVRSVPSLC PWRFKVYNYMSPTFCDHCGSLLWC-LVKQGLKCEDCGMNVHHKCREKVANLC PHKFGIHtJYKVPTFCDHCGSLLWG-LLR@LQCKVCKMNVHRRCETNVAPNC PHKFNVHNYKVPTFCDHCGSLLWG-IMRQGLQCKICKMNVHIRCQANVAPNC THRFKVYNYKSPTFCEHCGTLLWG-LARQGLKCDACGMNVHHRCQTKVANLC

151 151 150 280 292 295 281

rat

Consensus

PKC

Fig. 1. Alignment of PKD with related serine-threonine protein kinases. (A) Alignment of the catalytic domain of PKD and Dictyostelium myosin light chain kinase (MLCK). (B) Sequence difference between PKD and highly conserved regions characteristic of all members of the PKC family. (C) The cysteine-rich domains of PKD were aligned with the cysteine-rich domains of PKC isoforms. The invariant residues forming a consensus sequence are also shown.

XXDLKXXN/D in subdomain VI is of interest because it guides the peptide substrate into the correct orientation so that catalysis can occur (Knighton et al.. 1991). All members of the PKC family contain the sequence YRDLKLDN which differs from that of PKD (HCDLKPEN) in every variable residue (X).

We conclude that the protein kinase identified in the present study, named PKD, is a distinct kinase which does not belong to any of the previously identified subfamilies. The structural differences between the catalytic domains of PKD and PKCs suggested that these

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proteins could have important functional differences. We examined the ability of a bacterially expressed fusion protein containing the catalytic domain of PKD to phosphorylate a variety of potential substrates (Valverde et al., 1994). These studies revealed that the catalytic domain of PKD phosphorylates the synthetic peptide syntide-2, a substrate also utilised by calmodulin-dependent protein kinases (Mochizuki et al.. 1993; Lorca et al., 1993). PKD isolated from COS cells transfected with a PKD cDNA construct (pcDNA3-PKD) also phosphorylated syntide-2 (Fig. 2C and Van Lint et al., 1995). In contrast. PKD did not catalyse significant phosphorylation of a number of substrates utilised by

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PKCs such as histone, and a peptide substrate based on the sequence of the pseudosubstrate region of PKCE. Although PKCs cry. p and y utilise histone as a substrate much more effectively than the novel PKCs (Ohno et al., 1988; Osada et al., 1992). it has become apparent that this difference is not an intrinsic property of the catalytic domains but is conferred by the influence of the regulatory domain. Indeed, novel PKCs such as 6 or E that have been rendered constitutively active by proteolysis or mutation. phosphorylate histone as efficiently as the conventional PKCs (Schaap et al.. 1990; Pears et al.. 1991; Dekker et al., 1993). In this context, the inability of the catalytic domain of PKD to phosphorylate PKC substrates clearly emphasises the functional differences between these enzymes. I. I .2. The regulatory domain A salient feature of the deduced amino acid sequence of the NH,-terminal portion of PKD is a cysteine-rich repeat sequence (H-X, 2-C-X,-CmX , 3C-X,-C-X,-H-X,-C-X,-C) that exhibits homology with the cysteine-rich sequence tandemly repeated in the regulatory domain of all phorbol ester-sensitive

+

PS

+

Ca”

pcDNA3-PKD

pcDNA3

+

+

-

+

PS PDB

Fig. 2. Binding of [“HIPDB to PKD. (A) Binding of [jH]PDB to a bacterially expressed fusion protein containing the cysteine-rich domain. Binding was measured in the absence or presence of either Ca’+ (I mM) or PS (125 Kg/ml). The incubations were carried out with IO mM [‘H]PDB for 30 min at 3°C. The values of non-specific binding of [‘H]PDB. defined as that measured in the presence of 10 FM PDB, are also shown (closed bars). ALI other experimental details were as described by Valverde et al. (1991). (B) Expression of PKD in COS-7 cells confers increased [‘HIPDB binding. Control COS cells or COS cells tmnsfected with either pcDNA 3 vetor alone (pcDNA3) or with the pcDNA3PKD construct were incubated for 72 h and then washed and incubated at 37°C with medium containing IO nM [‘H]PDB for 30 min. Cell associated radioactivity was determined as described by Collins and Rozengurt (1982). Non-specific binding (hatched bars) was determined in the presence of 1 PM unlabelled PDB. (C) Lipid-regulation of PKD expressed in COS cells. COS cells transfected with pcDNA3 (open bars) or with pcDNA3-PKD (closed bars) were lysed and incubated with the PKD antibody PA-I described hy Valverde et al. (1993). Immunopreclpitateh were eked with the immunking peptide and then analysed in a syntide-2 phosphorylation assny. Kinase activity was measured either in the absence or presence of PS (I00 pg/ml). Each of the assays was performed in the absence or presence of PDB (200 nM). as indicated.

15 Razengurf

et af./Mutation

PKCs (Fig. 1C). Indeed, the first and second cysteine-rich motifs of PKD display 86% and 81% with a consensus sequence for identity phospholipid-dependent phorbol ester binding (Fig. 1C). Interestingly, the length of the sequence separating the cysteine-rich domains in PKD (95 residues) is substantially longer than that of conventional PKCs (28 amino acids) or novel PKCs (35-36 amino acids). These comparisons suggest that PKD could represent a novel target for DAG and biologically active phorbol esters. Other proteins that also contain a cysteine-rich. zinc finger-like motif with sequence identity to PKCs include the ~21” GTPase activating protein chimaerin (Ahmed et al.. 19931, the DAG kinase (Sakane et al., 1990,) the Cuenorhabditis elegans UNC-13 gene product (Maruyama and Brenner, 199 1) and the oncogene products RAF (Bruder et al., 1992) and VAV (Katzav et al., 1989). DAG kinase and RAF do not appear to bind phorbol ester with high affinity. Further, atypical PKCs which contain a single cysteine-rich motif, neither bind phorbol esters (One et al.. 1989b; Selbie et al., 1993) nor appear to be regulated by DAG (Ways et al., 1992: Nakanishi and Exton. 1992). Valverde et al. (1994) provided direct evidence indicating that PKD can serve as a novel phorbol ester receptor. Specifically, a bacterially expressed cysteine-rich domain of PKD binds [‘Hlphorbol 12,13-dibutyrate (PDB) in a specific manner and in a phospholipid dependent fashion (Fig. 2A). The DAG analogue 1-oleoyl-2-acetylglycerol (OAG) markedly reduced the binding of [’ H]PDB to the fusion protein. Specific binding of [3~]~~~ to the fusion protein containing the cystein-rich tandem was saturable (Valverde et al., 1994). Scatchard analysis of the data revealed a K, of 35 nM. This K, is similar to that recently determined for the binding of [“HIPDB to the cysteine-rich region of PKC-y (Quest et al., 1994). To substantiate that the binding measurements to the fusion protein in vitro reflect the physiological activity of PKD in intact cells, we verified that transient expression of PKD confers increased [3~]~~~ binding activity to COS cells as compared to either COS cells transfected with the vector (pcDNA3) or to untransfected cells (Fig. 2B and Van Lint et al., 1995). Furthermore, addition of PDB in

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the presence of L-a-phosphatidylserine (PSI markedly stimulated the activity of PKD measured in a syntide-2 phosphorylation assay (Fig. 20 and in an autophosphorylation assay (Van Lint et al.. 1995). Thus, our results provide evidence indicating that PKD is a novel target for the tumour promoters of the phorbol ester family as well as for the second messenger DAG. Johannes et al. (1994) recently cloned a human protein kinase termed PKCF with 92% homology to PKD. It is highly likely that the two kinases are functional homologs. However, Johannes et al. (1994) failed to demonstrate any phorbol ester binding and regulation and consequently concluded that this enzyme is an atypical PKC. These authors suggested that two amino acid substitutions in the cysteine rich region could be responsible for these results. In addition, they suggest that the spacing of the cysteine-rich motifs could result in an inappropriate conformation for efficient PDB binding. We conclude that PKD is not an atypical PKC since we clearly demonstrate that PKD binds phorbol esters and that the activity of this enzyme is markedly stimulated by PDB in the presence of PS. Moreover, we found fundamental differences in both substrate specificity and catalytic domain protein structure between PKD and PKCs. In addition, PKD can be distinguished from PKCs by other important structural features: (1) The regulatory domain of all known PKCs, including yeasts, Drosophila or mammalian isoforms, also contains a pseudosubstrate motif characterised by an alanine flanked by basic amino acids (Toda et al., 19931. In all PKCs, the pseudosubstrate motif is located 15 amino acids upstream from the beginning of the cysteine-rich domain. PKD does not contain sequences with homology to a typical PKC pseudosubstrate motif upstream of the cysteine rich region. The absence in PKD of a regulatory motif conserved in all PKCs further illustrates the divergence of PKD from the PKC family. (2) The NH?terminus of PKD possess a highly hydrophobic stretch of amino acids, suggesting a transmembrane domain, which is not found in any of the PKCs. (3) PKD contains a pleckstrin homology (PH) domain (Gibson et al., 1994) inserted between the cysteinerich motifs and the catalytic domain. PH domains have recently been identified in a variety of intracellular signalling and cytoskeletal proteins, but are not

PKD DOMAINS T.M

Cys-rich

PH

Catalytic

Fig. 3. A diagram of PKD structure incorporating a putative transmembrane (T.M) domain. a tandem repeat of cystein-rich regions (Cys-rich). a PH domain and a kinase catalytic domain. Additional explanations concerning the domains in PKD can he found in the text.

present in PKCs. Fig. 3 shows a diagram depicting the modular structure of PKD. We conclude that PKD cannot be classified in any PKC subfamily. 1.2. hnplicatiot2s One of the earliest responses of many cell types to extracellular stimuli is an increase in the synthesis of DAG, the physiological second messenger generated by action of the phospholipase C isoenzymes which are activated through tyrosine phosphorylation (e.g., PLC n> or G proteins (e.g.. PLC p) signal transduction pathways (Rhee and Choi. 1992). In addition. sustained DAG accumulation can be generated through other pathways involving phosphatidylcholine breakdown (Exton. 1990). Acting as a second messenger, DAG has been implicated in the signal transduction of many fundamental biological processes including the regulation of cell proliferation (Rozengurt et al.. 1984). PKD. a novel DAGregulated protein kinase, could be envisaged to play a role in mediating some of the cellular responses initiated by DAG-producing receptors. In this context, the biological effects elicited by neuropeptides provide an interesting example which is relevant for understanding growth regulation and carcinogenesis. In recent years an increasing number of small regulatory peptides or neuropeptides have been discovered in the neural and neuroendocrine cells of the gastrointestinal tract and central nervous system (Walsh. 1987). Some are localized in neurons and act as neurotransmitters in the central or peripheral nervous system, while others are released by endocrine cells and have effects both as systemic hormones circulating through the blood stream and by acting in a paracrine or autocrine fashion. Moreover, a number of peptides are found in both neuronal and endocrine cells. and a major effect of some regula-

tory peptides in vivo. e.g., bombesin/gastrin-releasing peptide (GRP). is to stimulate the release of other biologically active peptides. The role of these peptides as fast-acting neuro-humoral signallers has recently been expanded by the discovery that they also stimulate cell proliferation (Rozengurt, 1986; Zachary et al., 1987; Rozengurt. 1991). Furthermore. evidence is accumulating that the mitogenic effects of neuropeptides may be relevant for a number of normal and abnormal biological processes, and in particular tumorigenesis. For example, the peptides of the bombesin family including GRP bind to specific surface receptors and initiate a complex cascade of signalling events that culminates in the stimulation of DNA synthesis and cell division in Swiss 3T3 cells (Rozengurt. 199 I ; Zachary and Rozengurt. 1992). These peptides may also act as autocrine growth factors for certain small cell lung cancer cells (SCLC). The autocrine growth loop of bombesin-like peptides may be only a part of an extensive network of autocrine and paracrine interactions involving a variety of neuropeptides in SCLC including vasopressin. bradykinin, gastrin and neurotensin (Sethi et al.. 1992). In the context of the multistage evolution of cancer. neuropeptide mitogenesis may play a role at an early stage as tumour promoters in initiated cells or later as growth factors which sustain the unrestrained growth of the fully developed SCLC tumour. It is likely that a detailed understanding of the receptors and signal transduction pathways that mediate the mitogenic action of regulatory peptides will identify novel targets for therapeutic intervention. One of the earliest events to occur after the binding of bombesin to its specific receptor is the rapid stimulation of phospholipase C (PLC) catalysed hydrolysis of phosphatidyl inositol 4.5bisphosphate (PIP?) in the plasma membrane (Rozengurt. 1991). This reaction produces inositol I.45trisphosphate [Ins(l,4S)P,]. which. as a second messenger binds to an intracellular receptor and induces the release of Ca’+ from internal stores. PLC-mediated hydrolysis of PIP2 also generates I .2-diacylglycerol (DAG). DAG can also be generated from other sources. such as phosphatidylcholine hydrolysis. and acts as a second messenger in the activation of PKC by multiple extracellular stimuli including bombesin. The regulatory properties of

E. Rnzengurt et d/Mutation

PKD. specifically the stimulation of this enzyme by diacylglycerols (Van Lint et al., 1995) strongly suggests that PKD could also participate in mediating the cellular effects of neuropeptides. Another important implication of the results obtained with PKD is in the mechanism of action of phorbol esters. These agents act as potent tumour promoters and induce a variety of responses in many cultured cell types including effects on ionic channels. second messenger production, cell-cell communication, membrane transport, protein phosphorylation and cellular growth, morphology, differentiation and transformation (Kikkawa and Nishizuka, 1986; Rozengurt, 1986; Weinstein, 1988; Herschman, 199 1; Nishizuka, 1992). The PKC family of isoenzymes are widely believed to mediate most biological responses elicited by phorbol esters. However. the possibility that certain effects of phorbol esters are not mediated by PKC has also been suggested (e.g.. Ramchandran et al., 1994). The identification of a novel target for phorbol esters, PKD, whose mRNA is expressed in many organs and tissues as well as in cultured cells (Valverde et al., 1994 and unpublished results) raises the possibility that some of the actions of phorbol esters could be mediated partially or exclusively by PKD rather than by PKC. The elucidation of the precise role of PKD in mediating the biological effects of phorbol esters and in contributing to the signal transduction of many receptors that stimulate DAG accumulation emerges as an important problem that warrants further experimental work.

References Ahmed, S., J. Lee, R. Kozma. A. Best, C. Monfries and L. Lim (I 9931 A novel functional target for tumor-promoting phorbol esters and lysophosphatidic acid. J. Biol. Chem.. 268, 1070910712. Azzi, A., D. Boscoboinik and C. Hensey (19921 The protein kinase C family, Eur. J. B&hem.. 208. 547-557. Berridge. M.J. (19931 Inositol triphosphate and calcium signalling, Nature, 361, 315-325. Bruder. J.T., G. Heidecker and U.R. Rapp (1992) Serum-, TPAand Ras-induced expression from Ap- 1/E&driven promoters requires Raf-1 kinase. Genes Dev., 6, 545-556. Burns. D.J. and R.M. Bell (1991) Protein kinase C contains two phorbol ester binding domains, J. Biol. Chem.. 266. 1833018338.

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Collins. M. and E. Rozengurt (19821 Binding of phorbol esters to high affinity sites on murine fibroblastic cells elicits a mitogenie response, J. Ceil. Physiol.. 112. 42-50. Dekker, L.V., P. McIntyre and P.J. Parker (19931 Mutagenesis of the Regulatory domain of rat protein kinase C-7. J. Biol. Chem., 268, 19498-19504. Exton, J.H. (19901 Signaling through phosphatidylcholine breakdown. J. Biol. Chem.. 265. l-4. Garrett. S. and J. Broach (1989) Loss of Ras activity in Succharomyes cerel,isiue is suppressed by disruptions of a new kinase gene. YAKI. whose product may act downstream of the CAMP-dependent protein kinase. Genes Dev., 3, 1336- 1348. Gibson. T.J.. M. Hyvonen, Q, Musacchio and M. Saraste (1994) PH domain: the first anniversary. Trends Biochem. Sci. 19, 349-353. Hanks, SK. and A.M. Quinn (1991) Protein kinase catalytic domain sequence database: identification of conserved features of primary structure and classification of family members. Methods Enzymol.. 200, 38-62. Herschman, H.R. (19911 Primary response genes induced by growth factors and tumor promotors, Annu. Rev. Biochem.. 60, 281-319. Hubbard, S.R.. W.R. Bishop, P. Kirschmeier. S.J. George, S.P. Cramer and W.A. Hendrickson (19911 Identification and characterization of zinc binding sites in protein kinase C, Science, 254. 1776-1779. Hug, H. and T.F. Sarre (1993) Protein kinase C isoenzymes: divergence in signal transduction. Biochem. J., 29 I. 339-343. Johannes, F.J., J. Prestle. S. Eis, P. Oberhagemann and K. Pfizenmaier (I 994) PKCp is a novel, atypical member of the protein kinase C family, J. Biol. Chem.. 269, 6140-6148. Katzav, S.. D. Martin-Zanca and M. Barbacid (19891 ~‘a( . a novel human oncogene derived from a locus ubiquitously expressed in hematopoietic cells. EMBO J., 8, 2283-2290. Kikkawa, U. and Y. Nishizuka (19861 The role of protein kinase C in transmembrane signalling. Ann. Rev. Cell Biol., 2, 149-17s. Lorca. T., F.H. Cruzalegui. D. Fesquet, J.-C. Cavadore. J. M&y. A. Means and M. Doree (19931 Calmodulin-dependent protein kinase II mediates inactivation of MPF and CSF upon fettilization of Xerioprrs eggs, Nature. 366, 270-273. Majerus. P.W. (1992) Inositol phosphate biochemistry. Annu. Rev. Biochem.. 61. 225-250. Maruyama, I.C. and S. Brenner t 19911 A phorbol ester/diacylglycerol-binding protein encoded by the unc-13 gene of Caenorhabditis elegant, Proc. Natl. Acad. Sci. USA. 88, 5729-5733. Mochizuki. H., T. Ito and H. Hidaka (19931 Purification and characterization of Ca’+/calmodulin-dependent protem kinase V from rat cerebrum. J. Biol. Chem., 268. 9143-9147. Nakanishi. H. and J.H. Exton (19921 Purification and characterization of the < isoform of protein kinase C from bovine kidney. J. Biol. Chem., 267. 16347-16354. Nishizuka, Y. (19881 The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature, 334,661-665. Nishizuka. Y. (19891 Studies and prospectives of the protein

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