- Email: [email protected]
threonine protein kinases
Serine/threonine
protein kinases
John D. Scott and Thomas R. Soderling Vellum
Institute
for Advanced Biomedical
Signal transduction
in the nervous system is heavily dependent
multifunctional
serine/threonine
protein
Recent
have
our
studies
isoforms
of these
properties,
Research, Portland,
furthered
kinases
and substrate
and
kinases,
their
determinants
subcellular
of how localizations,
are important
USA
on the three
PKA, PKC, and
understanding
Oregon,
the
CaM-KII. multiple
regulatory
for the specificity
of
kinase functions.
Current
Opinion
in Neurobiology
Introduction Neural tissues contain very high concentrations of the three multifunctional serine/threonine protein kinases: cAh4P-dependent protein kinase (protein kinase A or PKA), Caz+/phospholipid-dependent protein kinase (protein kinase C or PKC), and Ca2+/calmodulin-dependent protein kinase II (CaM-KII). For example, CaM-KII constitutes up to 2 % of total protein in certain regions of the brain such as the hippocampus. Thus, it is not surprising that these kinases participate in many of the signal transduction pathways that are so important for providing modulation of such neural functions as neurotransmitter synthesis, release and receptor binding, ion channel properties, gene transcription, and synaptic plasticity. The purpose of this short review is to update selected aspects of the three kinases, and only recent papers will be referenced. Each of the kinases has been individually reviewed (PKA [ 11, PKC [ 21, and CaMK II [3] >, and the reader should consult these for background information.
CAMP-dependent Structure
protein
kinase
and function
The CAMP signaling pathway is composed of individual neurotransmitter receptors coupled through GTP-binding proteins to adenylate cyclase. Four molecules of CAMP bind each dormant PKA holoenzyme complex (R2C2), thereby causing the release of two active catalytic (C) subunits from the regulatory (R) subunit dimer. Three C subunit isoforms (Ccl, Co, and Cy) and four R subunit isoforms (RIa, RIP, RUa, and RI@) are expressed at different levels in various mammalian cells, and combine to form type I and type II holoenzymes. The pisoforms of each subunit are predominantly expressed in neural tissues.
1992, 2:2899295
The C subunit contains a catalytic core that is conserved in all protein kinases, with distinct sequence motifs or subdomains that are involved in ATP-binding, protein/peptide-binding and phosphotransferase activity. Recently, the crystal structure of the recombinant mouse Q-subunit complexed with a peptide inhibitor has been solved at a resolution of 2.7h; [4*-,5*]. These studies show that Ca is a bilobal molecule (Fig-l). The smallest lobe contains the sequences involved in ATP-binding while the larger lobe is responsible for peptide binding and catalysis, The cleft between the lobes is occupied by h4g2 + /ATP
Cellular
regulation
Both RI isoforms are primarily cytoplasmic, but neural tissues contain up to 75 % of either RII isoform associated with the plasma membrane, cytoskeletal components, dendrites, secretory granules, and the nuclear membrane. Type II PKA localization occurs through R subunit dimer interaction with specific-anchoring proteins. PKA localization in the cytoskeleton occurs through interaction of the amino-terminal 50 residues of RI1 with microtubule-associated protein 2 (MAP 2) [6,7]. A conserved amphipathic helix motif, which is involved in binding to RR, has been identified in MAP 2 and two other RII-anchoring proteins, AKAP79 and Ht31 [8]. Synthetic peptides which span the amphipathic helix region in Ht31 associate with RIIa or the type II holoenzyme with nanomolar affinity (D Carr and JD Scott, unpublished data). RI1 may also interact with the p34cdcz kinase [9], which is intriguing as inhibition of PKA plays a key role in the induction of mitosis and nuclear envelope breakdown [IO]. Furthermore, phosphorylation of RI1 at Thr211 by ~34~~~~ blocks activation of the type II holoenzyme by CAMP [ 111. While R&anchoring can account for localization of the PKA holoenzyme, the free C subunit can translocate into specific cellular compartments upon its activation.
Abbreviations C-catalytic; DAG4iacylglycerol;
CaM-KII-Ca*+/calmodulin-dependent IPS-inositol
1,4,5-trisphosphate;
NMDA-N-methyl-D-aspartate;
protein kinase II; CREW-CAMP-response-element LTP-long-term
PKA-protein
@
Current
Biology
potentiation;
kinase A; PKC-protein
Ltd ISSN 0959-4388
binding
MAP-Z-microtubule-associated
protein; protein
2;
kinase C; R-regulatory.
289
290
Signailing
mechanisms
pathetic regulation of this channel is due exclusively to phosphorylation of the channel by PKA
Ca2+/phospholipid-dependent Structure
Fig.1. A ribbon diagram illustrating the three-dimensional structure of the CAMP-dependent protein kinase catalytic subunit. The ATP phosphate anchor and catalytic loop are indicated. Reproduced with permission from WI.
Nuclear translocation of the C subunit has been demonstrated in both fibroblasts and neurons [ 12,13**,14]. Microinjection of R and C subunits with fluoresceine shows that type I PKA is cytoplasmic, but upon activation with Sbromo-cAMP the free C subunit translocates to the nucleus [ 121. This observation has been extended by an elegant series of experiments using fluorescent ram tio imaging to chart the migration of the PKA holoenzyme, labeled on both the C and R subunits, from its site of activation [I3e*,I4]. The microinjected fluores cent PKA holoenzyme remains in the cytoplasm when CAMP is low. After cAMP-induced dissociation of the holoenzyme, the C subunit translocates to the nucleus, while R remains in the cytoplasm. Upon lowering CAMP, the fluoresceine labeled C subunit leaves the nucleus and reforms the holoenzyme with the R subunit in the cytoplasm.
Substrates
The activation of a glutamate receptor ion channel is mediated by PKA phosphorylation [I5,161. Currents induced by glutamate or kainate are potentiated by agents which stimulate PKA activity in cultured hippocampal neurons or in excised patches, and are blocked by application of a peptide inhibitor of the kinase. Similar experiments have been performed on the cardiac voltagegated Ca2 + channel [ 171, and it was concluded that sym
protein
kinase
and function
PKC is a phospholipid-dependent protein kinase that can be activated by the second messenger diacylglycerol (DAG) or by phorbol esters. PKC is actually an isozyme family of nine structurally-related enzymes ranging in size from 7%90 kD [ 18,19]. Each isoform contains five variable subdomains (V,-Vg) interspersed with four conserved subdomains (C,C*) (see Fig. 2). The catalytic elements (C, and Cd) are similar to the catalytic core of other protein kinases and contain sequences responsible for ATI-binding and phosphotransferase activity. The amino-terminal V, domain contains an autoinhibitory pseudosubstrate sequence that occupies the substrate-binding site in the catalytic core and renders the enzyme inactive. Binding of DAG and a phospholipid cofactor, commonly phosphatidylserine, to the C, subdomain promotes conformational changes which displace the pseudosubstrate sequence and thereby activate the enzyme. Two repeated cysteine-rich sequences, homologous to the ‘zinc finger’ binding motif, form the C, subdomain and bind phorbol esters with nanomolar affinity [ 201. The k-isoform has only one zinc finger and does not bind phorbol esters [21]. Recent evidence suggests that two Zn2 + ions bind each zinc finger [ 221. The C, domain confers Ca2+ dependence on the CL,jYI,and y PKC isoforms, which require both phospholipid and Ca2+ for activation. In contrast, the 6, E, 5, q, and L isoforms are Ca2+ -independent and lack the C2 domain. These differences in activator requirements, as well as in substrate specificities and subcellular localizations (see below), suggest that each isoform may have a distinct role in signal transduction [ 23,241.
Cellular
regulation
Upon activation, PKC is translocated from the cytoplasm to particulate fractions, which localize the kinase to the cytoskeleton, postsynaptic densities, nuclei, and the plasma membrane. Selective translocation of PKC isoforms occurs in response to specific activators. For example, thyrotropin releasing hormone promotes translocation of PKC isoforms other than PKCs, while bryostatin 1 selectively translocates PKCy in GH4 rat pituitary cells [ 25,261. In HL 60 leukemia cells phorbol dibutyrate (PB2) promotes the translocation of both PKCcr and PII to the plasma membrane, while bryostatin 1 targets PKCBII to the nucleus [ 271. Persistent activation by phorbol esters also leads to down-regulation of the particulate-bound kinase by proteolysis. Furthermore, autophosphorylation may prime PKC for degradation as a mutant PKC~Llacking kinase activity is translocated but not proteolysed [ 281. Originally PKC targeting was thought to occur exclusively through association with lipids. The active kinase may also be localized through interactions with anchoring
Serine/threonine
a, 01, PII, and
lsoforms
Zinc
ATP-binding
I
I
domain
Catalytic domain
COOH
Fig.2. A diagram
Ht----lHHHl-----lH-I----I
Cl
mon subdomain v2
c2
v3
c3
v4
c4
6, F, 5, q and L Regulatory
H-
Vl
Cl
domain HH v2
v3
c4
The location of each below the figregions of con-
V, denotes
variable sequence. V5
com-
ure. C, denotes constant
pseudosubstrate v4
the
of the protein
is indicated
served sequences.
Catalytic domain HH-H c3
illustrating structure
kinase C isoforms.
V5
subdomain isoforms
Soderling
site
Pseudosubstrate site
Vl
Scott and
kinases
y
fingers
Regutatory
NH2
protein
motifs,
regions of
The positions
of the
site, zinc finger binding
and ATP-binding
sites are also in-
dicated.
proteins, however. Using a modified western blot technique, PKC PI has been shown to bind certain cytoskeletal elements, the PKC inhibitor protein (PKCI) and annexin 1 [29,30]. These PKC anchoring proteins may be structurally related as a conserved sequence of 16 amino acids is present in both. This sequence is believed to represent the PKC-binding site as peptides corresponding to it bind PKCPI, although with low affinity [ 311. PKC isoforms are regionally expressed in the central nervous system. For example, the u-, p-, and y-subtypes are differentially compartmentalized in retinal neurons with PKCy being specifically localized to the rod bipolar and amacrine cells [32]. This observation correlates with rem ports that PKC mediates spinule-type neurite outgrowth in the retina during light adaption, and that it is involved in the phospholylation of rhodopsin [33,34]. Furthermore, analysis of inaC mutants in Drosophila suggests that a photoreceptor-speci PKC homolog, eye-PKC, is required for deactivation and the rapid desensitization of the visual cascade [35*]. PKC activity has also been implicated in long-term potentiation (LTP). Neural imaging with 3[H]-PB2 demonstrates that PKC isoforms are redistributed within the CA3 hippocampal neurons upon acquisition of an associative-conditioned response [36]. In addition, microinjection of the protein kinase inhibitor staurosporin into Hermisrendu type B photoreceptors prevents expression of learning-induced changes in Ca2+ -activated K+ channel currents [37].
hydrazone, an inhibitor of PKC (ICsO = 20 l.tM) [41-l. An intriguing property of these compounds is that they as semble into an active PKC inhibitor in or near cells, and have been used to block kinase activity in cancer cells.
Ca2+/calmoduliwdependent Structure
protein
kinase II
and function
is also an isozyme family containing G12 subunits (5G6OkD each) per holoenzyme. The amino-terminal catalytic domain and the central regulatory region are highly conserved in isoforms sequenced from mammals, Drosophila [42], and yeast [43]. The variable carboxy1 terminus, responsible for subunit assembly, forms a central core in the holoenzyme surrounded by globular catalytic domains that are arranged like flower petals [ 441. CaM-KII
Studies over the past five years from a number of laboratories have led to the regulatory model depicted in Fig.3 1451. The binding of Caz+/CaM disrupts the autoinhibitory interaction with the catalytic domain, thereby activating the kinase and allowing phosphorylation of exogenous substrates as well as autophosphorylation of 67 sites within CaMKII. Autophosphorylation of Thr286 in the autoinhibitory domain occurs within seconds of l& nase activation and is uniquely responsible for formation of the Ca2+-independent form of CaMKII.
Substrates
Cellular
PKC activity appears to indirectly control calmodulin-mediated second messenger pathways in neurons. Two of the major PKC substrates, neuromodulin and the MARCKS protein, bind calmodulin with high affinity in the absence of Ca2+. Their function may be to concentrate calmodulin at specific sites within neurons, which is then released upon their phosphorylation by PKC [ 38,391. Pseudosubstrate inhibitor peptides have been designed from the MARCKS sequence and inhibit PKC activity with Ki values as low as 20 nM in vitro [40]. An alternative approach to the study of PKC inhibition involves the synergistic action of two prodrugs, an aldehyde and a hydrazine, which combine in situ to form a cytotoxic
It has been postulated that a transient elevation of intracellular Ca2+ may trigger prolonged physiological responses mediated by CaIv-KII through formation of the Ca2 + -independent species. Recent studies have demonstrated autophosphorylation and formation of Ca2+ -independent CaWKII activity in cultured cells and tissue slices [ 46*,47*,4&50]. Basal levels of Cal + -independent WV-KII (5-25% in the cerebellum and hippocampus) can be modulated by altering extracellular Ca2+, or by pre-treatment with protein kinase (H7) or protein phosphatase (okadaic acid) inhibitors. In all the preparations examined, except the cultured hippocampal slices [SO], K+ depolarization rapidly results in the formation
regulation
291
292
Signalling
mechanisms
290 300 MHRQETVDCLKKFNARRKLKGAILTTMLA
(a) Inactive P
(c) Fully active (100%)
!F!$Z$JfY&
0
Protein phosphatase P
(d) Partially
Cal’-independent
(50-
80 “/o)
Fig.3. A schematic model for the regulation of the Ca 2+/calmodulin-dependent protein kinase II (CaM-KII). The model depicts a single subunit anchored to the holoenzyme through the carboxyl terminus. (a) In the absence of CaM, CaM-KII is inactive as a result of (b) Interaction of Ca2+/CaM with its the interaction of the autoinhibitory domain (shaded area) with the catalytic domain (-wYh~). binding domain (darkly shaded area) conformationally inactivates the inhibitory domain, thereby activating the kinase, which can then bind Mg2+/ATP and (c) phosphorylate itself (Thr286). Exogenous proteins containing the consensus sequence can also be phosphorylated. (d) The autophosphorylated kinase remains active in the absence of Ca2+/CaM (i.e. is Ca 2+-independent), but it can be dephosphorylated and inactivated by protein phosphatases 1 and/or 2A. This model is modified from [451.
of Ca2+ -independent CaM-KII, which then slowly decays back to its basal value. In cerebellar granule cells [46*] the extent and duration of CaMKII activation is enhanced by okadaic acid, but these cells also contain protein phosphatases insensitive to this inhibitor. In PC12 and GH3 cells [47*,48], agents which trigger the phosphatidylinositol signaling system (i.e. formation of inositol 1,4,5-trisphosphate (IP3) and DAG) result in a very transient formation of Ca2 + -independent CaM-KII, consistent with the release of intracellular Ca2+ by IP3. PC12 cells have also been used to study a cell-permeable inhibitor, ~~62, which appears to be specific for CaMKU. Pre-treatment of PC12 cells with KN-62 (l-10pM) blocks 32P-labeling of CaM-KII [51*] and activation of tyrosine hydroxylase [52] in response to Ca2+ mob&ation. Postsynaptic injection of peptide inhibitors of Cti-KII or PKC block the induction of LTP, indicating that these two protein kinases may be involved in the regulation of synaptic plasticity. The induction of LTP in the hippocam pus requires a Caz+ influx through the N-methyl-D-aspartate (NMDA)-type glutamate receptor ion channel, so it is important to determine if activation of NMDA receptors activates CaMKII. In both cerebellar granule cells [4&] and cultured hippocampal neurons (K Fukunaga et al, unpublished data), glutamate treatment sign&
cantiy increases the Ca2+ -independent form of CaMKII through autophosphorylation of Thr286. This response is mediated via the NMDA receptor as it is enhanced by addition of ll.tM glycine or removal of extracellular Mg2+, and blocked by specific NMDA antagonists. The above in situ studies show that autophosphorylation and activation of CaM-KII is under dynamic regulation. The elevation of intracellular Ca2+ promotes the formation of the Ca2+ -independent form, and the extent and duration of this species depends on the activity of opposing protein phosphatases. It will be important to determine if subcellular localized CaM-KII (e.g. in the postsynaptic density) exhibits different kinetics of regulation. In none of the above studies was total CaM-KII activity, assayed in the presence of Caz+/CaM, altered. In the gerbil, however, forebrain ischemia, as a result of occlusion of the carotid arteries, produces an almost complete loss of soluble forebrain CaM-KII within 2-5 min [ 531. Particulate CaMKII, which would include that in the postsy naptic density, is more resistant with a loss of 3(r50 % in 2 h of reperfusion, after a 10 min ischemic insult. The loss of CaM-KII activity is in general paralleled by a loss of CaM-KII protein as assessed by Western blot analysis. PKC and PKA, however, show little or no decrease during the ischemic periods studied.
Serine/threonine
Substrates
influx can activate Cl- channels, and this activation is blocked by microinjection of the autoinhibitor domain peptide of CaM-KU [54*]. The Cl- channels can also be activated by CM-KII microinjected into the cell or by its application to excised patches. This observation may provide a therapeutic pathway for circumventing defective regulation of Cl- channels by PKA in patients with cystic fibrosis. Phosphorylation of the cardiac ryanodine receptor by CaM-KII reverses the inhibitory effect of CaM on Ca2+ release [55]. The neuronal IP3 receptor, which has some sequence homology to the ryanodine receptor and which also releases intracellular stores of Ca2+, is also phosphotylated by CaM-KII and PKC [56]. Another recently identified in vitro substrate of CM-KII and PKC is nitric oxide synthase. This enzyme is activated by the binding of Ca2+/Cah4 or by phosphorylation with PKC, but it is inactivated by CaM-KII [57]. Nitric oxide has been proposed as a putative retrograde synaptic messenger required for LTP. WV-KII may also be involved in desensitization of the epidermal growth factor receptor [58]. Ca2+
Studies of Ca2+-stimulated expression of c-fos have strongly implicated the involvement of the CaM-kinase family. Phosphotylation of Ser133 in the cAMP-responseelement binding protein (CREB), one of the major trans-activating proteins in the c-fm system, enhances its transcriptional activity. Elevation of intracellular Ca2+ in PC12 cells promotes 32P labeling of Ser133 in CREB, and purified CM-kinases I or II in vitro can phosphorylate CREB at this site [59*, 601. A study in rat pituitary tumor cells further demonstrates that transfection of an activated form of CaM-KII results in transcriptional activation of a reporter gene controlled by the RSV LTR promoter [61]. The role of Cti-kinases in Ca2+-stimulated gene expression, which appears to be important for several neuronal systems, will undoubtedly be an area of active investigation over the next several years. Little CaM-KII appears to be localized in the nucleus where CREB phosphorylation is thought to occur. A new member of the Cah4-kinase family, CaM-KIV or Gr, which is prominent in the nucleus and axons, has recently been described [62*,63]. This kinase contains most of the essential residues of CaIv-KII in its catalytic and regulatory domains, but its carboxyl terminus is highly acidic and the enzyme is monomeric. CaM-KIV phosphorylates many of the same proteins and sites as CaM-KII, and because of its nuclear localization, it will be important to determine if it phosphorylates CREB and other transcriptional regulatory proteins.
protein
kinases
Scott and Soderling
‘selective’ for a given protein kinase, and therefore must be used within defined concentration ranges, a feat difficult to obtain in intact cells. Appropriate inactive structural analogues of the inhibitor are essential controls for this approach. It is anticipated that inhibitors highly specific for individual protein kinases will be developed, and progress in this direction will be facilitated by computer design of inhibitors using the crystal structure of the PKA catalytic core. It has been gratifying to observe the progression over the past three decades of protein kinases from a novelty of glycogen metabolism into essentially all cellular systems. The ‘Decade of the Brain’ will undoubtedly witness continued progress in understanding the roles of protein kinases in such complex neural functions as transcriptional regulation and synaptic plasticity. While most attention to date has focused on the three multifunctional protein kinases, future studies will also emphasize important neuronal functions for existing and/or newly discovered specific protein kinases.
References
and recommended
Papers of particular interest, published view, have been highlighted as: . of special interest .. of outstanding interest 1.
reading
within the annual period of rep
SCOT JD: Cyclic
Pbarmacol
Nucleotide-Dependent Ther 1991, 50:123-145.
Protein
Kinases.
2.
MS, ODA T, BERRYN, SHINOMURA T, NISHIZUKAY, SHEARMAN A~AOKAY, OGITA K, KOIDEH, KIKKAWAU, KISH~MOTO A el al: Protein Kinase C Family and Nervous Function. Prog Brain Res 1991, 891125-141.
3.
SODERUNGTR, FUKUNAGAK, BRICKEYDA, FONG YL, RICH
DP, SMITH K, COLBRAN RJ: Molecular on Brain CaIcium/CaImoduIin-Dependent Prog Brain Res 1991, 89:16+183.
and
Cellular Studies Protein Kinase II.
KNIGHTON DR, ZHENG J, TENF(CK LF, ASHFORDVA, XUONG N, TAYLOR SS, SOWADSKIJA: Crystal Structure of Catalytic Subunit of Cyclic Adenosine Monophosphate-Dependent Protein Kinase. Science 1991, 253:407-414. The crystal structure of the Cu-subunit of PKA is resolved to 2.7& This is the first crystal structure of a protein kinase. 4.
..
KNIGHTONDR, ZHENGJ, TEN EYCK LF, ZUONG N, TAWR
SS, SOWADSKIJM: Structure of a Peptide Inhibitor Bound to the Catalytic Subunit of Cyclic Adenosine MonophosphateDependent Protein Kinase. Science 1991, 253414-420. The crystal sn-ucture of the peptide inhibitor (PKI) bound to Cu, is resolved to 2.7A 5. .
Luo 2, SHAFIT-ZAGARW B, ERLICHMAN J: Identification of the MAP2 and P75 Binding Domain in the Regulatory Subunit (RIIP) of Type II CAMP-Dependent Protein Kinase: Cloning and Expression of the cDNA for Bovine Brain RIIp. J Biol Chem 1990, 265:21804-21810.
JD, STOFKORE, MCDONALDJR, COMERJD, VITALISEA, MAivGWJA: Type II Regulatory Subunit Dhnerization Deter-
Scan
Prospective Dissecting the individual roles of the multifunctional protein kinases, which often have overlap of substrate specificities, in cellular systems is a challenge. A popular approach has been the use of cell permeable or microinjected inhibitors. These inhibitors are generally
mines the Subcellular Localization of the CAMP-Dependent Protein Kinase. J Rio1 Chem 1990, 265:21561-21566. CARR DW, STOFKO-HANRE, FRASERIDC, BISHOPSM, Acorr TS, BRENNANRG, Scan JD: Interaction of the Regulatory Subunit (RII) of cAMP-Dependent Protein KInase with RIIAnchoring Proteins Occurs Through an Amphipathic Helix Binding Motif. J Biol Cbem 1991, 26614188-14192.
293
294
Signalling mechanisms 9.
TOURNIER S, RAYNAUD F, GERBAUDP, LDHMANN SM, DO&E M, EVAIN-BRION D: Association of Type II cAMP-Dependent Protein Kinase with p34cdc2 Protein Kinase in Human Fibroblasts. J Biol &em 1991, 266:1901~19022.
10.
L%MBNJ, CAVADOREJC, LABBEJC, MAURERRA, FERNANDEZ A:
Inhibition of cAMP-Dependent Protein Kinase Plays a Key Role in the Induction of Mitosis and Nuclear Envelope Breakdown in Mammalian Cells. EMBO J1991, 10:1523-1533. 11.
HOCEVAR BA, FIELDS AP: Selective
28.
OHNO S, KONNO Y, ANITA Y, YANO A, SUZUKIK: A Point
Bicpbys
1991, 289:187-191.
Mutation
at the Putative ATP-Binding Site of Protein I&a.% the Kinase Activity and Renders it DownJ Biol C&m 1990, 265:6296-6300. Regulation-Insensitive. 29.
B&hem
MEINKOTHJL, Jr Y, TA~I~R SS, FERAMISCO JR: Dynamics of the
Edited by Mason WT, Relf
of Living Celka Practical Guide.
WANG LY, SALTER MW, MACDONALD JF: Regulation
Receptors phatases. 16.
17.
by cAMP-Dependent
Protein
Kinase
of Kainate and Phos-
1991, 253:1132-1134.
Science
sively
to cAMP-Dependent
Phosphorylation.
Nature 1991,
351:573-576. 18.
BURNS DJ, BL~~MENTHALJ, LEE MH, BELL RM: Expression of the a, PII, and y Protein Kinase C Isozymes in the Baculovirus-Insect Cell Expression System. J Biol Chem 1990, SCHAAF D, PARKERPJ: Expression,
acterization
of
Protein
Kinase
Purification, and CharC-E. J Biol Chem 1990,
BURNSDJ. BELLRM: Protein
Ester Binding 21.
22.
Domains.
Kinase
C Contains
J Biol Chem
34.
24.
and Characterization of PKC-L, a New Member of the Protein Kinase C-Related Gene Family Specifically Expressed in Lung, Skin, and Heart. Mol Cell Biol 1991, 11:126133. HUBBARDSR, BISHOPWR, KIRXHMEIERP, GEORGE SJ, CRAMER SP, HENDRICKSONWA: IdentiIication and Characterization of Zinc Binding Sites in Protein Kinase C. Science 1991, MESSING RO, PETERSEN PJ, HENRICH CJ: Chronic Ethanol Exposure Increases Levels of Protein Kinase C 6 and E and Protein Kinase C-Mediated Phosphorylation in Cultured Neural Cells. J Biol Chem 1991, 266:2342%23432. LEIBERSPERGER H, GSCHWENDTM, GERNOLDM, MARKSF: Im-
of Protein Hormone
RegKinase C Isozymes by Thyrotropinin GH4CI Cells. J Biol Cbem 1991,
36.
MACKANOS EA, PETI?TGR, RAMSDELL JS: Bryostatins
Selectively on GH4 Cell
Regulate Protein Kinase C-Mediated Effects Proliferation. J Biol Cbem 1991, 26611201-11212.
of Protein
Kinase
J Biol Cbem
C
1991,
SCHARENBERG AM, 0~~s JL, SCHREURS BG, CRAIGAM, ALKON DL:
Protein Kinase C Redistribution Within CA3 Stratum Oriens During Acquisition of Nictitating Membrane Conditioning in the Rabbit. Proc Nat1 Acud Sci K&4 1991, 886637441. 37.
FARLF(J, SCHUMANE: Protein Kinase C Inhibitors Prevent
Induction
and
Continued
Expression
Hermissenda Type B Photoreceptors. USA 1991, 88:20162020.
of Cell Memory
in
Proc Nat1 Acad Sci
38.
KA, NICOL~ONTA, STORMDR: CHAPMANER, Au D, ALEXANDER Characterization of the Cahnodulin Binding Domain of Neuromodulin. J Biol Cbem 1991, 266207-213.
39.
MCILLROY BK,
WALTERS JD,
Phosphorylation-Dependent KS Peptide to Cahnodulin. 40.
BUCKSHEARPJ, JOHNSONJD:
Binding
J Biol
of a Synthetic
MARC-
Chem 1991, 26649594964.
GRAFF JM, RAJANRR, RANDALL RR, NAIRNAC, BLACKSHEAR PJ:
Protein Kinase C Substrate and Inhibitor Characteristics Peptides Derived from the Myristoylated &nine-Rich Kinase Substrate (MARCKS) Protein Phosphorylation Domain. J Biol Cbem 1991, 266:14390-14398. 41. .
An in 42.
43.
CHO KO, WALL JB, PUGH, PC, ITO M, MUELLER SA, KENNEDY MB: The a Subunit of Type II Ca*+/Cahnodulin-Dependent
is Highly
Conserved
in Drosophila.
Neuron
PAUSCHMH, MAIMD, KUNI~AWA R, ADMONA, THORNER J: Mul-
tiple Ca*+/Cahnodulin-Dependent Protein Kinase Genes a Unicellular Eukaryote. EMBO J 1991, 10:1511-1522. 44.
of C Site
ROTENBERG SA, CALOGEROPOUL~U T, JAWORSK~ JS, WINSTEINIB, RIDEOUTD: A Self-Assembling Protein Kinase C Inhibitor. Proc Nat1 Acad Sci USA 1991, 88:249&2494. vitro analysis of a self-assembling PKC inhibitor.
Protein Kinase 1991, 7:439450.
26623761-23768. 26
of Rhodopsin.
R, HARDY RW, MARX J, TSUCHIDAT, SMITH DP, RANGANATHAN ZUKERCS: Photoreceptor Deactivation and Retinal Degeneration Mediated by a Photoreceptor-Specific Protein Kinase C. Science 1991, 254:1478-1483. The localization and characterization of eye PKC in photoreceptors shows specific functions for the enzyme.
KILEX SC, PARKERPJ, FABBROD, JAKENS: Differential
ulation Release
Phosphorylation
35. .
BACHERN, ZISMANY, BERENTE, LIVNEH E: Isolation
munological Demonstration of a Calcium-Unresponsive Protein Kinase C of the &Type in Different Species and Murine Tissues. J Biol G%em 1991, 26614778-14784. 25.
NNV~~N AC, WILUAMSDS: Involvement 266,:1772>17728.
254~17761779. 23.
Kinase C Mediates Transient Spinule-Type Neurite Outgrowth in the Retina During Light Adaptation. Proc Nat1 Acad Sci USA 1991, 88:3603-3607.
Two Phorbol
1991, 266:18330-18338,
88:39974000.
WEILERR, KOHLERK, JANSSEN U: Protein
265:7301-7307. 20.
of IntraKinase C.
33.
265:12044-12051. 19.
Protein
USUDA N, KONG Y, HAGIWARAM, UCHIDA C, TERA.SAWA M, NAGATAT, HIDAKAH: Differential Localization of Protein Kinase C Isozymes in Retinal Neurons. J Cell Biol 1991, 112:1241-1247.
GREENCARD P, JENJ, NA~RNAC, STEVENS CF: Enhancement
HARIZELL HC, MERYPF, FISCHMEISTER R, S~ABOG: Sympathetic Regulation of Cardiac Calcium Current is Due Exclu-
for Activated
32.
in the
of Glutamate Response by CAMP-Dependent Protein Kinase in Hippocampal Neurons. Science 1991, 253:113>1138.
Proteins
Proc Nat1 Acad Sci USA 1991,
G. Academic Press; 1991, in press. 15.
Receptor
MOCHLY-ROSEN D, KHANERH, LOPEZJ, SMITHBL: Intracellular Receptors for Activated Protein Kinase C. J Biol Cbem 1991, 26631486614868.
ADAMSSR, BACSKAIBJ, TA~OR SS, TS~ENRY: Optical
Probes for Cyclic AMP. In Fluorescent Probes fir Biological Activity
1990, 191:421429.
31.
13. ..
14.
Purification from Sheep Brain and Seto Lipocortins and 14-3-3 Protein. Eur J
M~CHLY-ROSEN D, KHANERH, LOPEZJ: Identification
celhdar
in
ADAMS SR, HAROOTUNVWAT, BUECHLER YJ, TAVL~RSS, TSIEN RY: Fluorescence Ratio Imaging of Cyclic AMP in Single Cells: Nature 1991, 3&X694-697. The construction and analysis of the fluorescent PKA analog FICRhRin libroblasts. A novel method to study PKA dynamics in viva.
TOKERA, ELLISCA, SELLERS lA, AITKENA: Protein Kinase C
Inhibitor Proteins quence Similarity 30.
Distribution of Cyclic AMP-Dependent Protein Kinase Living Cells. Proc Nat1 Acad Sci USA 1990, 87~9595-9599.
Translocation of PI,-Protein Kinase C to the Nucleus of Human Promyelocytic (HL60) Leukemia Cells. J Biol Cbem 1991, 266:28-33.
Ca Abolishes
BRAUNRK, VKJLLIET PR, CARBONARO-HALL DA, Hw FL: Phosphorylation of RII Subunit and Attenuation of cAMP-Dependent Protein Kinase Activity by Proline-Directed Protein Kinase.
Arch B&hem 12.
27.
in
KANASEKI T, 1KEUCHIY, SUGIURAH, YAMAUCHIT: Structural
Features
of Ca*+/Calmodulin-Dependent
Protein
Kinase
Serine/threonine
II Revealed by 115:104~1059. 45.
Electron
Microscopy.
J
Cell Biol 1991,
SODERUNG TR: Protein Kinases: Regulation
Domains.
J Biol @em
by Autoinbibitory 199Q, 265:1823%1826.
FUKUNAGAK, SODERUNG TR: Activation of Ca*+/CaIrnoduIinDependent Protein Kinase II in Cerebella Granule Cells by N-Methyl-D-Aspartate Receptor Activation. Mol Cell Neurosci 1990, 1:133-138. Demonstrates that activation of NMDA receptors by glutamate stimulates autophospholylation and activation of CaM-KII.
46. .
MACNICOLM, JEFFERSONAB, SCHULMANH: Ca*+/CaImodulin Kinase is Activated by the Phosphatidylinositol Signaling Pathway and Becomes Ca*+-Independent in PC 12 CeIIs. J Biol Cbem 19M, 265:18055-18058. Establishes that stimulation of the phosphatidylinositol system activates CaM-KII in addition to PKC. 47. .
48.
JEFFERSONAB, TRAVESSM, SCHULMANH: Activation functional Ca*+/CaImoduIin-Dependent Protein GHJ CeIIs. J Biol Cbem 1991, 266:1484-1490.
of MultiKinase in
49.
OCORR KA, SCHULMAN H: Activation of Multifunctional Ca*+/CaIrnoduIin-Dependent Kinase in Intact HippocampaI Slices. Neuron 1991, 6:907-914.
50.
of Type II MOLLOYSS, KENNEDY MB: Autophosphorylation Ca*+/CaImoduIin-Dependent Protein Kinase in Cultures of Postnatal Rat Hippocampal SIices. Proc Nat1 Acad Sci USA 1991, 88347564760.
51. .
TOKLJM~U H, CHIJNVAT, HAGIWAR~M, MIZUTANIA, TERASAWA M, HIDAKA H: KN-62, l-[N-O-Bis(S-IsoquinoIinesuIfonyl)-NMethyl-L-Tyrosyl]4-Phenylpiperazine, as Specific Inhibitor of Ca*+/CaImoduIin-Dependent Protein Kinase II. J Biol Cbem 1990, 265:431%320. Describes a cell-permeable inhibitor which appears to be highly specific for CaMKII. 52.
ISHII A, KIUCHI K, KOBAYASHIR, SUMI M, HIDAKAH, NAGATSU T: A Selective Ca*+/CaImodulin-Dependent Protein Kinase II Inhibitor, KN-62, Inhibits the Enhanced Phosphorylation and the Activation of Tyrosine Hydroxylase by 56 mM K+ in Rat Pheochromocytoma PC1 2H Cells. Biixbem Biopby Res Commun 1991, 176:1051-1056.
53.
YAMAMOTOH, FUKUNAGAK, LEE K, SODERLINGTR: IschemiaInduced Loss of Brain CaIcium/CalmoduIin-Dependent Protein Kinase IL J Neurocbem 1992, 58:111&1117.
54. .
WAGNERJA, COZENS AL, SCHULMANH, GRUENERTDC, STRYER L, GARDNER P: Activation of Chloride Channels in NormaI and Cystic Fibrosis Airway Epithellal Cells By Multifunctional Calcium/Calmodulin-Dependent Protein Kinase. Nature 1991, 349793-796.
protein
kinases
Scott and Soderling
Although Cl- channels in epithelial cells from cystic fibrosis patients cannot be activated by PKA or PKC, they can be activated by CaM-KII. 55.
WITCHER DR, KOVACSRJ, SCHULMANH, CEFALI DC, JONES LR: Unique Phosphorylation Site on the Cardiac Ryanodine Receptor Regulates Calcium Channel Activity. J Biol Chem 1991, 266:11144-11152.
56.
FERRISCD, HUGANIRRI, BREDT DS, CAMERONAM, SNYDERSH: Inositol Trisphosphate Receptor: Phosphorylation by Protein Kinase C and Calcium Calmodulin-Dependent Protein Kinase in Reconstituted Lipid Vesicles. Proc Nat1 Acud Sci USA 1991, 88:2232-2235.
57.
NAKANEM, MITCHELLJ, FORSTERMANN LJ, MUFZADF: Phosphotylation by Calcium Calmodulin-Dependent Protein Kinase II and Protein Kinase C Modulates the Activity of Nitric Oxide Synthase. B&hem Biopbys Res Commun 1991, 180:13961402.
58.
COUNTAWAY JL, NAIRNAC, DAVISRJ: Mechanism of Desensitization of the Epidermal Growth Factor Receptor ProteinTyrosine Kinase. J Biol Chem 1992, 267:112+1140.
59. .
SHENG M, THOMPSONMA, GREENBERGME: CREB: a Ca*+-Reg-
60.
DA.S PK, KARL KA, COLlCOS MA, PRYWEX R, KANDEL CAMP Response Element-Binding Protein is Activated Ca*+/CaImoduIin as weII as CAMP-Dependent Protein nase. Proc Nat1 Acud Sci USA 1991, 88:506-5065.
61.
KAPILOFF MS, MATHISJM, NELTON CA, LIN CR, ROSENFELDMG:
ulated Transcription Factor Phosphorylated by CalmodulinDependent Kinases. Science 1991, 252:1427-1430. Demonstrates that CaMKI and II can phosphorylate the transcriptional activating protein CREB.
Calcium/CaImoduIin-Dependent Pathway for Transcriptional USA 1991, 88:371@3714.
Protein Regulation.
ER by Ki-
Kinase Mediates a Proc Nat1 Acud Sci
62. .
JENSEN KF, OHMSTEDE CA, FISHER RS, SAHYOUN N: Nuclear and AxonaI Localization of Ca*+/CaImoduIin-Dependent Protein Kinase Type Gr in Rat CerebelIar Cortex. Proc Nat1 Acad Sci USA 1991, 88:28562853. Uses immunochemical and ultrastructural analysis to localize CaMKIV in the nucleus of cerebellar granule cells. 63.
MEANS AR,
CRUZALEGUI F,
LEMAGUERESSE B,
NEEDLEMAN DS,
T: A Novel Ca*+/Calmodulin-Dependent Protein Klnase and a Male Germ Cell-Specilic Calmodulin-Binding Protein are Derived from the Same Gene. Mol Cell Biol 1991, 11:396&3971. SIAUGHTER
GR,
ONO
JD Scott and TR Soderling, Vollum Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland, Oregon 97201, USA.
295
protein kinases
John D. Scott and Thomas R. Soderling Vellum
Institute
for Advanced Biomedical
Signal transduction
in the nervous system is heavily dependent
multifunctional
serine/threonine
protein
Recent
have
our
studies
isoforms
of these
properties,
Research, Portland,
furthered
kinases
and substrate
and
kinases,
their
determinants
subcellular
of how localizations,
are important
USA
on the three
PKA, PKC, and
understanding
Oregon,
the
CaM-KII. multiple
regulatory
for the specificity
of
kinase functions.
Current
Opinion
in Neurobiology
Introduction Neural tissues contain very high concentrations of the three multifunctional serine/threonine protein kinases: cAh4P-dependent protein kinase (protein kinase A or PKA), Caz+/phospholipid-dependent protein kinase (protein kinase C or PKC), and Ca2+/calmodulin-dependent protein kinase II (CaM-KII). For example, CaM-KII constitutes up to 2 % of total protein in certain regions of the brain such as the hippocampus. Thus, it is not surprising that these kinases participate in many of the signal transduction pathways that are so important for providing modulation of such neural functions as neurotransmitter synthesis, release and receptor binding, ion channel properties, gene transcription, and synaptic plasticity. The purpose of this short review is to update selected aspects of the three kinases, and only recent papers will be referenced. Each of the kinases has been individually reviewed (PKA [ 11, PKC [ 21, and CaMK II [3] >, and the reader should consult these for background information.
CAMP-dependent Structure
protein
kinase
and function
The CAMP signaling pathway is composed of individual neurotransmitter receptors coupled through GTP-binding proteins to adenylate cyclase. Four molecules of CAMP bind each dormant PKA holoenzyme complex (R2C2), thereby causing the release of two active catalytic (C) subunits from the regulatory (R) subunit dimer. Three C subunit isoforms (Ccl, Co, and Cy) and four R subunit isoforms (RIa, RIP, RUa, and RI@) are expressed at different levels in various mammalian cells, and combine to form type I and type II holoenzymes. The pisoforms of each subunit are predominantly expressed in neural tissues.
1992, 2:2899295
The C subunit contains a catalytic core that is conserved in all protein kinases, with distinct sequence motifs or subdomains that are involved in ATP-binding, protein/peptide-binding and phosphotransferase activity. Recently, the crystal structure of the recombinant mouse Q-subunit complexed with a peptide inhibitor has been solved at a resolution of 2.7h; [4*-,5*]. These studies show that Ca is a bilobal molecule (Fig-l). The smallest lobe contains the sequences involved in ATP-binding while the larger lobe is responsible for peptide binding and catalysis, The cleft between the lobes is occupied by h4g2 + /ATP
Cellular
regulation
Both RI isoforms are primarily cytoplasmic, but neural tissues contain up to 75 % of either RII isoform associated with the plasma membrane, cytoskeletal components, dendrites, secretory granules, and the nuclear membrane. Type II PKA localization occurs through R subunit dimer interaction with specific-anchoring proteins. PKA localization in the cytoskeleton occurs through interaction of the amino-terminal 50 residues of RI1 with microtubule-associated protein 2 (MAP 2) [6,7]. A conserved amphipathic helix motif, which is involved in binding to RR, has been identified in MAP 2 and two other RII-anchoring proteins, AKAP79 and Ht31 [8]. Synthetic peptides which span the amphipathic helix region in Ht31 associate with RIIa or the type II holoenzyme with nanomolar affinity (D Carr and JD Scott, unpublished data). RI1 may also interact with the p34cdcz kinase [9], which is intriguing as inhibition of PKA plays a key role in the induction of mitosis and nuclear envelope breakdown [IO]. Furthermore, phosphorylation of RI1 at Thr211 by ~34~~~~ blocks activation of the type II holoenzyme by CAMP [ 111. While R&anchoring can account for localization of the PKA holoenzyme, the free C subunit can translocate into specific cellular compartments upon its activation.
Abbreviations C-catalytic; DAG4iacylglycerol;
CaM-KII-Ca*+/calmodulin-dependent IPS-inositol
1,4,5-trisphosphate;
NMDA-N-methyl-D-aspartate;
protein kinase II; CREW-CAMP-response-element LTP-long-term
PKA-protein
@
Current
Biology
potentiation;
kinase A; PKC-protein
Ltd ISSN 0959-4388
binding
MAP-Z-microtubule-associated
protein; protein
2;
kinase C; R-regulatory.
289
290
Signailing
mechanisms
pathetic regulation of this channel is due exclusively to phosphorylation of the channel by PKA
Ca2+/phospholipid-dependent Structure
Fig.1. A ribbon diagram illustrating the three-dimensional structure of the CAMP-dependent protein kinase catalytic subunit. The ATP phosphate anchor and catalytic loop are indicated. Reproduced with permission from WI.
Nuclear translocation of the C subunit has been demonstrated in both fibroblasts and neurons [ 12,13**,14]. Microinjection of R and C subunits with fluoresceine shows that type I PKA is cytoplasmic, but upon activation with Sbromo-cAMP the free C subunit translocates to the nucleus [ 121. This observation has been extended by an elegant series of experiments using fluorescent ram tio imaging to chart the migration of the PKA holoenzyme, labeled on both the C and R subunits, from its site of activation [I3e*,I4]. The microinjected fluores cent PKA holoenzyme remains in the cytoplasm when CAMP is low. After cAMP-induced dissociation of the holoenzyme, the C subunit translocates to the nucleus, while R remains in the cytoplasm. Upon lowering CAMP, the fluoresceine labeled C subunit leaves the nucleus and reforms the holoenzyme with the R subunit in the cytoplasm.
Substrates
The activation of a glutamate receptor ion channel is mediated by PKA phosphorylation [I5,161. Currents induced by glutamate or kainate are potentiated by agents which stimulate PKA activity in cultured hippocampal neurons or in excised patches, and are blocked by application of a peptide inhibitor of the kinase. Similar experiments have been performed on the cardiac voltagegated Ca2 + channel [ 171, and it was concluded that sym
protein
kinase
and function
PKC is a phospholipid-dependent protein kinase that can be activated by the second messenger diacylglycerol (DAG) or by phorbol esters. PKC is actually an isozyme family of nine structurally-related enzymes ranging in size from 7%90 kD [ 18,19]. Each isoform contains five variable subdomains (V,-Vg) interspersed with four conserved subdomains (C,C*) (see Fig. 2). The catalytic elements (C, and Cd) are similar to the catalytic core of other protein kinases and contain sequences responsible for ATI-binding and phosphotransferase activity. The amino-terminal V, domain contains an autoinhibitory pseudosubstrate sequence that occupies the substrate-binding site in the catalytic core and renders the enzyme inactive. Binding of DAG and a phospholipid cofactor, commonly phosphatidylserine, to the C, subdomain promotes conformational changes which displace the pseudosubstrate sequence and thereby activate the enzyme. Two repeated cysteine-rich sequences, homologous to the ‘zinc finger’ binding motif, form the C, subdomain and bind phorbol esters with nanomolar affinity [ 201. The k-isoform has only one zinc finger and does not bind phorbol esters [21]. Recent evidence suggests that two Zn2 + ions bind each zinc finger [ 221. The C, domain confers Ca2+ dependence on the CL,jYI,and y PKC isoforms, which require both phospholipid and Ca2+ for activation. In contrast, the 6, E, 5, q, and L isoforms are Ca2+ -independent and lack the C2 domain. These differences in activator requirements, as well as in substrate specificities and subcellular localizations (see below), suggest that each isoform may have a distinct role in signal transduction [ 23,241.
Cellular
regulation
Upon activation, PKC is translocated from the cytoplasm to particulate fractions, which localize the kinase to the cytoskeleton, postsynaptic densities, nuclei, and the plasma membrane. Selective translocation of PKC isoforms occurs in response to specific activators. For example, thyrotropin releasing hormone promotes translocation of PKC isoforms other than PKCs, while bryostatin 1 selectively translocates PKCy in GH4 rat pituitary cells [ 25,261. In HL 60 leukemia cells phorbol dibutyrate (PB2) promotes the translocation of both PKCcr and PII to the plasma membrane, while bryostatin 1 targets PKCBII to the nucleus [ 271. Persistent activation by phorbol esters also leads to down-regulation of the particulate-bound kinase by proteolysis. Furthermore, autophosphorylation may prime PKC for degradation as a mutant PKC~Llacking kinase activity is translocated but not proteolysed [ 281. Originally PKC targeting was thought to occur exclusively through association with lipids. The active kinase may also be localized through interactions with anchoring
Serine/threonine
a, 01, PII, and
lsoforms
Zinc
ATP-binding
I
I
domain
Catalytic domain
COOH
Fig.2. A diagram
Ht----lHHHl-----lH-I----I
Cl
mon subdomain v2
c2
v3
c3
v4
c4
6, F, 5, q and L Regulatory
H-
Vl
Cl
domain HH v2
v3
c4
The location of each below the figregions of con-
V, denotes
variable sequence. V5
com-
ure. C, denotes constant
pseudosubstrate v4
the
of the protein
is indicated
served sequences.
Catalytic domain HH-H c3
illustrating structure
kinase C isoforms.
V5
subdomain isoforms
Soderling
site
Pseudosubstrate site
Vl
Scott and
kinases
y
fingers
Regutatory
NH2
protein
motifs,
regions of
The positions
of the
site, zinc finger binding
and ATP-binding
sites are also in-
dicated.
proteins, however. Using a modified western blot technique, PKC PI has been shown to bind certain cytoskeletal elements, the PKC inhibitor protein (PKCI) and annexin 1 [29,30]. These PKC anchoring proteins may be structurally related as a conserved sequence of 16 amino acids is present in both. This sequence is believed to represent the PKC-binding site as peptides corresponding to it bind PKCPI, although with low affinity [ 311. PKC isoforms are regionally expressed in the central nervous system. For example, the u-, p-, and y-subtypes are differentially compartmentalized in retinal neurons with PKCy being specifically localized to the rod bipolar and amacrine cells [32]. This observation correlates with rem ports that PKC mediates spinule-type neurite outgrowth in the retina during light adaption, and that it is involved in the phospholylation of rhodopsin [33,34]. Furthermore, analysis of inaC mutants in Drosophila suggests that a photoreceptor-speci PKC homolog, eye-PKC, is required for deactivation and the rapid desensitization of the visual cascade [35*]. PKC activity has also been implicated in long-term potentiation (LTP). Neural imaging with 3[H]-PB2 demonstrates that PKC isoforms are redistributed within the CA3 hippocampal neurons upon acquisition of an associative-conditioned response [36]. In addition, microinjection of the protein kinase inhibitor staurosporin into Hermisrendu type B photoreceptors prevents expression of learning-induced changes in Ca2+ -activated K+ channel currents [37].
hydrazone, an inhibitor of PKC (ICsO = 20 l.tM) [41-l. An intriguing property of these compounds is that they as semble into an active PKC inhibitor in or near cells, and have been used to block kinase activity in cancer cells.
Ca2+/calmoduliwdependent Structure
protein
kinase II
and function
is also an isozyme family containing G12 subunits (5G6OkD each) per holoenzyme. The amino-terminal catalytic domain and the central regulatory region are highly conserved in isoforms sequenced from mammals, Drosophila [42], and yeast [43]. The variable carboxy1 terminus, responsible for subunit assembly, forms a central core in the holoenzyme surrounded by globular catalytic domains that are arranged like flower petals [ 441. CaM-KII
Studies over the past five years from a number of laboratories have led to the regulatory model depicted in Fig.3 1451. The binding of Caz+/CaM disrupts the autoinhibitory interaction with the catalytic domain, thereby activating the kinase and allowing phosphorylation of exogenous substrates as well as autophosphorylation of 67 sites within CaMKII. Autophosphorylation of Thr286 in the autoinhibitory domain occurs within seconds of l& nase activation and is uniquely responsible for formation of the Ca2+-independent form of CaMKII.
Substrates
Cellular
PKC activity appears to indirectly control calmodulin-mediated second messenger pathways in neurons. Two of the major PKC substrates, neuromodulin and the MARCKS protein, bind calmodulin with high affinity in the absence of Ca2+. Their function may be to concentrate calmodulin at specific sites within neurons, which is then released upon their phosphorylation by PKC [ 38,391. Pseudosubstrate inhibitor peptides have been designed from the MARCKS sequence and inhibit PKC activity with Ki values as low as 20 nM in vitro [40]. An alternative approach to the study of PKC inhibition involves the synergistic action of two prodrugs, an aldehyde and a hydrazine, which combine in situ to form a cytotoxic
It has been postulated that a transient elevation of intracellular Ca2+ may trigger prolonged physiological responses mediated by CaIv-KII through formation of the Ca2 + -independent species. Recent studies have demonstrated autophosphorylation and formation of Ca2+ -independent CaWKII activity in cultured cells and tissue slices [ 46*,47*,4&50]. Basal levels of Cal + -independent WV-KII (5-25% in the cerebellum and hippocampus) can be modulated by altering extracellular Ca2+, or by pre-treatment with protein kinase (H7) or protein phosphatase (okadaic acid) inhibitors. In all the preparations examined, except the cultured hippocampal slices [SO], K+ depolarization rapidly results in the formation
regulation
291
292
Signalling
mechanisms
290 300 MHRQETVDCLKKFNARRKLKGAILTTMLA
(a) Inactive P
(c) Fully active (100%)
!F!$Z$JfY&
0
Protein phosphatase P
(d) Partially
Cal’-independent
(50-
80 “/o)
Fig.3. A schematic model for the regulation of the Ca 2+/calmodulin-dependent protein kinase II (CaM-KII). The model depicts a single subunit anchored to the holoenzyme through the carboxyl terminus. (a) In the absence of CaM, CaM-KII is inactive as a result of (b) Interaction of Ca2+/CaM with its the interaction of the autoinhibitory domain (shaded area) with the catalytic domain (-wYh~). binding domain (darkly shaded area) conformationally inactivates the inhibitory domain, thereby activating the kinase, which can then bind Mg2+/ATP and (c) phosphorylate itself (Thr286). Exogenous proteins containing the consensus sequence can also be phosphorylated. (d) The autophosphorylated kinase remains active in the absence of Ca2+/CaM (i.e. is Ca 2+-independent), but it can be dephosphorylated and inactivated by protein phosphatases 1 and/or 2A. This model is modified from [451.
of Ca2+ -independent CaM-KII, which then slowly decays back to its basal value. In cerebellar granule cells [46*] the extent and duration of CaMKII activation is enhanced by okadaic acid, but these cells also contain protein phosphatases insensitive to this inhibitor. In PC12 and GH3 cells [47*,48], agents which trigger the phosphatidylinositol signaling system (i.e. formation of inositol 1,4,5-trisphosphate (IP3) and DAG) result in a very transient formation of Ca2 + -independent CaM-KII, consistent with the release of intracellular Ca2+ by IP3. PC12 cells have also been used to study a cell-permeable inhibitor, ~~62, which appears to be specific for CaMKU. Pre-treatment of PC12 cells with KN-62 (l-10pM) blocks 32P-labeling of CaM-KII [51*] and activation of tyrosine hydroxylase [52] in response to Ca2+ mob&ation. Postsynaptic injection of peptide inhibitors of Cti-KII or PKC block the induction of LTP, indicating that these two protein kinases may be involved in the regulation of synaptic plasticity. The induction of LTP in the hippocam pus requires a Caz+ influx through the N-methyl-D-aspartate (NMDA)-type glutamate receptor ion channel, so it is important to determine if activation of NMDA receptors activates CaMKII. In both cerebellar granule cells [4&] and cultured hippocampal neurons (K Fukunaga et al, unpublished data), glutamate treatment sign&
cantiy increases the Ca2+ -independent form of CaMKII through autophosphorylation of Thr286. This response is mediated via the NMDA receptor as it is enhanced by addition of ll.tM glycine or removal of extracellular Mg2+, and blocked by specific NMDA antagonists. The above in situ studies show that autophosphorylation and activation of CaM-KII is under dynamic regulation. The elevation of intracellular Ca2+ promotes the formation of the Ca2+ -independent form, and the extent and duration of this species depends on the activity of opposing protein phosphatases. It will be important to determine if subcellular localized CaM-KII (e.g. in the postsynaptic density) exhibits different kinetics of regulation. In none of the above studies was total CaM-KII activity, assayed in the presence of Caz+/CaM, altered. In the gerbil, however, forebrain ischemia, as a result of occlusion of the carotid arteries, produces an almost complete loss of soluble forebrain CaM-KII within 2-5 min [ 531. Particulate CaMKII, which would include that in the postsy naptic density, is more resistant with a loss of 3(r50 % in 2 h of reperfusion, after a 10 min ischemic insult. The loss of CaM-KII activity is in general paralleled by a loss of CaM-KII protein as assessed by Western blot analysis. PKC and PKA, however, show little or no decrease during the ischemic periods studied.
Serine/threonine
Substrates
influx can activate Cl- channels, and this activation is blocked by microinjection of the autoinhibitor domain peptide of CaM-KU [54*]. The Cl- channels can also be activated by CM-KII microinjected into the cell or by its application to excised patches. This observation may provide a therapeutic pathway for circumventing defective regulation of Cl- channels by PKA in patients with cystic fibrosis. Phosphorylation of the cardiac ryanodine receptor by CaM-KII reverses the inhibitory effect of CaM on Ca2+ release [55]. The neuronal IP3 receptor, which has some sequence homology to the ryanodine receptor and which also releases intracellular stores of Ca2+, is also phosphotylated by CaM-KII and PKC [56]. Another recently identified in vitro substrate of CM-KII and PKC is nitric oxide synthase. This enzyme is activated by the binding of Ca2+/Cah4 or by phosphorylation with PKC, but it is inactivated by CaM-KII [57]. Nitric oxide has been proposed as a putative retrograde synaptic messenger required for LTP. WV-KII may also be involved in desensitization of the epidermal growth factor receptor [58]. Ca2+
Studies of Ca2+-stimulated expression of c-fos have strongly implicated the involvement of the CaM-kinase family. Phosphotylation of Ser133 in the cAMP-responseelement binding protein (CREB), one of the major trans-activating proteins in the c-fm system, enhances its transcriptional activity. Elevation of intracellular Ca2+ in PC12 cells promotes 32P labeling of Ser133 in CREB, and purified CM-kinases I or II in vitro can phosphorylate CREB at this site [59*, 601. A study in rat pituitary tumor cells further demonstrates that transfection of an activated form of CaM-KII results in transcriptional activation of a reporter gene controlled by the RSV LTR promoter [61]. The role of Cti-kinases in Ca2+-stimulated gene expression, which appears to be important for several neuronal systems, will undoubtedly be an area of active investigation over the next several years. Little CaM-KII appears to be localized in the nucleus where CREB phosphorylation is thought to occur. A new member of the Cah4-kinase family, CaM-KIV or Gr, which is prominent in the nucleus and axons, has recently been described [62*,63]. This kinase contains most of the essential residues of CaIv-KII in its catalytic and regulatory domains, but its carboxyl terminus is highly acidic and the enzyme is monomeric. CaM-KIV phosphorylates many of the same proteins and sites as CaM-KII, and because of its nuclear localization, it will be important to determine if it phosphorylates CREB and other transcriptional regulatory proteins.
protein
kinases
Scott and Soderling
‘selective’ for a given protein kinase, and therefore must be used within defined concentration ranges, a feat difficult to obtain in intact cells. Appropriate inactive structural analogues of the inhibitor are essential controls for this approach. It is anticipated that inhibitors highly specific for individual protein kinases will be developed, and progress in this direction will be facilitated by computer design of inhibitors using the crystal structure of the PKA catalytic core. It has been gratifying to observe the progression over the past three decades of protein kinases from a novelty of glycogen metabolism into essentially all cellular systems. The ‘Decade of the Brain’ will undoubtedly witness continued progress in understanding the roles of protein kinases in such complex neural functions as transcriptional regulation and synaptic plasticity. While most attention to date has focused on the three multifunctional protein kinases, future studies will also emphasize important neuronal functions for existing and/or newly discovered specific protein kinases.
References
and recommended
Papers of particular interest, published view, have been highlighted as: . of special interest .. of outstanding interest 1.
reading
within the annual period of rep
SCOT JD: Cyclic
Pbarmacol
Nucleotide-Dependent Ther 1991, 50:123-145.
Protein
Kinases.
2.
MS, ODA T, BERRYN, SHINOMURA T, NISHIZUKAY, SHEARMAN A~AOKAY, OGITA K, KOIDEH, KIKKAWAU, KISH~MOTO A el al: Protein Kinase C Family and Nervous Function. Prog Brain Res 1991, 891125-141.
3.
SODERUNGTR, FUKUNAGAK, BRICKEYDA, FONG YL, RICH
DP, SMITH K, COLBRAN RJ: Molecular on Brain CaIcium/CaImoduIin-Dependent Prog Brain Res 1991, 89:16+183.
and
Cellular Studies Protein Kinase II.
KNIGHTON DR, ZHENG J, TENF(CK LF, ASHFORDVA, XUONG N, TAYLOR SS, SOWADSKIJA: Crystal Structure of Catalytic Subunit of Cyclic Adenosine Monophosphate-Dependent Protein Kinase. Science 1991, 253:407-414. The crystal structure of the Cu-subunit of PKA is resolved to 2.7& This is the first crystal structure of a protein kinase. 4.
..
KNIGHTONDR, ZHENGJ, TEN EYCK LF, ZUONG N, TAWR
SS, SOWADSKIJM: Structure of a Peptide Inhibitor Bound to the Catalytic Subunit of Cyclic Adenosine MonophosphateDependent Protein Kinase. Science 1991, 253414-420. The crystal sn-ucture of the peptide inhibitor (PKI) bound to Cu, is resolved to 2.7A 5. .
Luo 2, SHAFIT-ZAGARW B, ERLICHMAN J: Identification of the MAP2 and P75 Binding Domain in the Regulatory Subunit (RIIP) of Type II CAMP-Dependent Protein Kinase: Cloning and Expression of the cDNA for Bovine Brain RIIp. J Biol Chem 1990, 265:21804-21810.
JD, STOFKORE, MCDONALDJR, COMERJD, VITALISEA, MAivGWJA: Type II Regulatory Subunit Dhnerization Deter-
Scan
Prospective Dissecting the individual roles of the multifunctional protein kinases, which often have overlap of substrate specificities, in cellular systems is a challenge. A popular approach has been the use of cell permeable or microinjected inhibitors. These inhibitors are generally
mines the Subcellular Localization of the CAMP-Dependent Protein Kinase. J Rio1 Chem 1990, 265:21561-21566. CARR DW, STOFKO-HANRE, FRASERIDC, BISHOPSM, Acorr TS, BRENNANRG, Scan JD: Interaction of the Regulatory Subunit (RII) of cAMP-Dependent Protein KInase with RIIAnchoring Proteins Occurs Through an Amphipathic Helix Binding Motif. J Biol Cbem 1991, 26614188-14192.
293
294
Signalling mechanisms 9.
TOURNIER S, RAYNAUD F, GERBAUDP, LDHMANN SM, DO&E M, EVAIN-BRION D: Association of Type II cAMP-Dependent Protein Kinase with p34cdc2 Protein Kinase in Human Fibroblasts. J Biol &em 1991, 266:1901~19022.
10.
L%MBNJ, CAVADOREJC, LABBEJC, MAURERRA, FERNANDEZ A:
Inhibition of cAMP-Dependent Protein Kinase Plays a Key Role in the Induction of Mitosis and Nuclear Envelope Breakdown in Mammalian Cells. EMBO J1991, 10:1523-1533. 11.
HOCEVAR BA, FIELDS AP: Selective
28.
OHNO S, KONNO Y, ANITA Y, YANO A, SUZUKIK: A Point
Bicpbys
1991, 289:187-191.
Mutation
at the Putative ATP-Binding Site of Protein I&a.% the Kinase Activity and Renders it DownJ Biol C&m 1990, 265:6296-6300. Regulation-Insensitive. 29.
B&hem
MEINKOTHJL, Jr Y, TA~I~R SS, FERAMISCO JR: Dynamics of the
Edited by Mason WT, Relf
of Living Celka Practical Guide.
WANG LY, SALTER MW, MACDONALD JF: Regulation
Receptors phatases. 16.
17.
by cAMP-Dependent
Protein
Kinase
of Kainate and Phos-
1991, 253:1132-1134.
Science
sively
to cAMP-Dependent
Phosphorylation.
Nature 1991,
351:573-576. 18.
BURNS DJ, BL~~MENTHALJ, LEE MH, BELL RM: Expression of the a, PII, and y Protein Kinase C Isozymes in the Baculovirus-Insect Cell Expression System. J Biol Chem 1990, SCHAAF D, PARKERPJ: Expression,
acterization
of
Protein
Kinase
Purification, and CharC-E. J Biol Chem 1990,
BURNSDJ. BELLRM: Protein
Ester Binding 21.
22.
Domains.
Kinase
C Contains
J Biol Chem
34.
24.
and Characterization of PKC-L, a New Member of the Protein Kinase C-Related Gene Family Specifically Expressed in Lung, Skin, and Heart. Mol Cell Biol 1991, 11:126133. HUBBARDSR, BISHOPWR, KIRXHMEIERP, GEORGE SJ, CRAMER SP, HENDRICKSONWA: IdentiIication and Characterization of Zinc Binding Sites in Protein Kinase C. Science 1991, MESSING RO, PETERSEN PJ, HENRICH CJ: Chronic Ethanol Exposure Increases Levels of Protein Kinase C 6 and E and Protein Kinase C-Mediated Phosphorylation in Cultured Neural Cells. J Biol Chem 1991, 266:2342%23432. LEIBERSPERGER H, GSCHWENDTM, GERNOLDM, MARKSF: Im-
of Protein Hormone
RegKinase C Isozymes by Thyrotropinin GH4CI Cells. J Biol Cbem 1991,
36.
MACKANOS EA, PETI?TGR, RAMSDELL JS: Bryostatins
Selectively on GH4 Cell
Regulate Protein Kinase C-Mediated Effects Proliferation. J Biol Cbem 1991, 26611201-11212.
of Protein
Kinase
J Biol Cbem
C
1991,
SCHARENBERG AM, 0~~s JL, SCHREURS BG, CRAIGAM, ALKON DL:
Protein Kinase C Redistribution Within CA3 Stratum Oriens During Acquisition of Nictitating Membrane Conditioning in the Rabbit. Proc Nat1 Acud Sci K&4 1991, 886637441. 37.
FARLF(J, SCHUMANE: Protein Kinase C Inhibitors Prevent
Induction
and
Continued
Expression
Hermissenda Type B Photoreceptors. USA 1991, 88:20162020.
of Cell Memory
in
Proc Nat1 Acad Sci
38.
KA, NICOL~ONTA, STORMDR: CHAPMANER, Au D, ALEXANDER Characterization of the Cahnodulin Binding Domain of Neuromodulin. J Biol Cbem 1991, 266207-213.
39.
MCILLROY BK,
WALTERS JD,
Phosphorylation-Dependent KS Peptide to Cahnodulin. 40.
BUCKSHEARPJ, JOHNSONJD:
Binding
J Biol
of a Synthetic
MARC-
Chem 1991, 26649594964.
GRAFF JM, RAJANRR, RANDALL RR, NAIRNAC, BLACKSHEAR PJ:
Protein Kinase C Substrate and Inhibitor Characteristics Peptides Derived from the Myristoylated &nine-Rich Kinase Substrate (MARCKS) Protein Phosphorylation Domain. J Biol Cbem 1991, 266:14390-14398. 41. .
An in 42.
43.
CHO KO, WALL JB, PUGH, PC, ITO M, MUELLER SA, KENNEDY MB: The a Subunit of Type II Ca*+/Cahnodulin-Dependent
is Highly
Conserved
in Drosophila.
Neuron
PAUSCHMH, MAIMD, KUNI~AWA R, ADMONA, THORNER J: Mul-
tiple Ca*+/Cahnodulin-Dependent Protein Kinase Genes a Unicellular Eukaryote. EMBO J 1991, 10:1511-1522. 44.
of C Site
ROTENBERG SA, CALOGEROPOUL~U T, JAWORSK~ JS, WINSTEINIB, RIDEOUTD: A Self-Assembling Protein Kinase C Inhibitor. Proc Nat1 Acad Sci USA 1991, 88:249&2494. vitro analysis of a self-assembling PKC inhibitor.
Protein Kinase 1991, 7:439450.
26623761-23768. 26
of Rhodopsin.
R, HARDY RW, MARX J, TSUCHIDAT, SMITH DP, RANGANATHAN ZUKERCS: Photoreceptor Deactivation and Retinal Degeneration Mediated by a Photoreceptor-Specific Protein Kinase C. Science 1991, 254:1478-1483. The localization and characterization of eye PKC in photoreceptors shows specific functions for the enzyme.
KILEX SC, PARKERPJ, FABBROD, JAKENS: Differential
ulation Release
Phosphorylation
35. .
BACHERN, ZISMANY, BERENTE, LIVNEH E: Isolation
munological Demonstration of a Calcium-Unresponsive Protein Kinase C of the &Type in Different Species and Murine Tissues. J Biol G%em 1991, 26614778-14784. 25.
NNV~~N AC, WILUAMSDS: Involvement 266,:1772>17728.
254~17761779. 23.
Kinase C Mediates Transient Spinule-Type Neurite Outgrowth in the Retina During Light Adaptation. Proc Nat1 Acad Sci USA 1991, 88:3603-3607.
Two Phorbol
1991, 266:18330-18338,
88:39974000.
WEILERR, KOHLERK, JANSSEN U: Protein
265:7301-7307. 20.
of IntraKinase C.
33.
265:12044-12051. 19.
Protein
USUDA N, KONG Y, HAGIWARAM, UCHIDA C, TERA.SAWA M, NAGATAT, HIDAKAH: Differential Localization of Protein Kinase C Isozymes in Retinal Neurons. J Cell Biol 1991, 112:1241-1247.
GREENCARD P, JENJ, NA~RNAC, STEVENS CF: Enhancement
HARIZELL HC, MERYPF, FISCHMEISTER R, S~ABOG: Sympathetic Regulation of Cardiac Calcium Current is Due Exclu-
for Activated
32.
in the
of Glutamate Response by CAMP-Dependent Protein Kinase in Hippocampal Neurons. Science 1991, 253:113>1138.
Proteins
Proc Nat1 Acad Sci USA 1991,
G. Academic Press; 1991, in press. 15.
Receptor
MOCHLY-ROSEN D, KHANERH, LOPEZJ, SMITHBL: Intracellular Receptors for Activated Protein Kinase C. J Biol Cbem 1991, 26631486614868.
ADAMSSR, BACSKAIBJ, TA~OR SS, TS~ENRY: Optical
Probes for Cyclic AMP. In Fluorescent Probes fir Biological Activity
1990, 191:421429.
31.
13. ..
14.
Purification from Sheep Brain and Seto Lipocortins and 14-3-3 Protein. Eur J
M~CHLY-ROSEN D, KHANERH, LOPEZJ: Identification
celhdar
in
ADAMS SR, HAROOTUNVWAT, BUECHLER YJ, TAVL~RSS, TSIEN RY: Fluorescence Ratio Imaging of Cyclic AMP in Single Cells: Nature 1991, 3&X694-697. The construction and analysis of the fluorescent PKA analog FICRhRin libroblasts. A novel method to study PKA dynamics in viva.
TOKERA, ELLISCA, SELLERS lA, AITKENA: Protein Kinase C
Inhibitor Proteins quence Similarity 30.
Distribution of Cyclic AMP-Dependent Protein Kinase Living Cells. Proc Nat1 Acad Sci USA 1990, 87~9595-9599.
Translocation of PI,-Protein Kinase C to the Nucleus of Human Promyelocytic (HL60) Leukemia Cells. J Biol Cbem 1991, 266:28-33.
Ca Abolishes
BRAUNRK, VKJLLIET PR, CARBONARO-HALL DA, Hw FL: Phosphorylation of RII Subunit and Attenuation of cAMP-Dependent Protein Kinase Activity by Proline-Directed Protein Kinase.
Arch B&hem 12.
27.
in
KANASEKI T, 1KEUCHIY, SUGIURAH, YAMAUCHIT: Structural
Features
of Ca*+/Calmodulin-Dependent
Protein
Kinase
Serine/threonine
II Revealed by 115:104~1059. 45.
Electron
Microscopy.
J
Cell Biol 1991,
SODERUNG TR: Protein Kinases: Regulation
Domains.
J Biol @em
by Autoinbibitory 199Q, 265:1823%1826.
FUKUNAGAK, SODERUNG TR: Activation of Ca*+/CaIrnoduIinDependent Protein Kinase II in Cerebella Granule Cells by N-Methyl-D-Aspartate Receptor Activation. Mol Cell Neurosci 1990, 1:133-138. Demonstrates that activation of NMDA receptors by glutamate stimulates autophospholylation and activation of CaM-KII.
46. .
MACNICOLM, JEFFERSONAB, SCHULMANH: Ca*+/CaImodulin Kinase is Activated by the Phosphatidylinositol Signaling Pathway and Becomes Ca*+-Independent in PC 12 CeIIs. J Biol Cbem 19M, 265:18055-18058. Establishes that stimulation of the phosphatidylinositol system activates CaM-KII in addition to PKC. 47. .
48.
JEFFERSONAB, TRAVESSM, SCHULMANH: Activation functional Ca*+/CaImoduIin-Dependent Protein GHJ CeIIs. J Biol Cbem 1991, 266:1484-1490.
of MultiKinase in
49.
OCORR KA, SCHULMAN H: Activation of Multifunctional Ca*+/CaIrnoduIin-Dependent Kinase in Intact HippocampaI Slices. Neuron 1991, 6:907-914.
50.
of Type II MOLLOYSS, KENNEDY MB: Autophosphorylation Ca*+/CaImoduIin-Dependent Protein Kinase in Cultures of Postnatal Rat Hippocampal SIices. Proc Nat1 Acad Sci USA 1991, 88347564760.
51. .
TOKLJM~U H, CHIJNVAT, HAGIWAR~M, MIZUTANIA, TERASAWA M, HIDAKA H: KN-62, l-[N-O-Bis(S-IsoquinoIinesuIfonyl)-NMethyl-L-Tyrosyl]4-Phenylpiperazine, as Specific Inhibitor of Ca*+/CaImoduIin-Dependent Protein Kinase II. J Biol Cbem 1990, 265:431%320. Describes a cell-permeable inhibitor which appears to be highly specific for CaMKII. 52.
ISHII A, KIUCHI K, KOBAYASHIR, SUMI M, HIDAKAH, NAGATSU T: A Selective Ca*+/CaImodulin-Dependent Protein Kinase II Inhibitor, KN-62, Inhibits the Enhanced Phosphorylation and the Activation of Tyrosine Hydroxylase by 56 mM K+ in Rat Pheochromocytoma PC1 2H Cells. Biixbem Biopby Res Commun 1991, 176:1051-1056.
53.
YAMAMOTOH, FUKUNAGAK, LEE K, SODERLINGTR: IschemiaInduced Loss of Brain CaIcium/CalmoduIin-Dependent Protein Kinase IL J Neurocbem 1992, 58:111&1117.
54. .
WAGNERJA, COZENS AL, SCHULMANH, GRUENERTDC, STRYER L, GARDNER P: Activation of Chloride Channels in NormaI and Cystic Fibrosis Airway Epithellal Cells By Multifunctional Calcium/Calmodulin-Dependent Protein Kinase. Nature 1991, 349793-796.
protein
kinases
Scott and Soderling
Although Cl- channels in epithelial cells from cystic fibrosis patients cannot be activated by PKA or PKC, they can be activated by CaM-KII. 55.
WITCHER DR, KOVACSRJ, SCHULMANH, CEFALI DC, JONES LR: Unique Phosphorylation Site on the Cardiac Ryanodine Receptor Regulates Calcium Channel Activity. J Biol Chem 1991, 266:11144-11152.
56.
FERRISCD, HUGANIRRI, BREDT DS, CAMERONAM, SNYDERSH: Inositol Trisphosphate Receptor: Phosphorylation by Protein Kinase C and Calcium Calmodulin-Dependent Protein Kinase in Reconstituted Lipid Vesicles. Proc Nat1 Acud Sci USA 1991, 88:2232-2235.
57.
NAKANEM, MITCHELLJ, FORSTERMANN LJ, MUFZADF: Phosphotylation by Calcium Calmodulin-Dependent Protein Kinase II and Protein Kinase C Modulates the Activity of Nitric Oxide Synthase. B&hem Biopbys Res Commun 1991, 180:13961402.
58.
COUNTAWAY JL, NAIRNAC, DAVISRJ: Mechanism of Desensitization of the Epidermal Growth Factor Receptor ProteinTyrosine Kinase. J Biol Chem 1992, 267:112+1140.
59. .
SHENG M, THOMPSONMA, GREENBERGME: CREB: a Ca*+-Reg-
60.
DA.S PK, KARL KA, COLlCOS MA, PRYWEX R, KANDEL CAMP Response Element-Binding Protein is Activated Ca*+/CaImoduIin as weII as CAMP-Dependent Protein nase. Proc Nat1 Acud Sci USA 1991, 88:506-5065.
61.
KAPILOFF MS, MATHISJM, NELTON CA, LIN CR, ROSENFELDMG:
ulated Transcription Factor Phosphorylated by CalmodulinDependent Kinases. Science 1991, 252:1427-1430. Demonstrates that CaMKI and II can phosphorylate the transcriptional activating protein CREB.
Calcium/CaImoduIin-Dependent Pathway for Transcriptional USA 1991, 88:371@3714.
Protein Regulation.
ER by Ki-
Kinase Mediates a Proc Nat1 Acud Sci
62. .
JENSEN KF, OHMSTEDE CA, FISHER RS, SAHYOUN N: Nuclear and AxonaI Localization of Ca*+/CaImoduIin-Dependent Protein Kinase Type Gr in Rat CerebelIar Cortex. Proc Nat1 Acad Sci USA 1991, 88:28562853. Uses immunochemical and ultrastructural analysis to localize CaMKIV in the nucleus of cerebellar granule cells. 63.
MEANS AR,
CRUZALEGUI F,
LEMAGUERESSE B,
NEEDLEMAN DS,
T: A Novel Ca*+/Calmodulin-Dependent Protein Klnase and a Male Germ Cell-Specilic Calmodulin-Binding Protein are Derived from the Same Gene. Mol Cell Biol 1991, 11:396&3971. SIAUGHTER
GR,
ONO
JD Scott and TR Soderling, Vollum Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland, Oregon 97201, USA.
295