calmodulin-dependent protein kinase II

W.H. Gispen and A. Routtenberg (Eds.) Progress in Brain Research, Vol. 89 0 1991 Elbevier Science Publishers B.V.

169 CHAPTER 12

Molecular and cellular studies on brain calcium/calmodulin-dependent protein kinase I1 T.R. Soderling, K. Fukunaga, D.A. Brickey, Y.L. Fong, D.P. Rich, K. Smith and R.J. Colbran Department of Molecular Physiology and Biophysics, Vatiderbilt University, Nashville, TN 37232-0615, U.S.A.

Introduction Elevation of intracellular free Ca2+ ([CaT+]) is a common cellular response in signal transduction pathways of many hormones and neurotransmitters. Of the multiple physiological responses triggered by alterations in [CaT+], phosphorylation/ dephosphorylation catalyzed by Ca2+-dependent protein kinases and phosphatase(s) is a frequently utilized mechanism. Several Ca2+-dependentprotein kinases from various tissues have been purified and characterized including phosphorylase kinase, myosin light chain kinase, protein kinase C, and Ca2+/calmodulin (CaM)-dependent protein kinases I, 11, and 111. Most of these Ca2+-dependent protein kinases phosphorylate a very restricted number of protein substrates. However, protein kinase C and Ca2+/CaM-dependent protein kinase II (CaM-kinase 11) phosphorylate numerous proteins, and these two multifunctional

Correspondence: Dr. T.R. Soderling, Vollurn Institute L-474, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Rd., Portland, OR 97201, U.S.A.

protein kinases are analogous in this respect to the well-studied CAMP-dependent protein kinase (CAMP-kinase). This article discusses brain CaM-kinase I1 with particular focus on its unqiue regulatory properties, its regulation in cultured brain cells, and its physiological functions. CaM-kinase I1 has widespread tissue distribution as oligomeric isozyme forms and is particularly abundant in brain (reviewed in Colbran and Soderling, 1990a; Schulman, 1988). In certain regions of the brain, such as hippocampus, it constitutes up to 2% of total protein (Erondu and Kennedy, 1985) which probably makes it the most abundant enzyme in these tissues. CaM-kinase I1 is localized presynaptically where it is involved in Ca2+-dependent regulation of neurotransmitter biosynthesis and exocytosis. At excitatory synapses in forebrain there is a thickening of the postsynaptic membrane called the postsynaptic density (PSD), and CaM-kinase I1 constitutes about 3050% of the protein in the PSD (Kennedy et al., 1983). These excitatory synapses are subject to a usage-dependent enhancement of synaptic transmission called long-term potentiation (LTP), a popular model for learning and memory. Recent

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results implicate CaM-kinase I1 in the mechanisms of LTP (Malenka et al., 1989; Malinow et al., 1989). Other neuronal functions of CaMkinase I1 will undoubtedly materialize as specific probes of its activity are developed.

Substrate specificity determinants

The primary sequence determinant in protein substrates of CaM-kinase I1 is -Arg-X,-X,Ser/Thr- (X, is any amino acid). A second basic residue at position X, is a negative determinant for CaM-kinase I1 (Soderling et al., 1986) whereas it is a strong positive determinant for CAMPkinase (Kemp et al., 1977). Although CaM-kinase I1 and CAMP-kinase often phosphorylate the same proteins, their primary sites of phosphorylation are often different (reviewed in Colbran et al., 1989a). The kinetic consequences of these different phosphorylation sites can either be the same (e.g., cardiac phospholamban, see Simmerman et al., 1986) or different (e.g., tyrosine hydroxylase; Atkinson et al., 1987). In addition to the primary sequence determinants, there are indications of higher order structural determinants. For example, most synthetic peptide substrates are not such good substrates as the protein from which they are derived. Direct evidence for higher order determinants comes from studies on phenylalanine hydroxylase, a protein which is phosphorylated on the same site by both protein kinases (Doskeland et aI., 1984). Binding of phenylalanine at an allosteric site enhances the phosphorylation rate by CAMP-kinase but inhibits the rate of phosphorylation by CaM-kinase 11. This result raises the interesting theoretical possibility that the rates of phosphorylation of the same site by these two protein kinases could be differentially altered by changes in the intracellular concentration of allosteric modulators.

Regulatory properties of CaM-kinase I1

A major effort of our laboratory over the past five years has been the elucidation of the regulatory properties of purified rat brain CaM-kinase 11. CaM-kinase 11, like most other serine/threonine protein kinases, undergoes intramolecular autophosphorylation at multiple serine and threonine residues. This autophosphorylation converts the kinase from an initial totally Ca2+/CaM-dependent enzyme to a form that expresses about 50-80% of its total activity (assayed with Ca2+/CaM) in the presence of EGTA (reviewed in Colbran and Soderling, 1990a). Biochemical analyses indicate this is due to autophosphorylation of Thr286.Our investigations of the regulation of CaM-kinase I1 by binding of Ca2+/CaM and by autophosphorylation were dependent on elucidation of the amino acid sequences, deduced from the cDNA sequences (Lin et al., 1987; Bulliet et al., 1988), of the three types of subunits (each of 50-60 kDa) of brain CaM-kinase I1 holoenzyme (650-700 kDa). These three polypeptides have 95% identical amino acid sequences over the NH,-terminal 2/3 of the subunits. They differ primarily by insertions/deletions within the COOH-terminal 1/3 of the subunits (Fig. 1). Homology comparisons to other protein kinases indicate that the catalytic domain is close to the NH ,-terminus, and a putative CaM-binding sequence is situated just NH,-terminal to the region of insertions/deletions. The COOHterminus is thought to be involved in subunit assembly and perhaps in subcellular localization of the kinase. Our approach to understanding the regulatory properties of CaM-kinase I1 has been to reconstitute the regulatory properties of the oligomeric holoenzyme using a monomeric 30-32 kDa fragment of the kinase in which the C-terminal 1/3 of the subunit, including the CaM-binding domain and the autophosphorylation site (i.e.,

171

residue 286; residue numbers will refer to the LY subunit sequence unless stated otherwise), has been proteolytically removed. This constitutivelyactive NH ,-terminal fragment of CaM-kinase TI is potently inhibited ( K i = 0.2 p M ) by a synthetic peptide containing the sequence of the a subunit from residues 281-309 (Colbran et al., 1988). Kinetic analyses of the inhibition showed that peptide 290-309 inhibits competitively with peptide substrates (Payne et al., 1988) whereas peptide 281-309 inhibits competitively with Mg2+/ATP (Colbran et al., 1989b). This suggests that residues 281-289 interact with the ATP-binding motif and residues 290-309 interact with the protein substrate binding elements of the catalytic domain. This conclusion was strengthened by the observation that peptide 290-309 did not protect the ATP-binding site from phenylglyoxal inactivation whereas peptide 281-309 afforded complete protection (Colbran et al., 1989b). Peptide 281-309 also contains the CaM-binding do-

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Fig. 1. Schematic representation of the subunits of brain CaM-kinase 11. The three highly homologous subunits (a,p, p ' ) of rat brain CaM-kinase I1 are depicted with the numbers on the right indicating the amino acids per subunit. Progressing from the NH ,-terminus to the COOH-terminus are the ATP-binding motif, the conserved amino acid triads of the putative catalytic domain, the multiregulatory domain including the CaM-binding and autoinhibitory elements and the positions of deletions in the a and p' subunits.

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main (residues 296-309), and as shown in Fig. 2 binding of Ca2+/CaM to peptide 281-309 cornpletely abolishes its inhibitory properties (Colbran et al., 1988). If Thr286is phosphorylated in peptide 281-309, its inhibitory potency is decreased by at least 10-fold (Fig. 2; Colbran et al., 1989b). In addition to the extremely rapid autophosphorylation of ThrZa6,which precedes phosphorylation of exogenous substrates (Kwiatkowski et al., 1988), there are at least 6-7 other sites of

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autophosphorylation. Some of these sites are only autophosphorylated by the Ca2+-independent form of the kinase in the absence of Ca2+/CaM (Hashimoto et al., 1987). This suggests that some of these sites may be within the CaM-binding domain, and this hypothesis is strengthened by the fact that this autophosphorylation results in a loss of CaM-binding. Recent studies have identified these sites of autophosphorylation as Thr 305/306 (Patton et al., 1990). These sites are located within the hydrophobic pocket of the

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4. MUY &'-MEPENDENT (5o-eoX) Fig. 3. Regulatory model for CaM-kinase 11. This model depicts the activation of CaM-kinase I1 by CaL+/CaM and by autophosphorylation. The amino acid backbone of a single subunit of the oligomeric enzyme is depicted by the solid line. The semi-circle and square blocks indicate the ATP- and protein-substrate binding elements of the catalytic domain and the\ denotes a putative flexible hinge region (residues 270-280). The COOH-terminus is thought to be involved in subunit assembly. The crosshatched and stippled bar represents the CaM-binding and autoinhibitory domains. As isolated the kinase is inactive due to the occupation of the catalytic domain by the autoinhibitory domain (species 1). Initial activation requires binding of Ca2+/CaM which induces a conformational change in the overlapping autoinhibitory domain which relieves its inhibitory interaction with the catalytic domain (species 2). In the presence of MgZ+/ATP the fully activated kinase catalyzes rapid autophosphorylation of ThrZs6 as well as phosphorylation of exogenous substrates containing the consensus recognition sequence -R-X-X-S/T- (species 3). Upon removal of Ca2+/CaM the kinase remains partially active (Ca*+-independent) due to the presence of the negative charge of the phosphorylated ThrZX6(species 4). The Ca'+-independent form of the kinase can autophosphorylate at Thr305/306which then prevents subsequent binding of Ca2+/CaM (species 5). Reproduced by permission from Soderling (1990). (LK)-(K)X)

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CaM-binding domain, and their phosphorylation in peptide 290-309 blocks CaM-binding (Colbran and Soderling, 1990b). It is somewhat surprising the these two Thr residues are autophosphorylated in the kinase since there is no Arg three residues NH ,-terminal, but they are probably held close to the catalytic domain by other structural restraints. This is consistent with the result obtained when the recognition determinant (Arg283) for Thr286 is mutated to Gln or Glu (see next section). Ser 314, which conforms to a consensus recognition sequence, is also autophosphorylated by the Ca2+-independentform of the kinase (Patton et a]., 1990), but its phosphorylation has little or no effect on CaM-binding since it lies outside the hydrophobic pocket of the CaM-binding domain (Colbran and Soderling, 1990b). The above results have been formulated into a regulatory model (Fig. 3) which is consistent with the existence of autoinhibitory domains in other protein kinases (Soderling, 1990). In the holoenzyme residues 281-302 constitute an autoinhibitory domain that interacts with and inhibits the NH,-terminal catalytic site elements (species 1, Fig. 3). Binding of Ca2+/CaM to residues 296-309 (species 2) disrupts the autoinhibitory domain, perhaps by inducing a-helical structure, and frees the catalytic domain which can then bind Mg2+/ATP and protein substrate in an active conformation. The activated kinase catalyzes phosphorylation of substrates at consensus recognition determinants as well as autophosphorylation at the consensus sequence Arg-Gln-GluThr 286 (species 3). Following autophosphorylation of Thr 286, the kinase remains partially active when Ca2+/CaM is removed (i.e., partially Ca'+/CaM-independent) since the negative charge prevents effective interaction with the catalytic domain (species 4). If Ca2+/CaM is removed from the kinase, additional sites of autophosphorylation within the CaM-binding domain are exposed. Ca2+-independent autophos-

phorylation of Thr 305/306 prevents subsequent binding of Ca2+/CaM (Colbran and Soderling, 1990b).

Site-directed mutagenesis studies The regulatory model of Fig. 1 is based largely on reconstitution experiments using proteolyzed kinase and synthetic peptides. We wanted to examine this model using site-directed mutagenesis of selective residues within the multiregulatory domain. The first two residues chosen were Arg283 and Thr286 since they are the recognition determinant and the regulatory autophosphorylation site, respectively. When Arg283was substituted by either Glu or Gln in synthetic peptide 281-309, the IC,, for inhibition of CaM-kinase I1 increased over 200-fold (Fong and Soderling, 1990), suggesting that Arg283 is important for the potency of the autoinhibitory domain. Furthermore, neither of these substituted peptides could be phosphorylated by CaM-kinase 11, consistent with the loss of the Arg recognition determinant. When these same mutations were made in the expressed kinase a-subunit, we expected a large increase in the Ca2+-independent activity of the mutants due to the decreased potency of the autoinhibitory domain. However, the mutant had essentially the same Ca2+-independence (2.15 k 0.62%) as the Arg283 wild-type (1.86 f 0.72%), but the G ~ mutant u ~ did ~show ~ a small but significant (p < 0.001) increase in Ca2+-independence to 5.46 f 0.90% (Fong and Soderling, 1990). Waxham et al. (1990) mutated Arg283 to IIe, and the mutant kinase also was completely Ca2+/CaM-dependent. Although there was autophosphorylation of Thr286 in the and G I U ~ 'mutants, ~ the rate was dramatically decreased (Fig. 4). The mutant did not exhibit any formation of Ca2+-independentactivity under autophosphorylation conditions (Waxham et al., 1990). We attribute the much larger effects of

174

1989; Waxham et al., 1990) and Leu286(Hanson substitutions in the synthetic peptides versus muet al., 1989) mutant kinases had the same basal tations in the intact kinase as a difference beCa2+-independence as the Thr 286 wild-type kitween intermolecular and intramolecular interacnase, and, unlike the wild-type kinase, their tions. There is good evidence (see below) that Ca2+-independence did not increase when subinteraction of the autoinhibitory domain with the jected to autophosphorylation even though sites catalytic site is intrasubunit. In the mutant kinase other than residue 286 were autophosphorylated. there are probably structural constraints which This result confirms the earlier biochemical studhold the autoinhibitory domain in close proximity ies which indicated that only autophosphorylation to the catalytic site. This may account for the of Thr286 is responsible for generation of the observed autophosphorylation of Thr2@ in the mutants when the substituted peptides were not Ca2+-independence of CaM-kinase 11. Most inphosphorylated at all. Furthermore, Arg283 is terestingly, when a negative charge was introprobably only one of several sites of interaction duced at position 286 by mutation to this between the autoinhibitory domain and the catmutant kinase exhibited about the same level of alytic site. When multiple mutations in the vicinCa’+-independence prior to autophosphorylation ity of Arg283 are made ( H i ~ - A r g ~ ~ ~ - G I n - G l u -as the wild-type kinase had after autophosphoryThr 286 to Asp-Gly 283-GIu-Glu-Thr286) to change lation (Fong et al., 1989; Waldmann et al., 1990). the positive charges to negative charges, the muThis result suggests that it is largely the negative tant kinase exhibited 67% Ca2+-independence charge at position 286 in the autophosphorylated (Waldmann et al., 1990). kinase which is responsible for the increase in Mutations have also been made at Thr286 in Ca2+-independence. the a-subunit. When Ala was substituted for These results from site-directed mutagenesis Thr286 in peptide 281-309, the IC,, was not strongly support the model of Fig. 1. Additional altered. As expected, the (Fong et al., mutations are in progress to further probe inter-

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Fig. 4. Site-directed mutants of CaM-kinase 11. The left panel illustrates that rates of formation of the Ca’+-independent form (i.e., ~ ~ The~ Ca*+-independence of these autophosphorylation of Thr2“) of the wild-type (Arg283, W), Gln283( 0 ) and G ~ ( Au) mutants. three species prior to autophosphorylation is shown in the inserted table. The right panel depicts the Ca2+-independenceof the wild-type (Thr 286), Ala286 and AspZs6 mutant kinases prior to (crosshatched bar) and after (solid bar) autophosphorylation. Reproduced by permission from Fong et al. (1989) and Fong and Soderling (1990).

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actions between the autoinhibitory domain and the catalytic site. All of our previous work on expressed a-subunit kinases was transcribed and translated in vitro. More recently we have successfully expressed the a-subunit and p-subunit kinases in Sf9 cells using the baculovirus system (Brickey et al., 1990). In agreement with Yamauchi et al. (19891, who expressed the kinases in CHO cells, we find that the expressed a-subunit is an oligomeric kinase of about 650 kDa whereas the expressed p-subunit is primarily a monomeric enzyme. Since the monomeric enzyme is completely Ca’+/CaM-dependent, this indicates that the interaction of the autoinhibitory domain with the catalytic site is intrasubunit. In contrast, the a subunit kinase in E. coli appears to be monomeric (Waxham et al., 1990). Regulation of CaM-kinase I1 in the postsynaptic density

Studies on the regulatory properties of CaMkinase I1 have utilized the purified cytosolic brain enzyme. It is important to establish whether CaM-kinase I1 in the PSD also exhibits these regulatory properties, and this has recently been demonstrated (Rich et al., 1989). Formation of the Ca’+-independent form of CaM-kinase I1 in the PSD through autophosphorylation of Thr286 is an attractive model for generating the constitutively-active protein kinase that appears to be important for induction of LTP in hippocampus (Malenka et al., 1989; Malinow et al., 1989). Another potential mechanism to generate a Ca’+-independent form of CaM-kinase I1 is through limited proteolysis by the Ca2+-dependent protease calpain (Kwiatkowski and King, 1989). This mechanism is attractive since infusion of the protease inhibitor leupeptin prevents induction of LTP (Staubli et a1.,1988) and calpain has been localized in the PSD (Perlmutter et al., 1988). Treatment of the isolated PSD with cal-

pain results in a 3-5 fold increase in CaM-kinase I1 activity due to proteolytic conversion of a small percentage (< 10%) of CaM-kinase I1 to a soluble, monomeric fragment of about 30 kDa which is fully active in the absence of Ca2+ (D.P. Rich et al., 1990). The fact that only a small fraction of the CaM-kinase I1 in the PSD is solubilized may be important since CaM-kinase 11 constitutes the major PSD protein and may be required for the structural integrety of the PSD. Since the 30 kDa constitutively-active fragment of CaM-kinase I1 is now soluble, it would have access to substrates (e.g., receptors and ion channels) other than those in the PSD. Reguiation of CaM-kinase I1 by gangliosides

Gangliosides, sialic acid containing glycosphingolipids, are found in high concentrations in nerve endings (up to 5-10% of total lipid; Ledeen, 1978) and are thought to regulate several enzymes including protein kinases (Chan, 1988). For this reason we investigated the effect of gangliosides on purified brain CaM-kinase I1 (Fukunaga et al., 1990a). Gangliosides (GTlb > G D l a > GM1) stimulate CaM-kinase I1 when assayed in the absence of Ca2+/CaM with little effect (up to 100 p M ) on activity in the presence of Ca2+/CaM. Although GTlb stimulates autophosphorylation of CaM-kinase 11, there is no formation of the stable Ca2+-independent form since Thr286 is not phosphorylated. GTlb appears to bind to the autoinhibitory domain of CaM-kinase 11 since it can reverse the inhibition by synthetic peptide 281-309 of the proteolyzed catalytic fragment of the kinase. The physiological role of ganglioside stimulation of CaM-kinase I1 is not clear. If CaM-kinase I1 is stimulated by gangliosides in vivo, the process would not result in formation of the Ca2+-independentform, as occurs with activation by Ca2+/CaM. These different mechanisms for stimulating CaM-kinase I1

176

may be important for different neuronal functions mediated by this multifunctional protein kinase. Regulation of CaM-kinase I1 in cultured brain cells All of the above studies have utilized in vitro approaches. We also wanted to examine regulation of CaM-kinase I1 in cultured brain cells and chose cerebellar granule cells (Fukunaga et al., 1990b). The hypothesis is that elevations in [Cat'] would trigger autophosphorylation of CaM-kinase I1 to its Ca2+-independent form which would remain active even when the [Ca?'] was resequestered to its basal value (Fig. 5). This could constitute an attractive mechanism for prolonging certain physiological functions in response to transient elevations of [CaT+]. The magnitude

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Fig. 5. Model of cellular response of CaM-kinase I1 to a transient Ca2+ signal. A stimulus which triggers a transient elevation in intracellular Ca2+ would trigger autophosphorylation of CaM-kinase I1 and formation of the Ca2+-independent form. This Ca2+-independent form would maintain CaM-kinase I1 activity, even when intracellular Ca2+ returned to its unstimulated value and prolong those physiological functions mediated by this multifunctional protein kinase. The duration of the Ca2+-independent form would depend on the relative activities of the CaM-kinase I1 autophosphorylation reaction and the opposing protein phosphatases.

and duration of the Ca'+-independent form would depend on the activity of the opposing protein phosphatases, which in turn may depend on the cell type or subcellular localization of CaM-kinase 11. Cultured cells were incubated under various conditions to alter the [Gaff] and then homogenized in the presence of kinase and phosphatase inhibitors to stablize the in situ level of CaMkinase I1 autophosphorylation (Fukunaga et al., 1990b,c). When extracellular Ca2+ was removed from the cells, only 1-2% of CaM-kinase I1 was in the Ca2+-independent form, and depolarization with 56 mM K + had no effect (Fig. 6). Addition of Krebs-Ringer HEPES buffer containing normal extracellular Ca2+ (2.7 mM) resulted in a new steady-state level of 4-5% Ca2+-independent CaM-kinase 11, and now Kf depolarization increased this to about 10% within 30 sec followed by a decline back to 4-5% by 5-10 min (Fig. 6). Ionomycin, a divalent cation ionophore, elicited 10% Ca2+-independencewhich remained elevated. Inclusion of 5 p M okadaic acid, a cell permeable protein phosphatase inhibitor, in the incubation medium potentiated the magnitude and duration of the Ca'+-independent activity of CaM-kinase 11 (Fig. 6). Phosphopeptide mapping of CNBr-cleaved 32 P-labeled 58-60 kDa subunits of CaM-kinase I1 revealed that under basal conditions the kinase contains 32P04 in many sites. Agents which promote formation of the Ca2+-independent species of the kinase increase 32P04incorporation into multiple sites of the kinase (Fig. 7). However, there was a good temporal correlation between "2-incorporation into CNBr peptide 1, which contains Thr,,,, and generation of the Ca*+-independentkinase activity. We have also examined the effects of glutamate treatment of cerebellar granule cells (Fukunaga et al., 1990~).Glutamate is a natural agonist for these cells and can act through either non-NMDA gated ion channels (e.g., kainate or

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Fig. 6. Response of CaM-kinase I1 in cerebellar granule cells to Ca2+-mobilizing conditions. In the top panel cultured cerebellar granule cells were preincubated in Krebs-Ringer HEPES buffer (KRH) minus Ca2+. At time 0 the following additions were made: 56 mM K + (*),KRH plus 2.7 mM Ca2+ alone ( A ) plus 56 mM K + and 2.7 mM Ca2+ ( 0 ) or 2.5 p M ionomycin and 2.7 mM Ca2+ ( W ) . At the indicated times cells were homogenized and assayed for CaM-kinase I1 activity in the presence of Ca2+/CaM (total activity) or EGTA (Ca*+-independent activity). In the bottom panel cells were preincubated for 15 min in normal KRH (plus Ca2+) in the absence ( 0 ) or presence (0) of 5 FM okadaic acid. At time zero 56 mM K + was added for 0.5 min (bar). Cells were homogenzied and assayed as in the top panel. Reproduced by permission from Fukunaga et al., 1990b.

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Fig. 7. Autoradiogram of 32P04-peptide mapping of CaMkinase I1 phosphorylated in situ. Granule cells were labeled for 5 h with 32P04 prior to treatment as follows: 45 min in Ca2+-free KRH plus another 15 min with 5 p M okadaic acid. The cells were then incubated for 0, 0.5, or 5 min in KRH plus 2.7 mM Ca2+, 56 mM KCI and 5 p M okadaic acid. Cells were homogenized, CaM-kinase I1 was immunoprecipitated and subjected to SDS/PAGE. The 32P04 subunits (58-60 kDa) of CaM-kinase I1 were cut out of the gel, subjected to CNBr hydrolysis and the CNBr peptides separated by ureaSDS/PAGE. Lane 8, CNBr peptides from 32P-labeled purified rat brain CaM-kinase 11; lane 7, synthetic peptide corresponding to residues 282-307 (CB1) containing the autophosphorylation site Thr286. Reproduced by permission from Fukunaga et al. (1990b).

quisqualate) or through NMDA gated ion channels (reviewed in Mayer and Westbrook, 1987). The latter category of ion channels have a permeability to Ca2+ (MacDermott et al., 1986; Connor et al., 1988) and are therefore of special interest. They are also of great interest because of the requirement for NMDA receptor activation and postsynaptic Ca2+ influx in the initiation of longterm potentiation (reviewed in Mayer and Westbrook, 1987; Gustafsson and Wigstrom, 1988). When cerebellar granule cells were treated with 100 p M of L-glutamate, kainate, quisqualate, NMDA, or L-aspartate, only kainate elicited an increase in the Ca2+-independenceof CaM-kinase 11. Since it is known that the NMDA-gated ion

channel is subject to a voltage-dependent Mg2+ blockage (Mayer et al., 1984), we removed extracellular Mg2+ and repeated the treatments. Under these conditions all agonists evoked increases (basal = 4.0%; stimulated = 6 4 % ) in the Ca2+independence of CaM-kinase 11. Since glutamate is the natural agonist, it was utilized for all additional experiments. We found that inclusion of 1 p M glycine in the extracellular incubation medium shifted the dose-response curve to the left such that now 10 p M glutamate was maximally effective rather than 100 p M. This is consistent with the known potentiation by glycine of the NMDA-gated ion channel (Johnson and Ascher, 1987). The increase in Ca2+-independence by 10 p M glutamate plus 1 p M glycine was blocked by specific antagonists (APV and CPP) of the NMDA receptor but not by the non-NMDA antagonist (GAMS) or nitrendipine, a blocker of voltage-dependent Ca2+ channels (Fig. 8). In the absence of extracellular Ca2+ there was no response of CaM-kinase I1 to glutamate plus glycine, but with extracellular Ca2+ the response was potentiated and prolonged by okadaic acid (Fig. 8). The increase in Ca2+-independence correlated quite well quantitatively and temporally with 32 PO, incorportion into Thr 287 (cerebellum contains predominantly the p subunit kinase) (Fig. 8). Several aspects of the cerebellar cell study provoke discussion. Firstly, we were surprised that under basal conditions, where intracellular Ca2+ is less than 100 nM, the kinase would be partially Ca2+-independent since in vitro studies suggested that CaM-kinase I1 is less sensitive to Ca2+/CaM (apparent K , of 20-100 nM CaM) than most other CaM-dependent enzymes (apparent K , of 1-10 nM CaM). Of course, it is possible there is a gradient (from the cell membrane inward) of [CaT+l and the basal value of 4-5% Ca2+-independence represents partial activation of CaM-kinase I1 by elevated Ca2+ near

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the membrane. Secondly, the magnitude (10%) and duration (5-10 min) of the increases in Ca2+-independence in response to Ca2+ influx and the potentiation by okadaic acid strongly suggests that protein phosphatases are limiting the autophosphorylation reaction. Autophosphorylation in the cell extract with ATP-y-S produced about 70% Ca2+-independent CaM-kinase I1 (Fukunaga et al., 1990b). Thiophosphorylated proteins are resistent to protein phosphatases. The fact that okadaic acid, which is a potent inhibitor of protein phosphatases 1 and 2A, only gave partial potentiation suggested the presence of both okadaic acid sensitive and insensitive phosphatases. This was directly demonstrated using the cell extract (Fukunaga et al., 1990b). The identity of the okadaic acid insensitive phosphatase as either type 2C or 2B (calcineurin) has not been established. In summary, these results indicate that formation of the Ca2+-independent species of CaM-kinase I1 is dynamically regulated in cerebellar granule cells by Ca2+-mobilizing agent and by protein phosphatase activity and is correlated with autophosphorylation of Thr287.

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Although it is clear from in vitro studies that CaM-kinase I1 is multifunctional, rigorously established substrates in brain, other than autophosphorylation of the kinase itself as discussed above, are very limited. In large part this is due to the lack of specific probes (inhibitors or activators) of CaM-kinase I1 that can be used in intact cell studies. This situation may soon change as specific peptide inhibitors of several protein kinases are known, and we are in the process of designing a specific peptide inhibitor of CaMkinase I1 (Smith et al., 1990). One might make synthetic genes for these peptides and express them in transfected cells using inducible promoters. Cell permeable organic molecules which selectively inhibit CaM-kinase I1 also offer great promise for probing the physiological functions of this kinase (Tokumitsu et al., 1990). Synapsin I is a neuron-specific protein associated with the outer surface of presynaptic vesicles (DeCamilli et al., 1983). It is one of the best

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Physiological substrates of CaM-kinase I1 in brain

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Fig. 8. Response of CaM-kinase I1 to glutamate and NMDA-antagonists. In the top panel granule cells were incubated in KRH with normal Ca2+ in the absence ( - ) or presence ( + ) of 10 p M L-glutamate and 1 p M glycine and either 100 p M NMDA antagonist (APV or CPP), non-NMDA antagonist (GAMS), or the voltage-dependent Ca" channel blocker nitrendipine (Nit). In the bottom panel cells were preincubated in KRH minus Ca2+. At time zero the following additions were made: 10 pM L-glutamate and 1 p M glycine alone ( A ) or with 2.7 mM Ca2+ in the absence ( 0 ) or presence (0) of 100 p M APV. The solid line gives Ca*+-independent activity of CaM-kinase 11. The dotted lines show 3zP0,-incorporation in the subunits of CaM-kinase I1 ( 0 ) or CNBr peptide CB1 ( D ) which contains Thr'". Reproduced by permission from Fukunaga et al. (1990~).

180

known in vitro substrates for CaM-kinase I1 and can be phosphorylated to a molar stoichiometry of about 1.8. Dephospho-synapsin binds to purified synaptic vesicles (Schiebler et al., 1986) and may link synaptic vesicles to the cytoskeleton. Phosphorylation of synapsin I by CaM-kinase 11, but not by CAMP-kinase or protein kinase C which phosphorylate different sites, reduces the affinity of synapsin I for the synaptic vesicles. It is proposed that phosphorylation of synapsin I by CaM-kinase I1 may promote the release of synaptic vesicles from the cytoskeleton for migration to the active zone and exocytosis. Considerable in situ evidence supports this model. Electrical stimulation or K+ depolarization leads to phosphorylation of synapsin I (Nestler and Greengard, 1982). In isolated synaptosomes K+ depolarization leads to autophosphorylation of CaM-kinase 11, transient formation of its Ca2+-independent form, phosphorylation of synapsin I (Gorelick et al., 1988), and increased neurotransmitter release. This increase in neurotransmitter release can be largely blocked by introduction of a peptide inhibitor of CaM-kinase I1 into the synaptosome (Nichols et al., 1990). Another established neuronal substrate for CaM-kinase I1 is tyrosine hydroxylase, the ratelimiting enzyme in catecholamine biosynthesis. Tyrosine hydroxylase can be phosphorylated by numerous protein kinases in vitro including CaM-kinase 11. Phosphorylation by CaM-kinase I1 increases the V,,, of tyrosine hydroxylase by promoting its interaction with an activator protein (Atkinson et al., 1987). In PC12 (Yanagihara et al., 1984) and adrenal chromafin cells (Pocotte et al., 1986) depolarizing agents result in a Ca2+dependent increase in the tyrosine hydroxylase V,,. The sites phosphorylated by CaM-kinase I1 in vitro are transiently phosphorylated in chromaffin cells with temporal correlation to the observed activation in tyrosine hydroxylase (Waymire et al., 1988). Thus, there is good evidence

that CaM-kinase I1 may be one of several protein kinases that can activate tyrosine hydroxylase in response to different agonists. There is accumulating evidence for the involvement of CaM-kinase 11 in the initiation of long-term potentiation and in kindling. CaMkinase I1 is an attractive candidate €or modulation of synaptic excitability (Lisman and Goldring, 1988) because of its localization in the postsynaptic density (Kennedy et al., 1983) and its potential for prolonged activation through autophosphorylation in response to transient elevations in [ca? I (see previous sections). It is known that initiation of long-term potentiation requires activation of NMDA-gated ion channels which are permeable to Ca2+ (reviewed in Mayer and Westbrook, 1987; Gustafsson and Wigstrom, 1988). When peptide inhibitors of either CaM-kinase I1 or protein kinase C are microinjected into the postsynaptic cell, they block the acquisition, but not the expression, of LTP (Malenka et al., 1989; Malinow et al., 1989). There is good evidence that initiation of LTP in the CA1 region of hippocampus, where CaM-kinase I1 is extremely abundant, occurs postsynaptically, but there is controversy concerning whether expression of LTP is presynaptic or postsynaptic (discussed in Science, 248: 1603-1605). The putative substrate of CaM-kinase I1 or protein kinase C in LTP is completely unknown, but the glutamate-gated ion channels (or associated regulatory proteins) which become potentiated in LTP, would be attractive candidates. As these ion channels are cloned (Hollman et al., 1989) the proteins can be directly tested as substrates for multiple protein kinases and effects on ion channel gating properties can be analyzed. CaM-kinase I1 may also be involved in the phenomena of kindling, an experimental model of epilepsy (Wada, 1982). When hippocampal membranes were isolated from control and kindled rats, the in vitro autophosphorylation of CaM-kinase I1 was decreased in membranes from +

181

kindled rats (Goldenring et al., 1986). This could be interpreted to suggest that the autophosphorylation state of CaM-kinase I1 was increased in vivo by the kindling, thus accounting for the decreased subsequent in vitro autophosphorylation. Alternative interpretations are possible which need to be examined.

Acknowledgements

Perspective

References

CaM-kinase I1 is of interest to neuroscientists due to (1) its abundance in neural tissues, (2) its strategic localization both pre- and postsynaptically, (3) its unique regulatory properties allowing for the potential to prolong responses to transient elevations in [Ca:+], and (4) the prevelance of signal transduction mechanisms in neural functions. Biochemical and molecular biological investigations from several laboratories over the past ten years have greatly advanced our understanding of the molecular properties of CaMkinase 11. The challenge for the next decade will be to utilize this understanding to further our knowledge of the physiological functions of CaM-kinase 11. Several avenues hold great promise. One of these is to develop specific inhibitors or activators of CaM-kinase I1 which can be used in intact cells. An example of this would be to express synthetic genes encoding a specific peptide inhibitor of CaM-kinase 11. We are currently working on the design of a specific peptide inhibitor. A second approach will be the use of transfected cells or transgenic mice containing mutant forms (e.g., constitutively active) of CaMkinase 11. As neuronal specific promoters become available this latter route will become more feasible. It is likely that these methods will establish the importance of CaM-kinase I1 in multiple neuronal functions (e.g., proto-oncogene regulation) alongside its counterparts CAMP-kinase and protein kinase C.

Atkinson, J., Richtand, N., Schworer, C.M., Kuczenski, R. and Soderling, T.R. (1987) Phosphorylation of purified rat striatal tyrosine hydroxylase by brain calmodulin-dependent protein kinase: effects of an activator protein. J. Neuruchem., 4 9 1241-1249. Brickey, D.A., Fong, Y.L. and Soderling, T.R. (1990) Expression and characterization of the a subunit of mouse brain Ca’+/CaM-dependent protein kinase I1 using the baculovirus expression system. Fed. Proc., 4: A2075. Bulleit, R.F., Bennett, M.K., Molloy, S.S., Hurley, J.B. and Kennedy, M.B. (1988) Conserved and variable regions in the subunits of brain type I1 Ca’+/CaM-dependent protein kinase. Neuron, 1: 63-72. Chan, K.F.J. (1988) Ganglioside-modulated protein phosphorylation: Partial purification and characterization of a ganglioside-inhibited protein kinase in brain. J. Biol. Chem., 263: 568-574. Colbran, R.J., Fong, Y.L., Schworer, C.M. and Soderling, T.R. (1988) Regulatory interactions between the CaM-binding, inhibitory and autophosphorylation domains of Ca’+/CaM-dependent protein kinase 11. J. Biol. Chem., 263: 18145-18151. Colbran, R.J., Schworer, C.M., Hashimoto, Y., Fong, Y.L., Rich, D.P., Smith, M.K. and Soderling, T.R. (1989a) Calcium/calmodulin-dependent protein kinase 11. Biuchem. J., 258: 313-325. Colbran, R.J., Smith, M.K., Schworer, C.M., Fong, Y.L. and Soderling, T.R. (1989b) Inhibitory domain of Ca2+/ calmodulin-dependent protein kinase 11: mechanism of action and regulation by phosphorylation. J. Biol. Chem., 264: 4800-4804. Colbran, R.J. and Soderling, T.R. (1990a) Ca’+/calmodulindependent protein kinase 11. Curr. Topics Cell. Regid., 31: in press. Colbran, R.J. and Soderling, T.R. (1990b) Ca’+/CaM-independent autophos-phorylation sites of Ca2+/CaM-dependent protein kinase 11: studies on the effect of phosphorylation of Thr-305/306 and Ser-314 on CaM-binding using synthetic peptides. J. Biol. Chem., 265: 11213-11219. Connor, J.A., Wadman, W.J., Hockberger, P.E. and Wong, R.K.S. (1988) Sustained dendritic gradients of CaZf in-

The authors would like to thank Martha Bass for excellent technical assistance and Dr. William Taylor for assistance with the mutagenesis studies. This work was supported by NIH grants GM41292 and NS27037.

182 duced by excitatory amino acids in CAI hippocampal neurons. Science, 240: 649-653. DeCamilli, P., Harris, S.M., Huttner, W.B. and Greengard, P. (1983) Synapsin I (protein I), a nerve terminal-specific phosphoprotein. 11. Its specific association with synaptic vesicles demonstrated by immunocytochemistry in agarose-embedded synaptosomes. J. Cell B i d , 96: 13551373. Doskeland, A.P., Schworer, C.M., Doskeland, S.O., Chrisman, T.D., Soderling, T.R., Corbin, J.D. and Flatmark, T. (1984) Phenylalanine 4-monooxygenase. Some aspects of its phosphorylation by a calcium and CaM-dependent protein kinase. Eur. J. Biochem., 145: 31-37. Erondu, N.E. and Kennedy, M.B. (1985) Regional distribution of type I1 Ca’+/CaM-dependent protein kinase in rat brain. J. Neurosci., 5: 3270-3277. Fong, Y.L. and Soderling, T.R. (1990) Studies on the regulatory domain of Ca’+/CaM-dependent protein kinase 11: functional analyses of Arg-283 using synthetic inhibitory peptides and site-directed mutagenesis of the (Y subunit. J. Biol. Chem., 265: 11091-11097. Fong, Y.L., Taylor, W.L., Means, A.R. and Soderling, T.R. (1989) Studies of the regulatory mechanism of Ca2+/ CaM-dependent protein kinase 11: mutation of Thr-286 to Ala and Asp. J. Biol. Chem., 264: 16759-16763. Fukunaga, K., Miyamoto, E. and Soderling, T.R. (1990a) Regulation of Ca*+/CaM-dependent protein kinase I1 by brain gangliosides. J. Neurochem., 54: 102-109. Fukunaga, K., Rich, D.P. and Soderling, T.R. (1990b) Generation of the Ca*+-independent form of Ca*+/CaM-dependent protein kinase I1 in cerebellar granule cells. J. Biol. Chem., 264: 21830-21836. Fukunaga, K. and Soderling, T.R. (1990~)Activation of CaZC/CaM-dependent protein kinase I1 in cerebellar granule cells by N-methyl-D-aspartate receptor activation. Mol. Cell. Neurosci., 1: 133-138. Goldenring, J.R., Wasterlain, C.G., Oestreicher, A.B., de Graan, P.N.E., Farber, D.B., Glaser, G. and DeLorenzo, R.J. (1986) Kindling induces a long-lasting change in the activity of a hippocampal membrane CaM-dependent protein kinase system. Brain Res., 377: 47-53. Gorelick, F.S., Wang, J.K., Lai, Y., Nairn, A.C. and Greengard, P. (1988) Autophosphorylation and activation of Ca2+/CaM-dependent protein kinase I1 in intact nerve terminals. J. Biol. Chem., 263: 17209-17212. Gustafsson, B. and Wigstrom, H. (1988) Physiological mechanisms underlying long-term potentiation. Trends Neurosci., 11: 156-162. Hanson, P.I., Kapiloff, M.S., Lou, L.L., Rosenfeld, M.G. and Schulman, H. (1989) Expression of a multifunctional Ca’+/CaM-dependent protein kinase and mutational analysis of its autoregulation. Neuron, 3: 59-70. Hashimoto, Y., Schworer, C.M., Colbran, R.J. and Soderling, T.R. (1987) Autophosphorylation of Ca*+/CaM-depen-

dent protein kinase 11: effects on total and Ca2+-independent activities. J. Biol. Chem., 262: 8051-8055. Hollmann, M., O’Shea-Greenfield, A., Rogers, S.W. and Heinemann, S. (1989) Cloning by functional expression of a member of the glutamate receptor family. Nature, 342: 643-648. Johnson, J.W. and Ascher, P. (1987) Glycine potentiates the NMDA response in cultured mouse brain neurons. Nature, 325: 529-531. Kemp, B.E., Graves, D.J., Benjamini, E. and Krebs, E.G. (1977) Role of multiple basic residues in determining the substrate specificity of CAMP-dependent protein kinase. J. Biol. Chem., 252: 4888-4894. Kennedy, M.B., Bennett, M.K. and Erondu, N.E. (1983): Biochemical and immuno-chemical evidence that the “major postsynaptic density protein” is a subunit of a CaM-dependent protein kinase. Proc. Natl. Acad. Sci. USA, 80: 7357-7361. Kwiatkowski, A.P., Shell, D.J. and King, M.M. (1988) The role of autophosphorylation in activation of the type I1 CaM-dependent protein kinase. J. Biol. Chem., 263: 64846486. Kwiatkowski, A.P. and King, M.M. (1989) Autophosphorylation of the type I1 CaM-dependent protein kinase is essential for a proteolytic fragment with catalytic activity: implications for long-term synaptic potentiation. Biochemistry, 28: 5380-5385. Ledeen, R.W. (1978) Ganglioside structures and distribution: Are they localized at the nerve ending? J. Suprumol. Struct., 8: 1-17. Lin, C.R., Kapiloff, M.S., Durgerian, S., Tatemoto, S., Russo, A.F., Hanson, P., Schulman, H. and Rosenfeld, M.G. (1987) Molecular cloning of a brain-specific CaZf/CaMdependent protein kinase. Proc. Natl. Acad. Sci. USA, 84: 5962-5966. Lisman, J.E. and Goldring, M.A. (1988) Feasibility of longterm storage of graded information by the Ca*+/CaM-dependent protein kinase molecules of the postsynaptic density. Proc. Natl. Acad. Sci. USA, 85: 5320-5324. MacDermott, A.B., Mayer, M.L. and Westbrook, G.L. (1986) NMDA-receptor activation increases cytoplasmic calcium in cultured spinal cord neurons. Nature, 321: 519-522. Malenka, R.C., Kauer, J.A., Perkell, D.J., Mauk, M.D., Kelly, P.T., Nicoll, R.A. and Waxham, M.N. (1989) An essential role for postsynaptic CaM and protein kinase activity in long-term potentiation. Nature, 340: 554-557. Malinow, R., Schulman, H. and Tsien, R.W. (1989) Inhibition of postynaptic protein kinase C or CaM-dependent protein kinase I1 blocks induction but not expression of long-term potentiation. Science, 245: 862-866. Mayer, M.L., Westbrook, G.L. and Guthrie, P.B. (1984) Voltage-dependent block by Mg*+ of NMDA responses in spinal cord neurons. Nature, 309: 261-263. Mayer, M.L and Westbrook, G.L. (1987) The physiology of

183 excitatory amino acids in the vertebrate central nervous system. Prog. Neurobiol., 28: 197-276. Nestler, E.J. and Greengard, P. (1982) Nerve impulses increase the phosphorylation state of protein I in rabbit superior cervical ganglion. Nature, 296: 452-454. Nichols, R.A., Sihra, T.S., Czernik, A.J., Nairn, A.C. and Greengard, P. (1990) Ca’+/CaM-dependent protein kinase I1 increases glutamate and noradrenaline release from synaptosomes. Nature, 343: 647-651. Patton, B.L., Miller, S.G. and Kennedy, M.B. (1990) Activation of type I1 Ca’+/CaM-dependent protein kinase by Ca2+/CaM is inhibited by autophosphorylation of threonine within the CaM-binding domain. J. Biol. Chem., 265: 11204-1 1212. Payne, M.E., Fong, Y.L., Ono, T., Colbran, R.J., Kemp, B.E., Soderling, T.R. and Means, A.R. (1988) Ca’+/calmodulin-dependent protein kinase 11: characterization of distinct CaM-binding and inhibitory domains. J. Biol. Chem., 263: 7190-7195. Perlmutter, L., Siman, R., Gall, C., Baudry, M. and Lynch, G. (1988) The ultrastructural localization of Ca*+-activated protease calpain in rat brain. Synapse, 2: 79-88. Pocotte, S.L., Holz, R.W. and Ueda, T. (1986) Cholinergic receptor-mediated phosphorylation and activation of tyrosine hydroxylase in cultured bovine adrenal chromaffin cells. J. Neurochem., 46: 610-622. Rich, D.P., Colbran, R.J., Schworer, C.M. and Soderling, T.R. (1989) Regulatory properties of Ca2+/CaM-dependent protein kinase I1 in rat brain postsynaptic densities. J. Neurochem., 53: 807-816. Rich, D.P., Schworer, C.M., Colbran, R.J. and Soderling, T.R. (1990) Proteolytic activation of Ca*+/CaM-dependent protein kinase 11: putative function in synaptic plasticity. Mol. Cell. Neurosci., 1: 107-116. Schiebler, W., Jahn, R., Doucet, J.P., Rothlein, J. and Greengard, P. (1986) Characterization of synapsin I binding to small synaptic vesicles. J. Biol Chem., 261: 8383-8390. Schulman, H. (1988) The multifunctional Ca’+/CaM-dependent protein kinase. Adu. Second Messenger Phosphoprotein Res., 22: 39- 111. Simmerman, H.K.B., Collins, J.H., Theibert, J.L., Wegener, A.D. and Jones, L.R. (1986) Sequence analysis of phospholamban: identification of phosphorylation sites and two major structural domains. J. Biol. Chem., 261: 1333313341.

Smith, M.K., Colbran, R.J. and Soderling, T.R. (1990) Specificities of auto-inhibitory domain peptides for four protein kinases: Implications for intact cell studies of protein kinase functions. J. Biol. Chem., 265: 1837-1840. Soderling, T.R., Schworer, C.M., Payne, M.E., Jett, M.F., Porter, D.K., Atkinson, J.L. and Richtand, N.M. (1986) Ca*+/calmodulin-dependent protein kinase 11. In J. Nunez, J.E. Dumont and R.J.B. King (Eds.), Hormones and Cell Regulation, Colloque INSERM, Vol. 139, John Libbey Eurotext, London, pp. 141-157. Soderling, T.R. (1990) Protein kinases: regulation by autoinhibitory domains. J. Biol. Chem., 265: 1823-1826. Staubli, U., Thibault, O., Baudry, M. and Lynch, G. (1988) Chronic administratioin of a thiol-proteinase inhibitor blocks long-term potentiation of synpatic responses. Brain Res., 444: 153-158. Tokumitsu, H., Chijiwa, T., Hagiwara, M., Mizutani, A,, Terawawa, M. and Hidaka, H. (1990) KN-62, a specific inhibitor of Ca’+/CaM-dependent protein kinase 11. J. Biol. Chem., 265: 4315-4320. Wada, J. (1982) Kindling 11. Raven Press, New York. Waldmann, R., Hanson, P.I. and Schulman, H. (1990) Multifunctional Ca’+/CaM-dependent protein kinase made Ca*+-independentfor functional studies. Biochemistry, 29: 1679- 1684. Waxham, M.N., Aronowski, J., Westgate, S.A. and Kelly, P.T. (1990) Mutagenesis of Thr-286 in monomeric Ca2+/ CaM-dependent protein kinase I1 eliminates Ca2+-independent activity. Proc. Natl. Acad. Sci. USA, 87: 1273-1277. Waymire, J.C., Johnson, J.P., Hummer-Lickteig, K., Lloyd, A,, Vigny, A. and Cravisco, G.L. (1988) Phosphorylation of bovine adrenal chromaffin cell tyrosine hydroxylase: temporal correlation of acetylcholine’s effect of site phosphorylation, enzyme activation and catecholamine synthesis. J. Biol. Chem., 263: 12439-12447. Yamauchi, T., Ohsako, S. and Deguchi, T. (1989) Expression and characterization of CaM-dependent protein kinase I1 from cloned cDNAs in Chinese hamster ovary cells. J. Biol. Chem., 264: 19108-19116. Yanagihara, N., Tank, A.W. and Weiner, N. (1984) Relationship between activation and phosphorylation of tyrosine hydroxylase by 56 mM K + in PC12 cells in culture. Mol. Pharmacol., 26: 141-147.