Definition of Optimal Substrate Recognition Motifs of Ca2+-Calmodulin-dependent Protein Kinases IV and II Reveals Shared and Distinctive Features*

Ronald R. WhiteDagger , Young-Guen Kwon§, Meng Taing, David S. Lawrence, and Arthur M. EdelmanDagger par

From the Dagger  Department of Pharmacology and Toxicology and § Department of Chemistry, State University of New York, Buffalo, New York 14214 and  Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The substrate recognition determinants of Ca2+-calmodulin-dependent protein kinase (CaMK) IV and CaMKIIalpha were investigated using peptide substrates modeled on the amino acid sequence encompassing Ser-9 of synapsin I. For both kinases, hydrophobic residues (Leu or Phe) at the -5 position, are well tolerated, whereas non-hydrophobic residues (Arg, Ala, or Asp) decrease Vmax/Km by 55- to >4000-fold. At the -3 position, substitution of Ala for Arg leads to decreases of 99- and 343- fold in Vmax/Km for CaMKIV and CaMKIIalpha , respectively. For both kinases, the nature of the residues occupying the -4, -1, and + 4 positions exerts relatively little influence on phosphorylation kinetics. CaMKIV and CaMKIIalpha respond differently to substitutions at the -2 and +1 positions. Substitution of Arg at the -2 position with non-basic residues (Gln or Ala) leads to 6-fold decreases in Vmax/Km for CaMKIV, but 17-28-fold increases for CaMKIIalpha . Additionally, peptides containing Leu, Asp, or Ala at the +1 position are phosphorylated with similar efficiencies by CaMKIV, whereas the Leu-substituted peptide is preferred by CaMKIIalpha (by a factor of 5.8-9.7-fold). Thus, CaMKIV and CaMKIIalpha preferentially phosphorylate substrates with the motifs: Hyd-X-Arg-X-X-Ser*/Thr*, and Hyd-X-Arg-NB-X-Ser*/Thr*-Hyd, respectively, where Hyd represents a hydrophobic, X any, and NB a non-basic amino acid residue. The different specificities of the two kinases may contribute to their targeting to distinct physiological substrates during Ca2+-dependent cellular events.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Ca2+-calmodulin-dependent protein kinases (CaMKs)1 are recognized as crucial mediators of the physiological effects of stimuli-induced elevations of intracellular Ca2+ (1, 2). Within the CaMK group, CaMKII and CaMKIV are distinguished by their abilities to phosphorylate a range of phosphoaccepting substrates both in vitro and in vivo, leading to their designation, along with CaMKI, as "multifunctional" CaM kinases. Recently, there has been intense experimental interest in the roles played by these CaM kinases in the Ca2+-dependent enhancement of synaptic efficacy and in transcriptional activation, although the physiological substrates underlying these effects remain to be fully identified (2-4).

An approach that has proven to be of great utility for identifying potential targets of protein kinases is definition of their substrate recognition motifs (consensus sequences) using synthetic phosphoaccepting peptides (5). Work by Pearson et al. (6) confirmed an earlier proposal (7) that an Arg residue at the -3 position relative to the phosphorylatable Ser or Thr2 is essential for substrate recognition by CaMKII. This "minimal" motif, R-X-X-S*/T*,3 has been detected in the phosphorylation site sequences of many of the protein substrates of CaMKII (8, 9). However, it was also noted by Pearson and co-workers (6) that, of several peptides that incorporated an Arg at the -3 position, RRATSNVFA and RKASGPPV were not appreciably phosphorylated by CaMKII, and that another, LRRASLG (Kemptide), was an extremely poor substrate. Thus, determinants in addition to the "essential arginine" must be required for substrate recognition. More recent reports have suggested that, in addition to the -3 Arg, particular residues are preferred by CaMKII, specifically at the -5 (10), -2 (11, 12), +1 (10, 12), and +2 (13) substrate positions. Moreover, for some substrates, for example, vimentin (13) or Ser-142 of CREB (14), an Arg at the -3 position may be non-essential for recognition. These more recent studies on the substrate specificity of CaMKII utilized different substrates and kinetic methods and examined different positions within the respective substrates (10-13). Hence, the importance for CaMKII of these additional proposed specificity determinants relative to each other, and relative to the -3 Arg, has remained unclear. As a result, R-X-X-S*/T* is still the only generally accepted consensus sequence for CaMKII (2).

Compared with CaMKII, the substrate specificity of CaMKIV has received relatively little study. CaMKIV was demonstrated to require in its substrates an Arg, three residues amino-terminal to the phosphorylated Ser or Thr (15). However, like CaMKII, CaMKIV phosphorylates Kemptide (LRRASLG) poorly, despite the presence of an Arg at the -3 position in this peptide (15). These results suggest that the recognition motif of CaMKIV, like that of CaMKII, has yet to be fully defined. The idea that the substrate specificities of CaMKII and IV are more complex than is currently appreciated raises the possibility that these enzymes may exhibit differences in the patterns of their responses to specific substrate sequence elements. This hypothesis is consistent with observations that, despite overlap in the set of proteins phosphorylated by the two kinases (15, 16), certain sequences, for example, Ser-9 of synapsin (17, 18) or Ser-142 of CREB (14), are preferentially phosphorylated by CaMKIV or CaMKII, respectively.

The present study was therefore conducted to: 1) define an optimal substrate recognition motif for CaMKIV; 2) assess the relative importance of additional specificity determinants of CaMKII, and define its optimal motif; and 3) compare the motifs to define distinguishing features of substrate recognition by these two important signal-transducing protein kinases.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Synthetic Peptides-- Syntide-2 (PLARTLSVAGLPGKK) was purchased from Life Technologies, Inc. All other peptides were synthesized as COOH-terminal amides according to Merrifield's methodology (19) and purified by high performance liquid chromotography as described previously (20). The concentrations of peptides were determined spectrophotometrically by the quantitative ninhydrin reaction (epsilon  = 2.53 × 104 M-1 cm-1, 570 nm) (20, 21), with the exception of syntide-2, LRRRLSDDNF, and LRRQLSDANF. Concentrations of the latter peptides were quantified by stoichiometric phosphorylation using CaMKIIalpha .

Enzymes-- Recombinant CaMKIV and CaMKIIalpha were expressed using the baculovirus system and were the generous gifts of Kristin Anderson, Jon Schreiber, and Anthony Means (Duke University). CaM kinase kinase (CaMKK), utilized to phosphorylate and activate CaMKIV prior to kinetic experiments was purified from rat brain as an approximately equimolar mixture of the CaMKKalpha and -beta isoforms (22). Protein concentrations were determined by the Lowry procedure (23) as described previously (24) using bovine serum albumin as standard.

Peptide Kinase Assays-- CaMKIV requires phosphorylation at Thr-196 for expression of its catalytic activity (25). Therefore, the enzyme was preincubated with a maximally stimulating concentration of purified CaMKKalpha /beta at 30 °C for 10 min in the presence of 10 mM MgCl2, 0.2 mM ATP, 1 mM CaCl2, and 1 µM CaM. Peptide kinase activity was then measured at 30 °C in a reaction mix containing 50 mM Tris, pH 7.6, 0.5 mM dithiothreitol, 0.5 mg/ml bovine serum albumin, 0.2 mM [gamma -32P]ATP (~0.2 × 103 cpm/pmol), 10 mM MgCl2, 1 mM CaCl2, 1 µM calmodulin, various concentrations of peptides, and residual buffer components from protein storage buffers. CaMKIIalpha was assayed under the same reaction conditions but without preincubation with CaMKK. 32P incorporation was determined by adsorption of phosphorylated peptides onto P-81 filter paper, followed by washing in 75 mM H3PO4 and ethanol and liquid scintillation spectrometry as described (24). In control reactions, all peptides were tested, but none were found to be detectably phosphorylated by CaMKK in the absence of CaMKIV. Other control reactions were performed with CaMKIIalpha in the absence of peptide substrate to control for apparent activity that might be due to CaMKIIalpha autophosphorylation. Although this activity was routinely subtracted as a "blank" for CaMKIIalpha assays, it was only detectable at the highest enzyme concentrations used (which were needed to phosphorylate the poorest substrates).

Calculation of Kinetic Parameters-- For each experiment, the substrate concentrations (typically five) were chosen to bracket the Km, with the rate at each concentration determined at multiple time points to ensure both linearity of activity and utilization of <= 20% of substrate during the course of the reaction. The rate at each concentration/time point was determined in duplicate or triplicate. Km and Vmax values were computed by non-linear regression analysis using the program, ENZFITTER (Elsevier-BioSoft, Cambridge, United Kingdom). Kinetic parameters, listed in Tables I and II, represent the mean ± S.E. of two or three independent experiments.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

The synaptic vesicle-associated protein synapsin I is efficiently phosphorylated by CaMKII at two COOH-terminal sites, termed sites 2 and 3, but only very slowly phosphorylated at the NH2-terminally located site 1 (Ser-9) (17, 18). By contrast, CaMKIV phosphorylates either or both of sites 2 and 3 as well as site 1 (17). By peptide mapping, the ratio of 32P incorporation by CaMKIV is approximately 1:2 (site 1:sites 2/3), suggesting that within about a factor of 2 the sites are equivalent as substrates for the latter enzyme. These observations are consistent with the determinants of substrate recognition by CaMKII and IV as being overlapping but nonidentical. Consequently, it may be concluded that the sequence in the vicinity of site 1 discriminates between the two kinases, either by the absence of positive determinants and/or the introduction of negative determinants, for CaMKII. Therefore, in the present study, we examined the substrate specificities of both kinases using the same set of peptides, modeled on site 1 of synapsin, and synthesized with systematic variation in the sequence: LRRRLSDANF. This peptide serves as a reference against which the effects of deletions and substitutions can be gauged and is referred to here as the synapsin site 1 "parent" peptide.

Kinetics of Peptide Phosphorylation by CaMKIV-- As shown in Table I, the parent peptide is an excellent substrate for CaMKIV, with Km and Vmax values of 0.18 µM and 1.64 µmol/min/mg, respectively, and is actually a better substrate (due to a 6.2-fold lower Km and 1.7-fold higher Vmax) than one commonly used in CaMKIV assays (16), syntide-2. Table I also presents, in systematic fashion in an NH2- to COOH-terminal direction, kinetic parameters for 16 additional peptides incorporating deletions and substitutions in the sequence of the parent peptide.

                              
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Table I
Kinetics of peptide phosphorylation by CaMKIV
Determination of kinetic parameters for each experiment was as described under "Experimental Procedures." Values shown represent the means of two or three experiments ± S.E. Substituted residues are underlined and in boldface type.

Since the positions of importance for CaMKIV were unknown (except for the -3 Arg), initial experiments were performed with peptides incorporating sequential NH2-terminal deletions. Deletion of Leu at the -5 position dramatically raises the Km, resulting in a 285-fold decrease in the Vmax/Km ratio. Thus, -5 is a critical position for substrate recognition. Absence of a residue at the -5 position would therefore explain the inability of Kemptide to be significantly phosphorylated by CaMKIV (15). Deletion of the residues at both the -5 and -4 positions results in an additional, although modest, decrease in Vmax/Km, suggesting that the -4 position may have some influence on substrate recognition. Since an effect of deletion could conceivably be due to reduction in peptide length, the kinetic parameters of substituted peptides were then determined. At the -5 position, the aliphatic hydrophobic Leu, was replaced by either the aromatic hydrophobic Phe, the basic Arg, the non-polar, relatively non-hydrophobic Ala, or the acidic Asp. Whereas the Phe-substituted peptide is a good substrate, displaying only a 3.6-fold decrease in Vmax/Km relative to that of the parent peptide, the other three substituted peptides are poorly phosphorylated with from 55- to >4000-fold reductions in Vmax/Km. Thus, a hydrophobic amino acid residue at the -5 position is important for substrate recognition by CaMKIV. The small decrease in Vmax/Km after substitution of Leu with Phe suggests that either an aliphatic or an aromatic side chain is accommodated in the active site, although the former may be slightly favored.

The role of Arg residues at the -4, -3, and -2 positions on phosphorylation kinetics by CaMKIV was then assessed. Peptides with Ala substituted for Arg at the -4 position or with Ala or Gln at the -2 position are relatively good substrates, although these substitutions lead to decreases in Vmax/Km of 3.9-fold and 5.7-6.1-fold, respectively. By contrast, substitution of Arg at the -3 position with Ala results in an approximately 100-fold decrease in Vmax/Km. Thus, an Arg at the -3 position is of approximately similar importance as is a hydrophobic amino acid at the -5 position. The modest inhibitory effects of replacement of Arg at the -4 and -2 positions may help explain why syntide-2 (which lacks these determinants) is a somewhat poorer substrate than the synapsin site 1 parent peptide, LRRRLSDANF.

The nature of the amino acids at the -1, +1, and +2 positions exerts relatively little influence on substrate recognition by CaMKIV since non-conservative substitutions at these positions lowers Vmax/Km by only 1.8-5.1-fold. Similarly, the residue at the +4 position appears to be of minor importance since its deletion or substitution (Ala for Phe) lowers Vmax/Km by only 5-fold.

Fig. 1 (panel A) is a plot of the effects of all substitutions on Vmax/Km values relative to those of the parent peptide. Based on these results, the optimal substrate recognition motif for CaMKIV is Hyd-X-Arg-X-X-Ser*/Thr*. Although residues at other positions, specifically -4, -2, -1, and +4 do exert some influence on phosphorylation kinetics, their overall effect is exceedingly modest and they are therefore not incorporated as determinants in this motif.


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Fig. 1.   Plot of the influence of substrate determinants on the kinetics of peptide phosphorylation by CaMKIV, CaMKIIalpha , and CaMKI. The effect on the Vmax/Km of CaM kinase activity as a result of the indicated amino acid substitutions in the parent sequence (LRRRLSDANF) are calculated as the -fold change relative to the latter (in either the positive or negative direction). Panel A, CaMKIV, data are from Table I. Panel B, CaMKIIalpha , data are from Table II. Panel C, CaMKI, data are from Ref. 20.

Kinetics of Peptide Phosphorylation by CaMKIIalpha -- Syntide-2 is phosphorylated by CaMKIIalpha with a 35-fold higher Vmax/Km than the synapsin site 1 parent peptide (Table II). The relatively poor substrate efficacy of the latter peptide is consistent with the inability of CaMKII to phosphorylate this site in the context of the intact protein (18). This strong preference of CaMKIIalpha for syntide-2 is in marked contrast to CaMKIV, for which syntide-2 is actually an approximately 10-fold poorer substrate than the synapsin site 1 parent peptide (Table I). Thus, the two enzymes demonstrate differential substrate selectivity. The basis for this selectivity was explored with peptides based on the synapsin site 1 parent peptide (Table II).

                              
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Table II
Kinetics of peptide phosphorylation by CaMKIIalpha
Determination of kinetic parameters for each experiment is as described under "Experimental Procedures." Values shown represent the means of two or three experiments ± S.E. Substituted residues are underlined and in boldface type.

Since the nature of the amino acid residue at the -5 position is critical for substrate recognition by CaMKIV, peptides substituted at this position were evaluated as substrates for CaMKIIalpha . Whereas another hydrophobic residue (Phe) is well tolerated at this position (displaying only a 4.5-fold decrease in Vmax/Km), substitution of basic (Arg), non-polar (Ala), or acidic (Asp) residues leads to decreases of 79-172-fold in their respective Vmax/Km values. Therefore, as is the case with CaMKIV, the presence of a hydrophobic residue at the -5 position is a strong positive determinant for CaMKIIalpha . This requirement explains the inability of some peptides, which have an Arg at -3 but lack a residue at the -5 position, to serve as substrates for CaMKII (6). The role of Arg residues at the -4, -3, and -2 positions was then examined. The -4 position has little influence on phosphorylation kinetics since a non-conservative substitution (Ala for Arg) changes Vmax/Km by only 2.6-fold. By contrast, Ala substitution at the -3 position drops Vmax/Km by 343-fold.

At the -2 position, substitutions yield changes in kinetic parameters using CaMKIIalpha clearly distinct from those observed with CaMKIV. Here, replacement of Arg with the non-polar amino acids, Ala or Gln, leads to 6-fold decreases in Vmax/Km for CaMKIV (Table I), whereas these substitutions result in increases of 17-fold and 28-fold, respectively, for CaMKIIalpha (Table II). These results indicate that, while a positively charged Arg residue at the -2 position may be a (modest) positive determinant for CaMKIV, it is a strong negative determinant for CaMKIIalpha .

The next position examined (-1) does not discriminate between the kinases since substitution of Ala for Leu, lowers Vmax/Km by about the same extent (4.3-fold) with CaMKIIalpha as with CaMKIV (5.1-fold). However, at the +1 position, CaMKIIalpha phosphorylates the peptide containing a hydrophobic Leu residue with a Vmax/Km value 5.8-9.7-fold higher than those of the Asp- and Ala-substituted peptides, respectively. This is in clear contrast to CaMIV for which substitution of Leu at the +1 position creates, if anything, a slightly poorer substrate than either the Asp- or Ala-substituted peptides (Table I). It may also be noted that the presence of a hydrophobic residue at the +1 position in combination with a non-basic residue at the -2 position fully accounts for the improved ability of syntide-2 to serve as substrate for CaMKIIalpha , compared with the synapsin site 1 parent peptide. Finally, non-conservative substitutions at the +2 (Asp for Ala) and +4 (Ala for Phe) positions, respectively, have little effect on kinetic parameters with CaMKIIalpha . The lack of influence of the +4 position is also demonstrated by the similar kinetics exhibited by a COOH-terminally deleted peptide as compared with the parent peptide. Fig. 1 (panel B) is a plot of the effects of all substitutions on Vmax/Km values relative to that of the parent peptide. It indicates an optimal substrate recognition motif for CaMKIIalpha of Hyd-X-Arg-NB-X-Ser*/Thr*-Hyd.

We have delineated in this report optimal substrate recognition motifs for CaMKIV and CaMKIIalpha . These motifs explain the previously demonstrated abilities of these kinases to discriminate between highly similar sequences in protein substrates. Furthermore, the identification of substrate determinants that differentiate between the two kinases may assist in the identification of potential in vivo phosphorylation sites, allowing the distinct physiological roles of these enzymes to be discerned.

    DISCUSSION
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Of the eight positions evaluated in this study, determinants at -5 and -3 are the most critical and are of approximately equivalent importance for both kinases (Tables I and II). For CaMKIIalpha , the >300-fold decrease in Vmax/Km upon replacement of the -3 Arg with Ala is consistent with the classification of CaMKII as "arginine-requiring" by Pearson et al. (6). Additionally, based on the ~100-fold lower Vmax/Km of LRARLSDANF relative to the parent peptide, an Arg residue at the -3 position is also important for CaMKIV. However, it should be noted that CaMKIV phosphorylates the former peptide with a Km of 9.9 µM and Vmax of 0.91 µmol/min/mg, i.e. a Vmax/Km 2-3 orders of magnitude higher than that of CaMKIIalpha . Thus, it is conceivable that in vivo there may be target substrates for CaMKIV lacking the -3 Arg, which are, nevertheless, CaMKIV-selective.

Of the three multifunctional CaM kinases (I, II, and IV), the only one for which there is x-ray crystallographic data is CaMKI (26). However, since the kinases are 45-50% identical at the amino acid level in their catalytic domains, it is likely that their structures will prove to be similar. By analogy to CaMKI, the -3 Arg of the substrate can therefore be predicted to interact with Glu-121 of CaMKIV and with Glu-96 of CaMKII, residues equivalent to Glu-102 of CaMKI (26). The decreases in Vmax/Km values of both kinases upon replacement of the -5 Leu with non-hydrophobic residues are striking. For CaMKII, the kinetic parameters are consistent with the initial rate data of Stokoe et al. (10). This strong preference for a -5 hydrophobic residue has also been observed for CaMKI (20). In the case of CaMKI, a hydrophobic pocket (formed by Ile-210, Pro-216, and Phe-104) appears to be appropriately positioned to accommodate the substrate -5 residue (26). All three of these residues are conserved in CaMKII, and two of three are conserved (Ile-229 and Phe-123) in CaMKIV, suggesting the possibility of a similar mechanism for the latter kinases. Such a mechanism is also consistent with a molecular modeling study of CaMKIIalpha (27). In this model, the hydrophobic Leu-299 of the pseudosubstrate domain occupies a position equivalent to the -5 position in an exogenous substrate. It should be noted, however, that for CaMKIV, the effects of substitutions at the -5 and -3 positions are primarily manifested as Km changes (consistent with the loss of binding interactions), whereas for reasons that are unclear, the same substitutions yield mainly Vmax changes with CaMKIIalpha . Understanding the basis for this difference in the affected kinetic parameter will therefore require, in future studies, solution of the kinetic mechanisms and quantification of the affected individual microscopic rate constants in both cases.

CaMKIV and CaMKIIalpha respond differently to substitutions at the -2 and +1 positions. A Gln at -2 creates an excellent substrate for CaMKIIalpha (Table II) and was strongly selected by CaMKII using a degenerate peptide library technique (12). However, a peptide containing an Ala at -2 is also effectively phosphorylated by CaMKIIalpha , indicating that although a Gln at this position is advantageous, it is by no means obligatory. In contrast to CaMKII, substitution of either Gln or Ala at the -2 position leads to no increase in substrate efficacy for CaMKIV (Table I). At the +1 position, CaMKIIalpha (but not CaMKIV) responds positively to a hydrophobic residue (Tables I, II). In cAMP-dependent protein kinase (PKA), a hydrophobic residue is preferred at the +1 position as a consequence of its interaction with a hydrophobic groove lined by Leu-198, Pro-202, and Leu-205 (28). In both CaMKIV and CaMKII, hydrophobic residues are located equivalently to Leu-198 of PKA. Additionally, proline residues homologous to Pro-202 are also present in both CaM kinases. However, whereas CaMKII has a Leu at position 180 homologous to Leu-205 of PKA, CaMKIV has Cys, suggesting a possible weakened interaction in the latter, which may explain why CaMKIV fails to demonstrate a +1 hydrophobic preference. Interestingly, CaMKI, which also responds positively to a substrate hydrophobic residue at the +1 position, has a Val residue homologous to Leu-205 of PKA (20).

Finally, we did not find an Asp residue at the +2 position to be a positive determinant for CaMKIIalpha . This was unexpected in that, by the degenerate library technique, CaMKII selects an acidic residue (preferably Asp) at this position (12). It has been observed that CaMKII has the capacity to phosphorylate seryl or threonyl residues in the absence of an Arg at the -3 position in a number of substrates including vimentin, CREB (Ser-142), caldesmon, and others (29). In the case of vimentin, an Asp residue at the +2 position is a positive determinant (13). Thus, it is possible that CaMKII recognizes an Asp at the +2 position only in the absence of an Arg at the -3 position.

The optimal substrate motifs constructed for CaMKIV and CaMKIIalpha , using these identified determinants, exhibit a number of interesting features. Somewhat surprisingly, the motif for CaMKIV is actually more "degenerate" than that of CaMKIIalpha in its incorporation of only two important determinants (-3 and -5 positions) as opposed to four (-5, -3, -2, and +1 positions) in the case of the latter. This reinforces the appropriateness of the designation of CaMKIV as a multifunctional CaM kinase. However, compared with CaMKII, relatively few protein substrates of CaMKIV have been identified, including CREB (14, 15, 30, 31), serum response factor (SRF) (15, 32), Rap1b (33), oncoprotein-18 (34), and synapsin I (17). Based upon the substrate recognition motif of CaMKIV delineated here, it will be of interest to see if additional protein substrates can now be identified. Conversely, scores of proteins (~50 were compiled in a recent review; see Ref. 2) have been reported to be phosphorylated by CaMKII, although some have only been demonstrated to be phosphorylated in vitro. Thus, for CaMKII, examination of phosphorylation site sequences for the presence of positive determinants at the -5, -2, and +1 positions, in addition to the -3 Arg, may allow enhanced predictive accuracy of which potential targets have the greatest likelihood of being preferred substrates in vivo.

The availability in the literature of a large number of identified protein phosphorylation sites for CaMKII, as well as some for CaMKIV, permits a comparison of these sequences with the optimal substrate recognition motifs of the kinases as defined here using synthetic peptides (Table III). As can be seen, there is excellent agreement with the motifs, although, at least in the case of CaMKII, the correspondence is not perfect. There are two probable explanations for this. First, since for CaMKII and, to a lesser extent, CaMKIV substrate efficacy is a function of multiple determinants, lack of a single or even several positive determinants may lead to a significant decrease in the rate of phosphorylation, but not a complete inability of the protein to serve as substrate. For example, pyruvate kinase and phenylalanine hydroxylase contain basic residues at the -2 position; the autophosphorylation sites at Thr-305/Thr-306 are missing a hydrophobic residue at the -5, and a basic residue at the -3, positions. However, it has been observed that the rates of phosphorylation of these substrates are 1-2 orders of magnitude slower than that of a more preferred substrate(s), for example, glycogen synthase or syntide-2 (35, 36). Similarly, a peptide based on Ser-142 of CREB (a sequence missing Arg and hydrophobic residues at the -3 and +1 positions, respectively), could be phosphorylated by CaMKIIalpha but with a Vmax/Km 2-4 orders of magnitude less than is observed with synapsin site 1 parent peptide and syntide-2.4 Second, it is also possible that secondary or tertiary orders of structure might enhance substrate ability of selected proteins, a factor that could be of particular importance for the "non-arginine requiring" substrates listed in Table III.

                              
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Table III
Phosphorylation sites of protein substrates of CaMKIV and CaMKII
Residues that are positive determinants for each kinase (or are conservative replacements, e.g. Lys for Arg) are shaded. Pro is considered acceptable at -5 for CaMKII based on Ref. 10. Substrates of CaMKII shown below the line are examples of those that may not require an arginine at the -3 position.

The motifs of CaMKII and CaMKIV are similar to that of CaMKI, for which a minimal motif of Hyd-X-Arg-X-X-S*/T*-X-X-X-Hyd was defined (20, 37). CaMKI differs prominently from CaMKIV in its requirement for a hydrophobic residue at the +4 position, and from CaMKII in both the latter positional requirement and in its lack of the need for a non-basic residue at the -2 position (Fig. 1, panel C). The three multifunctional CaM kinases are therefore clearly distinguishable on the basis of substrate specificity. Consequently, in concert with other variables in which they differ, such as tissue and subcellular localizations and modes of regulation (1-3), their ability to recognize and respond to distinct substrate sequence elements may contribute importantly to their individual physiological roles.

    ACKNOWLEDGEMENTS

We greatly appreciate the generosity of Kristin Anderson, Jon Schreiber and Anthony Means in supplying the recombinant CaMKIV and CaMKIIalpha enzymes used in these studies. We also thank Elaine Goldstein and Michele Selbert for excellent technical assistance.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants NS24738 (to A. M. E.) and GM45989 (to D. S. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

par To whom correspondence should be addressed. Tel.: 716-829-3491; Fax: 716-829-2801; E-mail: aedelman{at}ubmede.buffalo.edu.

1 The following abbreviations are used: CaMK, Ca2+-CaM-dependent protein kinase; CaM, calmodulin; CaMKKalpha /beta , CaMK kinase (alpha  and beta  isoforms; Ref. 22); CREB, cAMP response element-binding protein; synapsin site 1 parent peptide, LRRRLSDANF (a synthetic peptide modeled on the sequence of phosphorylation site 1 (Ser-9) of synapsin I); syntide-2, PLARTLSVAGLPGKK (a synthetic peptide based on the sequence of phosphorylation site 2 of glycogen synthase); Kemptide, LRRASLG; PKA, cAMP-dependent protein kinase.

2 The convention utilized in this study designates the phosphorylated residue as the 0 position, and the other amino acids in the NH2- and COOH-terminal directions numbered negatively and positively, respectively.

3 Hyd represents a hydrophobic, X any, and NB a non-basic, amino acid residue.

4 R. R. White, Kwon, Y.-G., Taing, M., Lawrence, D. S., and Edelman, A. M., unpublished observations.

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

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