From the 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
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ABSTRACT |
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The substrate recognition determinants of
Ca2+-calmodulin-dependent protein kinase
(CaMK) IV and CaMKII 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
CaMKII
, 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 CaMKII
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 CaMKII
. 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 CaMKII
(by a factor of 5.8-9.7-fold). Thus, CaMKIV and
CaMKII
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.
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INTRODUCTION |
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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.
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EXPERIMENTAL PROCEDURES |
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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 ( = 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 CaMKII
.
Enzymes--
Recombinant CaMKIV and CaMKII 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 CaMKK
and -
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 CaMKK/
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
[
-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. CaMKII
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
CaMKII
in the absence of peptide substrate to control for apparent
activity that might be due to CaMKII
autophosphorylation. Although
this activity was routinely subtracted as a "blank" for CaMKII
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.
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RESULTS |
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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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Kinetics of Peptide Phosphorylation by CaMKII--
Syntide-2 is
phosphorylated by CaMKII
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 CaMKII
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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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 CaMKII
, 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 CaMKII
. 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 CaMKII
(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
CaMKII
. 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 CaMKII respond differently to substitutions at the
2
and +1 positions. A Gln at
2 creates an excellent substrate for
CaMKII
(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 CaMKII
, 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, CaMKII
(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 CaMKII. 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 CaMKII,
using these identified determinants, exhibit a number of interesting
features. Somewhat surprisingly, the motif for CaMKIV is actually more
"degenerate" than that of CaMKII
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 CaMKII
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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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.
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ACKNOWLEDGEMENTS |
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We greatly appreciate the generosity of
Kristin Anderson, Jon Schreiber and Anthony Means in supplying the
recombinant CaMKIV and CaMKII enzymes used in these studies. We also
thank Elaine Goldstein and Michele Selbert for excellent technical
assistance.
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FOOTNOTES |
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* 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.
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; CaMKK/
, CaMK kinase (
and
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.
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REFERENCES |
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