(Received for publication, March 17, 1995; and in revised form, July 5, 1995)
From the
The mechanism for the regulation of
Ca/calmodulin-dependent protein kinase I (CaM kinase
I) was investigated using a series of COOH-terminal truncated mutants.
These mutants were expressed in bacteria as fusion proteins with
glutathione S-transferase and purified by affinity
chromatography using glutathione Sepharose 4B. A mutant (residues
1-332) showed complete Ca
/CaM-dependent
activity. Truncation mutants (residues 1-321, 1-314, and
1-309) exhibited decreasing affinities for
Ca
/CaM and also exhibited decreasing
Ca
/CaM-dependent activities. Truncation mutants
(residues 1-305 or 1-299) were unable to bind
Ca
/CaM and were inactive. In contrast, truncation
mutants (residues 1-293 or 1-277) were constitutively
active at a slightly higher level (2-fold) than fully active CaM kinase
I. These results indicate the location of the
Ca
/CaM-binding domain on CaM kinase I (residues
294-321) and predict the existence of an autoinhibitory domain
near, or overlapping, the Ca
/CaM-binding domain.
These conclusions were supported by studies which showed that a
synthetic peptide (CaM kinase I(294-321)) corresponding to
residues 294-321 of CaM kinase I inhibited the fully active
kinase in a manner that was competitive with Ca
/CaM
and also inhibited the constitutively active mutant (residues
1-293) in a manner that was competitive with Syntide-2, a peptide
substrate, (K
= 1.2
µM) but was non-competitive with ATP. Thus, these results
suggest that CaM kinase I is regulated through an intrasteric mechanism
common to other members of the family of
Ca
/CaM-dependent protein kinases.
Calcium (Ca) is widely recognized as an
essential intracellular second messenger in eukaryotic systems
regulating processes such as muscle contraction, neurotransmitter
release, gene expression, and cell proliferation (for reviews, see
Campbell, 1983; Davis, 1992). In a number of cases the effects of
Ca
are believed to be mediated by the ubiquitously
distributed Ca
receptor, calmodulin (CaM) (
)(for reviews, see Manalan and Klee, 1984). Strong
evidence, in turn, indicates that the effects of
Ca
/CaM are often achieved through the regulation of
protein phosphorylation (for reviews, see Nairn et al., 1985;
Hanson and Schulman, 1992; Nairn and Picciotto, 1994). A family of
Ca
/CaM-dependent protein kinases has been identified:
phosphorylase kinase, myosin light chain kinase, and EF-2 kinase (CaM
kinase III) that are highly specific enzymes, while CaM kinases II and
IV are multifunctional enzymes. CaM kinase I was first purified from
bovine brain based on its ability to phosphorylate site 1 of the
neuronal protein synapsin I (Nairn and Greengard, 1987). Since then the
enzyme has been found to be expressed in both neuronal and non-neuronal
tissues (Ito et al., 1994a; Picciotto et al., 1995),
thus necessitating a re-evaluation of the kinase as a potential
multifunctional kinase. In this respect, two other good substrates for
CaM kinase I have been identified in vitro: CREB, a
cAMP-response element-binding protein (Sheng et al., 1991),
and CF-2, a portion of the R-domain of the cystic fibrosis
transmembrane conductance regulator (Picciotto et al., 1992).
CaM kinase I is active as a monomer (Nairn and Greengard, 1987),
although various molecular masses of 37-43 kDa have been assigned
to the enzyme. A preparation from bovine brain, purified using synapsin
I as a substrate, was found to consist of two major polypeptides of M 37,000 and 39,000 and a minor polypeptide of M
42,000 (Nairn and Greengard, 1987). Two other
Ca
/CaM-dependent protein kinases, termed CaM kinases
Ia (M
43,000) and Ib (M
39,000), have been purified from rat brain using a synthetic
peptide based on site 1 of synapsin I as a substrate (DeRemer et
al., 1992a, 1992b). Another preparation, termed CaM kinase V (M
41,000), has been purified from rat brain using
the synthetic peptide, Syntide-2, as a substrate and its partial amino
acid sequences determined (Mochizuki et al., 1993). The
full-length cDNA for rat CaM kinase I has been cloned and a M
of 41,643 deduced from the amino acid sequence
(Picciotto et al., 1993; Cho et al., 1994). Based on
a high identity of amino acid sequences, CaM kinase V appears to be
closely related to, though distinct from CaM kinase I (Picciotto et
al., 1993). These results, together with recent immunological
studies of CaM kinase I and V (Ito et al., 1994a, 1994b;
Picciotto et al., 1995), suggest the possibility that CaM
kinase I, Ia, Ib, and V form a family of related isoforms.
Comparison of the deduced amino acid sequence of CaM kinase I with
that of the subunit of rat brain CaM kinase II revealed a high
identity (approximately 42%) throughout the catalytic domain (Picciotto et al., 1993). In addition, a second domain was identified
near the COOH terminus of CaM kinase I that showed limited identity
(approximately 32%) to the Ca
/CaM-binding domain of
the CaM kinase II subunit. In the present study, the structural basis
for the Ca
/CaM-dependent regulation of the enzyme has
been established. The results obtained indicate that CaM kinase I is
regulated through an intrasteric mechanism common to other members of
the family of Ca
/CaM-dependent protein kinases.
CaM was purified from bovine brain as
described (Endo et al., 1981). Smooth muscle myosin light
chain kinase (smMLCK) was purified from chicken gizzard as described
(Adelstein and Klee, 1981). The catalytic subunit of cAMP-dependent
protein kinase (PKA) was purified from bovine heart as described (Beavo et al., 1974). Ca-activated
phospholipid-dependent protein kinase was partially purified from
rabbit brain essentially as described (Inagaki et al., 1987).
The peptides, Syntide-2, and smooth muscle myosin light chain (smMLC) peptide were purchased from Peptide Institute Inc. (Osaka, Japan). The amino acid sequence of the smMLC peptide, KKRAARATSNVFA, corresponds to that surrounding the site phosphorylated in smMLC by smMLCK, except that the residues, AA, replaced for the original residues, PQ (Pearson et al., 1982). The peptide, CaM kinase I(294-321), based on the amino acid sequence of residues 294-321 of rat CaM kinase I (IKKNFAKSKWKQAFNRTAVVRHMRKLQL) was synthesized with a model 431A peptide synthesizer (Applied Biosystems Inc.). CaM kinase I(294-321) was purified by preparative reversed-phase HPLC (C-18). Aliquots of CaM kinase I(294-321) were subjected to analytical reversed-phase HPLC (C-18) and Pico-tag amino acid analysis (Waters Associates). The peptide was further characterized by amino acid sequencing with a model 473A protein sequencer (Applied Biosystems Inc.) (data not shown).
Oligo-1 s. was made with a BamHI site added upstream of the
position 1 (``start codon'') of a cDNA encoding rat CaM
kinase I. Oligo-277a. s., Oligo-293a. s., Oligo-299a. s., Oligo-305a.
s., Oligo-309a. s., Oligo-314a. s., Oligo-321a. s., and Oligo-332a. s.
correspond to complements of the coding strand with an EcoRI
site included upstream of the positions 831, 879, 897, 915, 927, 942,
963, and 998, respectively. Oligo-1 s. and Oligo-277a. s., Oligo-1 s.
and Oligo-293a. s., Oligo-1 s. and Oligo-299a. s., Oligo-1 s. and
Oligo-305a. s., Oligo-1 s. and Oligo-309a. s., Oligo-1 s. and
Oligo-314a. s., Oligo-1 s. and Oligo-321a. s., and Oligo-1 s. and
Oligo-332a. s. were used to amplify 855-, 903-, 921-, 939-, 951-, 966-,
987-, and 1022-base pair DNA fragments, respectively, from 5 ng of the
cDNA using Taq DNA polymerase (Promega). The conditions for
polymerase chain reaction (PCR) were 25 cycles of the following: 94
°C for 1 min, 65 °C for 2 min, and 72 °C for 2 min. The
amplified DNAs were extracted with phenol-chloroform,
ethanol-precipitated, and digested with BamHI and EcoRI. The purified fragments were subcloned into the BamHI and EcoRI sites of pGex-2T (Pharmacia P-L
Biochemicals Inc.) using Ligation Pack (NIPPON GENE Ltd., Tokyo, Japan)
according to the manufacturer's instructions. The sequences of
the DNA products encoding mutant 1-332 and mutant 1-293
were determined on both strands using Sequenase Ver. 2.0 (U. S.
Biochemical Corp.) and found to be correct. The complete sequences of
the other DNAs produced by PCR were not determined. While it is
possible that errors in DNA sequences were introduced based on the
error rate of Taq DNA polymerase (2
10
/base pair, Saiki et al., 1988), the fact
that the molecular weights of the protein products of the other mutants
were as expected, and the fact that many of the mutants expressed
activity suggested that no critical nonsense mutation was introduced by
the PCR technique. Recombinants carrying the insert of interest were
isolated using JM109 as a bacterial host and induced essentially as
described (Maruta et al., 1991). Briefly, 500 ml of LB plus 50
µg/ml ampicillin was inoculated with a 50-ml overnight culture of
the clone of interest and allowed to grow to OD
of 0.8 at
25 °C (approximately 2 h necessary for the growth). IPTG was added
to a final concentration of 0.1 mM and the culture allowed to
grow overnight. Cells were harvested by centrifugation at 4,000
g for 10 min and resuspended in 50 ml of lysis buffer
containing 50 mM Tris (pH 7.5), 25% saccharose, 2 mM dithiothreitol, and 50 µg/ml leupeptin. All further steps were
carried out at 0-4 °C. Nonidet P-40 and MgCl
were
added to a final concentration of 0.5% and 5 mM, respectively,
and cells lysed by sonication using first a 1-min step at mid-power
followed by four 1-min steps at high power. Debris was pelleted by
centrifugation at 12,000
g for 20 min. The supernatant
was recovered and applied onto a column of glutathione Sepharose 4B (10
ml wet volume) pre-equilibrated with wash buffer containing 20 mM Tris (pH 7.5), 2 mM MgCl
, 1 mM
-mercaptoethanol, and 100 µM phenylmethylsulfonyl fluoride. The column was washed extensively
with wash buffer and eluted with elution buffer containing 50 mM Tris (pH 8.8), and 5 mM glutathione. 3-ml fractions were
collected in tubes containing 1 ml of 200 mM Tris (pH 7.5) for
pH neutralization. Protein concentrations were determined by the
Bradford assay(1976) and fractions containing peak protein values were
pooled (approximately 12 ml in all cases), dialyzed against wash
buffer, adjusted with glycerol to a final concentration of 20% and
stored in aliquots at -70 °C. CaM kinase I fusion proteins
were essentially homogeneous as judged by staining of
SDS-polyacrylamide gels with Coomassie Brilliant Blue and the protein
concentration of the pure protein was used to determine the specific
activity (see below).
Figure 1: Construction and expression of a series of COOH-terminal truncated mutants of CaM kinase I as GST-fusion proteins. A, a series of DNAs was amplified from a cDNA encoding rat CaM kinase I using PCR as described under ``Experimental Procedures.'' The DNA fragments were subcloned into pGEX-2T and expressed in E. coli as GST-fusion proteins. Mutants 1-314 and 1-309 have an Arg residue resulting from a sequencing error instead of the correct Ala residue at position 309. A highly conserved catalytic domain (open box residues 1-270) and a non-conserved regulatory domain (shadowed box residues 271-332) are shown. B, aliquots (0.7-2.4 µg of protein/lane) of recombinant kinases purified by affinity chromatography on a column of glutathione Sepharose 4B as described under ``Experimental Procedures'' were analyzed by SDS-PAGE (6% acrylamide) and staining with Coomassie Brilliant Blue. The top and bottom of the gel were removed to highlight the differences in the mobility of the mutants.
All
preparations were purified to apparent homogeneity as demonstrated by
staining of SDS-polyacrylamide gels using Coomassie Brilliant Blue (Fig. 1B). Mutant 1-332 exhibited a molecular
mass of 62 kDa, consistent with the predicted value of 37 kDa of rat
CaM kinase I plus 26 kDa of GST. A proportional increase in mobility on
SDS-PAGE was observed with removal of increasing amounts of the
COOH-terminal region (i.e. mutants 1-321, 1-314,
1-309, 1-305, and 1-299, Fig. 1B). In
contrast, mutant 1-293 migrated slower than mutant 1-299
although residues 294-299 had been deleted. In addition, mutant
1-277, which was expected to migrate with the fastest mobility,
co-migrated with mutant 1-299. The basis for the anomalous
migrations of mutant 1-293 and 1-277 is not known. Previous
studies of mitogen-activated protein kinase (Matsuda et al.,
1992) and cdc25-C (Hoffmann et al., 1993) have indicated that
phosphorylation by their respective activating kinases resulted in a
decreased mobility on SDS-PAGE. Recent studies have indicated that CaM
kinase I is also phosphorylated at an active site Thr residue by one or
more kinases (Sugita et al., 1994), and that this results in a
slower mobility on SDS-PAGE. ()However, there is no evidence
that any of the recombinant proteins were phosphorylated during
preparation.
Figure 2:
Ca-dependent CaM binding
and Ca
/CaM independent and dependent activities of
recombinant kinases. A, aliquots (0.9-1.7 µg of
protein/lane) of recombinant kinases were subjected to SDS-PAGE (10%
acrylamide). The gels were incubated with
I-CaM (5
10
cpm/ml) in a buffer containing 1 mM CaCl
(upper panel) or 1 mM EGTA (lower panel), washed in the corresponding buffer, and
exposed, without drying, to x-ray film prior to development as
described under ``Experimental Procedures.'' B,
aliquots (0.3-0.39 mg) of mutants 1-332, 1-314,
1-309, and 1-293 were adjusted to 1 mM CaCl
, applied onto a column of CaM-coupled Sepharose
HP (indicated by Apply), washed extensively, and proteins
eluted with 1 mM EGTA (indicated by EGTA) as
described under ``Experimental Procedures.'' Protein in the
eluate was monitored by absorbance at 280 nm. C, protein
kinase activities of mutants were measured in the presence of 1 mM EGTA (solid bars), or 0.5 mM CaCl
plus 1.5 µM CaM (open bars) as described
under ``Experimental Procedures.'' The enzymes were added to
a final concentration of 5.5-12 µg/ml. Mean values and
standard errors were determined from three independent experiments,
each performed in duplicate.
Wild-type recombinant CaM
kinase I was completely dependent on Ca/CaM (Fig. 2C), as previously demonstrated (Picciotto et
al., 1993). Using Syntide-2 as a substrate, its specific activity
was 73.8 ± 7.6 nmol/min/mg. The activity of recombinant CaM
kinase I is significantly lower than the fully active form of the
enzyme that requires phosphorylation by an activating kinase (Sugita et al., 1994). However, recent studies have indicated that the
substrate specificity and CaM dependence of recombinant CaM kinase I
are the same as the fully activated enzyme. (
)Mutants
1-332 and 1-321 displayed similar properties to wild-type
kinase (Fig. 2C). However, the
Ca
/CaM-dependent activities of mutants 1-314
and 1-309 were significantly decreased, and mutants 1-305
and 1-299 were not activated at all by Ca
/CaM.
Mutants 1-293 and 1-277 were fully active in the absence of
Ca
/CaM. Notably, they were 2-fold more active than
the Ca
/CaM-activated mutant 1-332. In fact,
these two mutants were slightly inhibited by Ca
in
the absence or presence of CaM (data not shown).
Taken together,
these results suggest that residues TAVVR
play a pivotal role in the binding of CaM kinase I to
Ca
/CaM. The Ca
/CaM-binding domain
of the enzyme contains at least residues 310-314 and may extend
further toward the NH
terminus of the enzyme. Mutant
1-293 acquired Ca
/CaM-independent activity when
residues I
KKNFA
were deleted, suggesting
the existence of an autoinhibitory domain, which may extend toward the
COOH terminus from residues 294-299.
Figure 3:
Inhibition of Ca/CaM
dependent activities of CaM kinase I and smMLCK by the synthetic
peptide. A, mutant 1-332 (8.7 µg/ml) was incubated
(30 °C, 10 min) with either 0 nM (
), 100 nM (
), or 200 nM (
) CaM kinase I(294-321)
using 30-750 nM CaM. B, smMLCK (3.0 µg/ml)
was incubated (30 °C, 10 min) with either 0 nM (
), 20
nM (
), or 40 nM (
) CaM kinase
I(294-321) using 3-300 nM CaM.
P
incorporation into Syntide-2 and smMLC peptide were determined as
described under ``Experimental Procedures.'' The results are
representative of three independent experiments, each performed in
duplicate. Protein kanase activities are plotted as a function of CaM
concentration, taking the Ca
/CaM-dependent activities
of mutant 1-332 and smMLCK at 250 and 30 nM CaM,
respectively, in the absence of CaM kinase I(294-321) as a value
of 100%.
Figure 4:
Inhibition of Ca/CaM
independent activity of mutant 1-293 by CaM kinase
I(294-321): kinetic analyses. A, mutant 1-293 (5.5
µg/ml) was incubated (30 °C, 10 min) with either 0
µM (
), 0.5 µM (
), or 1.0 µM (
) CaM kinase I(294-321) using 25-1000
µM Syntide-2. B, the enzyme (5.5 µg/ml) was
incubated (30 °C, 10 min) with either 0 µM ([circlo]), 0.5 µM (
), or 1.0
µM (
) CaM kinase I(294-321) using
25-1000 µM [
-
P]ATP.
P incorporation into Syntide-2 was determined as described
under ``Experimental Procedures.'' The results are
representative of three independent experiments, each performed in
duplicate, and presented as double-reciprocal plots. Kinetic constants
were estimated by fitting the data to the Michaelis-Menten equation
using the method of least-squares. Inset, the apparent K
/V
is plotted as
a function of CaM kinase I(294-321)
concentration.
Figure 5:
Comparisons of two amino acid sequences of
CaM kinase I and of their recombinants on SDS-PAGE. A, the
original (I) and corrected (II) COOH-terminal amino
acid sequences of CaM kinase I are shown. In the correct sequence,
Arg is replaced by Ala
, and the deletion of
one nucleotide, G, in the third position of the codon GGG encoding
Gly
causes a frameshift resulting in an extension of the
COOH-terminal amino acid sequence. B, wild-type kinase was
expressed with the cDNA subcloned into pGex-2T by Picciotto et
al.(1993) and purified as described under ``Experimental
Procedures.'' Aliquots (10.4 µg of protein/lane) of mutant
1-332 and wild-type recombinant protein were treated with or
without 0.1 unit of thrombin at 37 °C for 30 min prior to analysis
by SDS-PAGE (10% acrylamide). Mutant 1-332 and wild-type protein
are indicated by the arrow and arrowhead,
respectively.
In the present study, we have directly analyzed the
structural basis for the regulation of CaM kinase I by analyzing the
activity and Ca/CaM-binding of a series of
COOH-terminal truncated mutants. Analysis of the binding of
Ca
/CaM to the mutants indicated that the
Ca
/CaM-binding domain of the kinase is located
between residues 294-321. This conclusion was supported by the
demonstration that a synthetic peptide encompassing these residues
competed with Ca
/CaM in the activation of either CaM
kinase I or smMLCK. Deletion of residues 322-332 reduced the
binding of Ca
/CaM as measured by
I-CaM
overlay, but had little effect on binding of Ca
/CaM
as determined by affinity chromatography on CaM-Sepharose or activity
measurements. Truncation of residues T
AVVR
had the greatest effect on binding of Ca
/CaM,
particularly as measured by CaM-Sepharose chromatography. Further
truncation of residues K
SKWKQ
completely
abolished binding of Ca
/CaM. Notably, analysis of
binding of Ca
/CaM by CaM overlay, CaM-Sepharose
chromatography and activation of kinase activity indicated that there
is not an absolute correlation between all measurements of binding of
Ca
/CaM and stimulation of kinase activity.
The
Ca/CaM-binding domains of numerous proteins have been
shown to have a propensity for the formation of amphipathic
-helices (O'Neil and DeGrado, 1990). Helical wheel analysis
of residues 299-316 indicated that these residues of CaM kinase I
are predicted to contain a hydrophobic domain and a basic, hydrophilic
domain (Fig. 6B). In addition, analysis of residues
303-316 by the method of Chou and Fasman(1978) predicted an
-helical conformation (data not shown). Direct structural analysis
of the binding of Ca
/CaM to synthetic peptides
encompassing Ca
/CaM-binding domains have indicated
several common features that are critical for high affinity binding
(Ikura et al., 1992; Meador et al., 1992, 1993) (Fig. 6A). Upon binding of Ca
to the
four binding sites in CaM, the two lobes of the protein fold around the
helical structure making contact between the NH
-terminal
domain of Ca
/CaM and the COOH-terminal part of the
peptide, and between the COOH-terminal domain of
Ca
/CaM and the NH
-terminal part of the
peptide. Two hydrophobic amino acids, 8-12 residues apart
(Trp
and Phe
in skMLCK), are necessary to
bind to hydrophobic pockets in Ca
/CaM, although
extensive contacts between many other hydrophobic residues (Phe
and Val
in skMLCK) are apparent. In addition,
specific basic residues (Arg
in smMLCK) are believed to
act as ``fulcrums'' to initiate the interaction between the
central helix of Ca
/CaM and the target enzymes
(Meador et al., 1992). The variability in the exact location
of the principal hydrophobic amino acids in the different structure is
tolerated by the fact that the central helix of
Ca
/CaM is highly flexible and extends to accommodate
the particular binding peptide (Meador et al., 1993).
Figure 6:
Comparison of
Ca/CaM-binding domains and
-helical wheel
analysis. A, amino acid sequences of
Ca
/CaM-binding domains were compared.
Trp
, Phe
, Val
, and
Phe
of skMLCK as well as Arg
of smMLCK are
believed to be important for binding to Ca
/CaM
(positions indicated by asterisks). Conserved amino acid
residues of CaM kinase I (residues 294-321) and other
Ca
/CaM-binding domains were aligned with these key
positions. Amino acid sequences known to be directly involved in
binding Ca
/CaM with structural studies (Meador et
al., 1992, 1993; Ikura et al., 1992) are underlined. Ca
/CaM-binding domains: skMLCK
(Ikura et al., 1992), smMLCK (Meador et al., 1992),
CaM kinase II (Meador et al., 1993), CaM kinase IV (Sikera et al., 1987), calcineurin (Kincaid et al., 1988),
plasma membrane Ca
pump (Kataoka et al.,
1991), and constitutive nitric oxide synthase (cNOS) (Zhang et
al., 1994). B, the amino acid residues 299-316 of
CaM kinase I were plotted as an
-helical wheel. The diagram
represents an end-on view of this region being in a right-handed
-helical conformation viewed from the NH
end. A basic,
hydrophilic face and a hydrophobic face are underlined with a
corresponding note.
Comparison of the proposed Ca/CaM-binding domain
of CaM kinase I with that of other Ca
/CaM-binding
proteins indicated several conserved features (Fig. 6A). Residues T
AVV
of
CaM Kinase I are likely to represent the COOH-terminal end of the
proposed Ca
/CaM-binding domain. These residues are
essential for binding to Ca
/CaM and are predicted
from the method of Kyte and Doolittle(1982) to be the most hydrophobic
region of the proposed Ca
/CaM-binding domain.
Residues K
SKWKQ
are also critical for
binding Ca
/CaM and likely represent the
NH
-terminal end of the Ca
/CaM-binding
domain. Notably, Trp
of CaM kinase I aligns with
conserved tryptophan residues in several other
Ca
/CaM-binding proteins (Fig. 6A).
Thus, the results from the present study, together with the results
obtained from the crystallographic analyses (Meador et al.,
1993), suggest that the Ca
/CaM-binding domain of CaM
kinase I contains at least residues 302-314. However, the results
from the CaM binding and activity studies (Fig. 2, A and C) suggest that the presence of some part or all of
residues 315-321 (HMRKLQL) contribute to binding of CaM, possibly
by stabilization of the structure of the CaM-binding domain. In
addition, the precise NH
-terminal limit of the CaM-binding
domain cannot be assigned based on the present experimental results.
Full-length wild-type CaM kinase I is completely dependent on
Ca/CaM for activity. Deletion of residues
294-332 resulted in a fully active enzyme. In contrast, a
slightly longer CaM kinase I mutant (residues 1-299) was basally
inactive and could not be activated by addition of
Ca
/CaM. In addition, the constitutively active kinase
was inhibited by a synthetic peptide corresponding to residues
294-321 in a manner competitive with respect to peptide
substrate. Together, these results indicate that an autoinhibitory
domain exists within residues 294-321 and that the presence of
residues 294-299 is sufficient to inactivate CaM kinase I
catalytic activity.
The elucidation of the molecular basis for the
regulation of protein kinases has been the subject of a number of
recent studies (Knighton et al., 1991, 1992; Cruzalegui et
al., 1992, 1993; Ito et al., 1991; Fitzsimons et
al., 1992). The structure of the catalytic subunit of PKA in a
complex with the high affinity pseudosubstrate inhibitor
(PKI(5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24) )
has been determined by x-ray crystallography (Knighton et al.,
1991, 1992). Arg in PKI (the P(-3) position)
interacts with several carboxyl side chains of the enzyme in a manner
that places a non-phosphorylatable alanine residue in the place of the
normally phosphorylated serine or threonine residue at the P(0)
position (Fig. 7). Furthermore, a phenylalanine residue
(Phe
) that interacts with a hydrophobic pocket formed by
residues Y
PPHH
of PKA is critical for the
high affinity of the inhibitor.
Figure 7: Comparison of amino acid sequences of CaM kinase I (residues 294-321), the inhibitor peptide of PKA (PKI (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24) ), and peptide substrates for CaM kinase I. The amino acid sequences surrounding the phosphorylation sites of site 1 of synapsin I (Czernik et al., 1987), the cyclic AMP-response element-binding protein (CREB) (Sheng et al., 1991), and Syntide-2 were aligned with that of PKI(5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24) . P(0) represents the position in PKI(5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24) that is occupied by a phosphorylatable amino acid residue in a substrate for PKA (Knighton et al., 1991). P(-11), P(-5), P(-3), and P(+4) are the positions relative to P(0). Conserved amino acid residues at these positions are boxed. Three alternative comparisons of CaM kinase I (residues 286-321) are shown in which potentially important basic residues (at P(-3)) and hydrophobic residues (at P(-11),(-5), and (+4)) are aligned.
The best characterized substrates
for CaM kinase I are the members of the synapsin family. Synapsin I and
II are both phosphorylated at NH-terminal serine residues (Fig. 7) that are also phosphorylated by PKA (Czernik et
al., 1987). The substrate specificity of CaM kinase I has recently
been investigated and compared with that of PKA using a series of
peptide substrates based on the sequence of site 1 of synapsin I (Lee et al., 1994). These studies indicate that the second of three
consecutive arginine residues corresponds to the basic amino acid
residue found in substrates for PKA at the P(-3) position (Fig. 7). In addition, the studies by Lee et al. indicated that hydrophobic amino acids are required at the
P(-5) and P(+4) positions (Fig. 7). With the
exception of the synapsins, other physiological substrates for CaM
kinase I remain to be identified. In the present study, we have found
that Syntide-2 is an effective substrate for CaM kinase I. In addition,
we have found that a peptide (LSRRPSYRKILNDL) corresponding to the
amino acid sequence surrounding the PKA site phosphorylated in the
cAMP-response element-binding protein (CREB) is also a very good
substrate for CaM kinase I.
Comparison of the amino acid
sequence of residues 294-321 of CaM kinase I with that of
PKI(5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24) and
the phosphorylation sites of synapsin I, syntide-2, and CREB (Fig. 7) revealed several common features that support the
possibility that the autoinhibitory domain of CaM kinase I interacts
with the catalytic domain through a pseudosubstrate mechanism. Three
alignments of the autoinhibitory domain that support a pseudosubstrate
mechanism were possible. In the first (which required insertion of two
gaps in the sequence), Lys of CaM kinase I at the
P(-3) position would place Trp
and Ala
at the P(-5) and P(+4) positions, respectively. These
two positions would correspond to the critical hydrophobic amino acids
present in each of the known substrates for the enzyme; however,
alanine is not normally considered a hydrophobic residue. In this
alignment, Phe
would replace the phosphorylatable residue
at P(0). Notably, in this alignment Phe
is placed at the
P(-11) position in an analogous position to that of the critical
phenylalanine found in PKI (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24) .
In PKI, Phe
interacts with the hydrophobic domain
Y
PPFF
in PKA catalytic subunit (Knighton et al., 1991). By analogy, Phe
may interact with
the corresponding hydrophobic domain Y
PPFY
in CaM kinase I. However, this model is weakened by the fact that
the alignment required insertion of gaps, the presence of an alanine
residue at the P(+4) position, and the fact that the
pseudosubstrate domain would correspond to
W
KQAFNATA
, which is not supported by the
observation that residues I
KKNFA
are
sufficient to maintain the enzyme inactive form. An alternative
alignment that maintains the necessary features of the pseudosubstrate
places residues I
KKNFAKSKW
in the
P(-5) to P(+4) positions. Important features of this
alignment are the lack of insertions of gaps in the sequence, the
presence of alanine instead of phenylalanine at the P(0) position, and
the excellent alignment of the hydrophobic amino acids at P(-5)
and P(+4) positions. Finally, a third alignment that also
maintains all the necessary features of a pseudosubstrate places
Phe
and Phe
in the P(-5) and
P(+4) positions. In either alignment two or three, elements of the
CaM-binding domain overlap the proposed pseudosubstrate domains,
suggesting that while distinct regions of the regulatory domain of CaM
kinase I are primarily responsible for mediating autoinhibition and
CaM-binding, the two domains are closely associated and probably
overlap.
Comparison of the results obtained in this study with that
of other CaM kinases reveals a number of similarities, as well as a
number of differences. There is general agreement concerning the
position of the Ca/CaM-binding domain of the various
enzymes (Fig. 6A), although the exact features of the
mechanism of autoinhibition vary. A number of studies of MLCK,
including detailed molecular modeling (Ito et al., 1991;
Knighton et al., 1992), have suggested that this enzyme may be
regulated by a strict pseudosubstrate mechanism. However, other studies
using site-directed mutagenesis and/or preparations of truncated MLCK
mutants have suggested that the original pseudosubstrate model requires
modification (Fitzsimons et al., 1992; Yano et al.,
1993). Recently, the crystal structure of a fragment of twitchin
kinase, which shares a high level of identity with smMLCK, has been
elucidated (Hu et al., 1994). While the structure clearly
establishes the regulation of twitchin kinase by an intrasteric
autoinhibitory mechanism, the lack of amino acid sequence identity of
the regulatory domain of twitchin kinase with MLCK and the absence of
information from site-directed and deletion mutagenesis make it
difficult to evaluate the existence of a strict pseudosubstrate
mechanism. Studies of CaM kinase II (Cruzalegui et al., 1992)
and CaM kinase IV (Cruzalegui et al., 1993) have also
suggested regulation by an autoinhibitory mechanism. In the case of CaM
kinase II, the presence of residues K
KFN
(Fig. 6A) was, like CaM kinase I, sufficient to
maintain the enzyme in an inactive state. However, replacement of KKFN
by AAAL had little effect on Ca
/CaM-dependent
activation of the enzyme, although deletion of the amino acids
generated a partially Ca
/CaM-independent kinase.
Based on these results, it was therefore concluded that autoinhibition
of CaM kinase II required determinants in addition to residues
291-294. In the case of CaM kinase IV, truncation at Leu
(K
KL
, Fig. 6A)
generated a fully active kinase that did not require
Ca
/CaM. Notably, truncation of CaM kinase I, II, and
IV at an analogous hydrophobic residue (Fig. 6A)
resulted in an active enzyme; however, the position of the truncation
placed a pair of lysine residues either as part (CaM kinase I and II)
of the autoinhibitory domain or not (CaM kinase IV). These results,
together with the observations that the substrate specificity of CaM
kinase I depends on hydrophobic amino acids, suggest that at least in
the case of CaM kinase I, the mechanism of autoinhibition is highly
dependent on hydrophobic amino acids.
As discussed above, there was
not an absolute correlation between binding of Ca/CaM
and stimulation of kinase activity. These results suggest, therefore,
that although Ca
/CaM may bind efficiently to a
limited domain in the regulatory domain of the kinase, the complete
regulatory domain is necessary (including COOH-terminal amino acid
residues) to allow the removal of the autoinhibitory domain from the
active site of the enzyme upon binding of Ca
/CaM. The
models presented above for the position of the proposed pseudosubstrate
domain also raise additional questions concerning the exact role of the
binding of Ca
/CaM in the activation of the kinase. In
the case of the second or third models, Ca
/CaM would
be predicted to bind directly to the pseudosubstrate domain and release
it from the active site of the kinase. In contrast, in the first model
binding of Ca
/CaM would be accompanied by both
removal of the pseudosubstrate domain from the active site and an
alteration in the interaction of residues 294-299 with the
catalytic domain of the kinase.
In summary, these various studies suggest that the CaM kinases are not all regulated by exactly the same structural mechanism. CaM kinase I appears to be the smallest of the autoinhibited kinases, shows an overlapping substrate specificity with PKA, and appears to be regulated by a pseudosubstrate mechanism that shares more features with PKA than the other CaM kinases. In contrast, the autoinhibitory mechanism used by MLCK and CaM kinase II may have evolved from the pseudosubstrate mechanism, perhaps as a reflection of the changes in substrate specificity of these enzymes. The present studies, together with future elucidation of the structure of CaM kinase I and other CaM kinases, should help to resolve these issues.