From the Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710
Received for publication, October 17, 2002, and in revised form, December 23, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The multifunctional
calcium/calmodulin-dependent protein kinases I and IV
(CaMKI and CaMKIV) are closely related by primary sequence and
predicted to have similar substrate specificities based on peptide
studies. We identified a fragment of p300-(1-117) that is a
substrate of both kinases, and through both mutagenesis and Edman
phosphate (32P) release sequencing, established
that CaMKI and CaMKIV phosphorylate completely different sites.
The CaMKI site, Ser89
(84LLRSGSSPNL93), fits the
expected consensus whereas the CaMKIV site, Ser24
(19SSPALSASAS28), is novel. To
compare kinase substrate preferences more generally, we employed a
proteomic display technique that allowed comparison of complex cell
extracts phosphorylated by each kinase in a rapid in vitro
assay, thereby demonstrating substrate preferences that overlapped but
were clearly distinct. To validate this approach, one of the proteins
labeled in this assay was identified by microsequencing as HSP25,
purified as a recombinant protein, and examined as a substrate for both
CaMKI and CaMKIV. Again, CaMKI and CaMKIV were different, this time in
kinetics and stoichiometry of the phosphorylation sites, with CaMKI
preferring Ser15
(10LLRTPSWGPF19) to
Ser85 (80LNRQLSSGVS89)
3:1, but CaMKIV phosphorylating the two sites equally. These differences in substrate specificities emphasize the need to consider these protein kinases independently despite their close homology.
The calcium (Ca2+) receptor calmodulin
(CaM)1 translates changes in
intracellular calcium concentration to changes in biochemical functions
of a variety of proteins that participate in a wide range of cellular
processes, including transcription, cell cycle regulation, and
differentiation (1). Ca2+/CaM binds target enzymes to
elicit alterations in their confirmations and activities (2). Among the
Ca2+/CaM-regulated enzymes, the multifunctional
Ca2+/calmodulin-dependent protein kinases
(CaMKs) occupy positions of influence because they communicate the
Ca2+ signal via phosphorylation to a broad range of
substrates. Within this kinase family, CaMKII clearly has distinct
regulation and function, whereas CaMKI and CaMKIV are more difficult to
discriminate as they share substantial sequence homology and behave
similarly in many biochemical analyses. Nevertheless, there are many
indications that at a physiological level their functions are nonredundant.
Differences in CaMKI and CaMKIV localization probably play important
roles in the regulation of these kinases. Although CaMKI is a
ubiquitously expressed, primarily cytoplasmic enzyme, CaMKIV is found
in the nuclei of only a select group of cells including cells in the
thymus, brain, testes, and ovaries. However, these expression and
subcellular localization differences are not sufficient to account
either for the distinct behaviors of CaMKs I and IV in vitro
or for their lack of redundancy in vivo. All cells that express CaMKIV also contain CaMKI, and CaMKI is capable of nuclear entry in some contexts (3). The only identified Caenorhabditis elegans CaMKI/IV homologue, ceCaMKI, further blurs this
distinction. Although this enzyme is more highly homologous to CaMKI by
primary sequence, like CaMKIV it is nuclear and only expressed in a
limited set of cells (4, 5). As we sought to clarify the ambiguity of
the homology of ceCaMKI, we observed biochemical differences between
CaMKI and CaMKIV that led us to evaluate more thoroughly the substrate
preferences of the two mammalian enzymes.
The similarities between CaMKI and CaMKIV relative to CaMKII extend to
substrate preferences to the degrees that they have been characterized.
All known CaMKI substrates (but not all CaMKII substrates) can be
phosphorylated by CaMKIV. For example, CREB Ser133 can be
phosphorylated by CaMKs I, IV, and II, while only the later kinase also
phosphorylates Ser142 (6); C/EBP Few studies compare the catalytic activities of CaMKI and CaMKIV toward
substrates other than short synthetic peptides. Whereas the
peptide-derived consensus sequences appear to reflect the behavior of
the kinases toward the few known protein substrates, their limited
predictive value is demonstrated in our analysis of the phosphorylation
by CaMKIV of an N-terminal fragment (1-117) of the transcriptional
cointegrator, p300. N-terminal fragments of p300 have previously been
shown to be in vitro substrates for PKC, AMP kinase, and
CaMKI (13-15), and here we show that it is also a CaMKIV substrate.
Among the 20 serine and threonine residues in the first 117 amino
acids, a single serine, Ser89, emerges as a likely
candidate phosphoacceptor site based on CaMKI/IV consensus comparisons.
Indeed, mutation of Ser89 to Ala abolished the ability of
CaMKI to phosphorylate the protein. However, despite the overlapping
consensus determinants of CaMKI and CaMKIV, the mutant remained a
robust substrate of CaMKIV. This observation, and the repeated failure
of efforts to identify new substrates through degenerate
consensus-directed searching, indicate that determinants must exist
beyond the known consensus, either hidden within the primary sequence
or in the higher level folded structure. We undertook further
investigations of CaMKI and CaMKIV protein substrate preferences to try
to define their degree of overlap and to develop methods for
identifying new substrates.
Here we compare the preferences of the mammalian kinases CaMKI (human
Recombinant DNA and Protein Expression--
Vectors for
production of human glutathione S-transferase-CaMKI and rat
glutathione S-transferase-CaMKIV and myelin basic protein-CaMKK
CeCaMKI cDNA was independently isolated from mixed stage
C. elegans RNA by reverse transcriptase-PCR,
sequenced on both strands, and subcloned into
pGEX-2T,2 and recombinant
protein was prepared according to the vector protocols from the manufacturers.
The vector pQE9-HSP25 was the gift of L. Cooper (20).
His6-HSP25 was prepared under denaturing conditions as
previously described, and renatured by dialysis (3500 Da MWCO) at
4 °C against gradually decreasing concentrations of urea in 50 mM Tris, 125 mM NaCl, 0.5 mM
dithiothreitol, pH 7.5. The dialyzed protein was concentrated ~8-fold
with Centricon centrifugal filter units (10 kDa MWCO, Millipore) and
quantified by standard methods.
Standard Kinase Assays--
Kinase reactions were carried out at
30 °C in 50 mM HEPES, pH 7.5, 1 mM
dithiothreitol, 0.1% Tween 20, and 10 mM
MgCl2. Unless otherwise specified, 50 nM
kinase, 200 µM ATP, 1 mM CaCl2
and 1 µM CaM were incubated for 5 min with 2 µM recombinant substrate. For calcium-free reactions, 1 mM EGTA was included in place of CaCl2. In
activation assays, the kinase and 50 nM kinase kinase (or
mock controls) were preincubated in the reaction buffer 20 min before
the addition of [ Determination of Phosphorylation Sites--
Recombinant,
His6-tagged proteins were labeled for analysis of
phosphorylation sites using 50 µM kinase and 20 µM substrate incubated for 30 min at 30 °C, with 400 µM ATP and specific activities of 5000-10000 cpm/pmol.
To purify the substrates away from the kinases, the reaction was
diluted 1:1 with 20 mM Tris, pH 7.5, containing 20 µl of
packed Ni2+-chelating resin, and allowed to bind for 60 min
at 4 °C. The resin was washed in batch with 2× 200 µl of 20 mM Tris, 0.5 M NaCl, 5 mM imidazole
and then with 2× 200 µl of 20 mM Tris, 10 mM
imidazole. Protein was eluted in 2× 50 µl of 100 mM
EDTA, which was diluted 1:1 with 20 mM Tris, pH 7.5, and
run over a BioSpin-6 column (Bio-Rad) to exchange buffers and remove
any remaining unincorporated ATP. Labeled proteins were digested with
endo-Lys-C, cyanogen bromide, or trypsin (as indicated), and acidified
by the addition of trifluoroacetic acid. In some cases, the peptide mixture was bound directly to Immobilon membrane, and the site of
phosphorylation determined by Edman sequencing with up to 20 cycles of
pulsed liquid chemistry to determine 32P released (23, 24).
In other cases (as indicated), the resulting peptide mixture was
resolved on a reverse phase column (Zorbax SB-C18, 4.6 × 250 mm)
by a linear acetonitrile gradient in 0.1% trifluoroacetic acid. Peak
fractions of radioactivity were analyzed on Immobilon membrane by Edman
cycle 32P release. In some cases, a portion of the
phosphopeptide sample was analyzed by TOFNEG mass spectroscopy using a
precursor ion scan in negative mode to identify parent ions that
liberated the characteristic 78.997 m/z phosphate
ion. TOFPOS was then used to measure the masses of peptide fragments
produced by collision-induced dissociation (MS/MS), and predict the
primary sequence of the unknown phosphopeptide.
MEF Extract Assays--
The mouse embryonic fibroblasts were the
generous gift of Christina Kahl, who had derived them from wild type
C57/B6 mice as previously reported (25). The cells were stored at
passage 2 under liquid nitrogen until thawing and expansion by standard methods in Dulbecco's modified Eagle's medium supplemented to 10%
with fetal bovine serum. All cells were collected by trypsinization and
centrifugation at or before passage 6. After two washes with Hanks'
saline solution, cell pellets were flash frozen and stored at
MEF extract was prepared by resuspending ~2 × 108
previously frozen MEF cells in extract lysis buffer (20 mM
Tris, 0.1 M NaCl, 1 mM EDTA, 1 mM
dithiothreitol, pH 8.0, with protease inhibitors: 200 µg/ml
Pefablock, 1 µg/ml pepstatin A, 1 µg/ml leupeptin, 2 µg/ml
aprotinin), sonication, and addition of Triton X-100 to 0.5%. After
rocking at 4 °C for 2 h, extracts were diluted 5-fold with
lysis buffer bringing the Triton X-100 concentration to 0.1%. Following clarification by centrifugation at 15,000 × g, the extract was filtered (0.45 µM),
aliquoted, and flash frozen for storage at
For phosphorylation of cell extracts, reactions were carried out at
30 °C in reaction buffer (50 mM Tris, pH 7.5 with 1 mM dithiothreitol and 0.1% Triton X-100). The indicated
kinases, with or without kinase kinase, each at 2 µM (for
0.5 µM final), were preincubated 20 min in 50 µl of
reaction buffer supplemented with 20 mM MgCl2,
5 mM CaCl2, 100 µM ATP, and 8 µM purified bovine CaM. This mixture was diluted to 200 µl by the addition of 50 µCi of [ Substrate Identification--
Samples for mass spectrometry
could be recovered directly from wet silver-stained gels using well
stained, radioactive extract protein landmarks for alignment. Samples
from up to eight duplicate gels or membranes were pooled to increase
sample quantity. Gel fragments were treated in series with 50 mM ammonium bicarbonate, 100 mM sodium
hyposulfate, and 30 mM potassium ferrocyanide for 15 min
per solution. The gel slice was then soaked in 50 mM
ammonium bicarbonate for 5-min washes until transparent, then incubated in trypsin overnight at 37 °C. Resulting peptides were extracted with several washes of 5% formic acid, 60% acetonitrile, and
subjected to electrospray mass spectrometry on a QSTAR-Pulsar mass
spectrometer (Applied Biosystems). TOFPOS was used to measure the
peptide fingerprint, and then to determine primary sequence of specific
peptides by collision-induced dissociation (MS/MS). Matches were made
using the FASTS program to search public data bases.
p300-(1-117) Is Differentially Phosphorylated by the
Multifunctional CaMKs--
The ability of hsCaMKI to phosphorylate the
N-terminal 1-117-amino acid fragment of p300 was previously reported
(15), but the phosphorylation site was not identified. We characterized this substrate in more detail, to compare the multifunctional kinases
and further define their substrate requirements. First, purified
bacterial expressed, His6-tagged p300-(1-117) (referred to
as 117(wt), Fig. 1A) was used
as a substrate for recombinant ceCaMKI, hsCaMKI, and rnCaMKIV (Fig.
1B). 117(wt) was phosphorylated in vitro by all
three of these CaMKs, demonstrated by 32P incorporation.
Because 32P incorporation was dramatically enhanced by
preincubation of the kinases with recombinant rnCaMKK
Molar phosphate incorporation into 117(wt) was determined relative to
recombinant CREB in our assay conditions, to estimate stoichiometry of
117 phosphorylation by the kinases based on the well characterized CaMK
phosphorylation profile of CREB. By this method, CaMKI incorporated
0.84 ± 0.13 mol/mol of phosphate into 117(wt), whereas CaMKIV
incorporated 1.14 ± 0.16. These results, together with the S89A
observations, suggested that each kinase phosphorylated a single
separate site: Ser89 for CaMKI and an undetermined site for
CaMKIV.
To identify the CaMKIV site we analyzed the 117 primary sequence for
less stringent "consensus sites." Mutation of additional potential
phosphoacceptor sites (Ser19, -20, -82, and -90) in the
context of S89A still did not prevent phosphorylation by CaMKIV. Taking
a biochemical approach to site identification, 117(wt) was
32P-labeled by CaMKI and CaMKIV. Sites of phosphorylation
were identified by determining the cycles in which 32P was
released when 32P-labeled fractions of 117(wt) tryptic
digests were subjected to sequential Edman degradation under conditions
that optimized 32P recovery (24). Analysis of 117(wt)
phosphorylated by CaMKI confirmed Ser89 as the target site
(Fig. 1C, upper). In contrast, phosphorylation by
CaMKIV was consistent only with incorporation into Ser24,
even when considering partial digestion products (Fig. 1C,
lower). Mutation of Ser24 to Ala, 117(S24A), had
no effect on phosphorylation by CaMKI, but it dramatically reduced
phosphorylation by CaMKIV, confirming the biochemical results (Fig.
1B). This site had not been among the candidate sites
identified; it contained none of the amino acid determinants normally
associated with CaMK phosphorylation. This fragment of p300, therefore,
contains two atypical CaMK sites; Ser89 is the only known
site phosphorylated by CaMKI but not by CaMKIV, whereas
Ser24 is a nonconsensus CaMKIV site.
CaMKI and CaMKIV Phosphorylate Distinct Subsets of Proteins in
Complex Mixtures--
To evaluate more thoroughly the degree to which
CaMKI and CaMKIV protein substrate sets are coincident, an assay was
developed to visualize in vitro substrate proteomes within
complex cell extracts generated from MEF or mixed stage N2
C. elegans. In MEF extract, the patterns observed with
activated ceCaMKI and hsCaMKI were remarkably similar, although the
ceCaMKI pattern was fainter, and they were quite different from that of
rnCaMKIV (Fig. 2). In particular, a group
of high molecular weight acidic proteins was phosphorylated exclusively
by ceCaMKI and hsCaMKI. In contrast, activated rnCaMKIV phosphorylated
several low molecular weight basic proteins that were only weak CaMKI
targets. Less dramatic results were obtained when performing the assay
with nematode extract (Fig. 2). A few proteins were phosphorylated by
mammalian CaMKI and not by CaMKIV (most easily seen in the acidic, low
Mr region), and vice versa (particularly in the
middle of the gels). Interestingly, several proteins were
phosphorylated by both mammalian kinases but not by ceCaMKI. In both
extract types, the pattern of proteins phosphorylated by the downstream
kinases alone, hsCaMKI, ceCaMKI, and rnCaMKIV, without the addition of
rnCaMKK HSP25 Is a CaMK Substrate in MEF Extracts--
The identity of one
low molecular weight protein phosphorylated by the CaMKs, and favored
by CaMKIV, was assigned by mass spectrometry. The two-dimensional gel
spot (Fig. 3A) was excised, digested with trypsin, and the primary sequence was obtained by analysis of the collision-induced dissociation spectra. The peptide, AVTQSAEITIPVTFEAR (Fig. 3B), was determined through FASTS
data base alignment comparison (27) to be an exact match for the mouse
25-kDa heat shock protein (mmHSP25). The molecular mass (25 kDa)
and pI values (5.7, 5.9) previously observed for the related rat HSP27
(28) agree with the position of the unknown substrate on the
two-dimensional gel.
To confirm that the assays we developed in fact reflected patterns of
substrate preference, phosphorylation of recombinant His6-mmHSP25 by hsCaMKI and rnCaMKIV was analyzed. Both
CaMKs could phosphorylate the protein efficiently. The apparent
Km for phosphorylation by CaMKI was 36 ± 4 µM and by CaMKIV was 45 ± 5 µM (Fig.
4B). The observed
kcat for CaMKI was twice that for CaMKIV, and
the stoichiometry was low even after 2 h; incorporation reached
44% with CaMKIV and 27% with CaMKI (data not shown). Radiation release by Edman degradation of trypsin-digested, labeled HSP25 indicated that the major site(s) of phosphate incorporation for both
kinases was three residues C-terminal to a basic residue, whereas radiation released from CNBr-digested peptides indicated that
Ser15 was at least one site (data not shown). Reverse phase
high performance liquid chromatography purification of CaMKI or CaMKIV
32P-labeled tryptic peptides from HSP25 revealed two peaks
(Fig. 4C), and MS/MS analysis of these peptides identified
the specific sites of phosphate incorporation as Ser15 and
Ser85 (data not shown). Correlating the peptide
identification with radioactive incorporation by quantitative high
performance liquid chromatography, it can be seen that two sites are
phosphorylated equally well by CaMKIV, but that CaMKI preferentially
phosphorylated Ser15 over Ser85 in a 3:1 ratio
(Fig. 4C). These experiments confirm that HSP25 is an
in vitro CaMK substrate, and identify two new sites that are differentially phosphorylated by CaMKI and CaMKIV.
We have compared kinase specificity profiles of mammalian CaMKI
and CaMKIV both by identification of specific sites in individual proteins and by evaluating the set of proteins in a homogenized cell
extract that can be in vitro substrates for a particular kinase. These approaches are complimentary, and they lead to the same
overall conclusion, the substrates preferred by CaMKI and CaMKIV are
different, and do not always follow the peptide-derived consensus sequences.
In the extract analyses, unlike combinatorial peptide library screens
that restrict one to iterating from defined sequence parameters, the
kinases are presented with native cellular proteins. Reaction
conditions were designed to provide the best possible definition of the
substrate preference within the given extract mixture. They allowed
reactions to be performed in solution using high concentrations of
exogenous kinase and short reaction times to approximate initial rate
conditions and thereby minimize bias toward detection of abundant
substrates. Importantly, these assays were not designed to identify
solely physiologic substrates. Whereas the proteins phosphorylated
should represent the best rates of phosphorylation among the proteins
available to the kinase, the use of whole extract and exogenous kinase
makes it impossible to attribute physiologic relevance to any
particular phosphorylation in this assay. Because local concentrations
of kinase and substrate in a cell are likely to be regulated, and vary
widely from the total cellular concentrations, the relevance of a
phosphorylation cannot be determined by in vitro kinetics
alone. Nonetheless, the comparison between different kinases allows
some prediction as to how the kinases might behave given the same
opportunity, and may narrow the field of candidate kinases responsible
for a given physiologic phosphorylation event. Furthermore, by
comparing the pattern similarities and differences between substrate
sets in whole proteomes arrayed in two dimensions, we assess the degree to which the catalytic functions of these enzymes are interchangeable. For example, if CaMKI and CaMKIV exhibited nearly identical substrate preferences, differences in function could be wholly attributed to
differences in localization and regulation by other pathways.
Using autoradiograms aligned with the Melanie software package based on
landmark phosphoproteins present in the extract control, the patterns
for CaMKK Given the highly degenerate definition of substrate consensus sequence
currently available, it is not surprising that some proteins with the
"required" minimal characteristics are not CaMK substrates as
predicted, and the patterns of substrate choice are not simply subsets
of each other. p300-(1-117) is one of those proteins. Among its many
serines and threonines, Ser89 meets the consensus
requirements for phosphorylation by any of the CaMKs, but while clearly
a CaMKI site for both C. elegans and human CaMKI homologues,
it is not the predominant CaMKIV site. In agreement with these results,
another p300 fragment, p300-(74-163), was not phosphorylated by CaMKIV
in vitro, although it was a PKC substrate (13). The CaMKIV
site we have identified in p300, Ser24, lacks any of the
determinants found in previous studies. It is sufficiently different
from peptides used as starting points that it could represent a
completely different type of substrate, with different primary sequence
determinants, not likely to be identified through systematic variation
of a known sequence. We also considered but ruled out tertiary
structure as a determinant to explain this difference from previous
peptide data as denaturing the 117(wt) by boiling had no apparent
effects on phosphorylation. Interestingly, both CaMKI and CaMKIV
stimulate transcription driven by wt, S89A, or S24A forms of a
Gal4-p117 fusion protein in transfection assays.3 Experiments with
p300 in cultured cells have demonstrated
phospho-Ser89-dependent repression of
transcription induced by activation of PKC (13), whereas CaMKIV
stimulation of the related cointegrator CBP has been demonstrated
through phosphorylation of Ser301 (29). Ser301
is not conserved in p300, but both Ser89 and
Ser24 appear to be conserved in CBP, albeit with
substantial sequence differences C-terminal to the sites, yet neither
was identified as a target in the CBP experiments. Such differences in
phosphorylation sites may help explain differences between p300 and
CBP, as well as between CaMKI and CaMKIV effects, but much work remains
to sort out potential biological relevance.
HSP25, identified directly from our proteomic approach as a CaMK
substrate, follows the peptide consensus sequences more closely. Of the
two phosphorylation sites identified, Ser15 most closely
resembles the CaMKI-phosphorylated synapsin site-1 peptide sequence
with a hydrophobic residue at P+4, and it is, in fact, preferred by
CaMKI. CaMKIV is able to phosphorylate both sites, which would be
predicted based on reported preferences. Interestingly, the primary
sequence context surrounding Thr146 would indicate that it
is equally suited to be a CaMKIV phosphorylation site, but is not
phosphorylated to a significant degree by either of these CaMKs.
Because both tested CaMKs were capable of phosphorylating recombinant
His-HSP25 in vitro, we analyzed phosphorylation kinetics in
more detail to determine the source of differences observed in the
initial extract phosphorylation. The Km for initial rates with CaMKI and CaMKIV were essentially identical in
vitro, but kcat of CaMKI was almost twice
that of CaMKIV for this substrate. Based on these kinetics alone, CaMKI
would be predicted to outpace CaMKIV in our short term extract
phosphorylation assay. However, CaMKI preferred Ser15
whereas CaMKIV phosphorylated both sites equally, and in a 30-min assay
phosphorylation by CaMKIV outpaced CaMKI. This could result from
incorporation into a slow phosphoacceptor site or from changes in
oligomerization of HSP25 known to follow phosphorylation. In this
light, differences between tissue-purified and recombinant HSP25, such
as post-translational modification and pre-formed oligomerization, may
be important to selection as a CaMK substrate and could result in
differential phosphorylation.
Although our methods do not necessarily indicate a physiological
substrate relationship, they do not exclude this possibility. The small
HSPs form complex quaternary structures that are regulated at least in
part by phosphorylation, and can act as chaperones, regulate actin
dynamics, and block apoptosis (30). Because they respond to a variety
of extracellular signals, many kinases may be capable of regulating
their function. Several have already been implicated, including
mitogen-activated protein kinase AP-2 (31-33) and
p38-regulated/activated protein kinase (30). Interestingly, CaMKIV has been associated with regulation of cytoskeletal dynamics through phosphorylation of oncoprotein 18 (34), which correlated with
increased microtubule formation. Immunohistochemical studies of HSP25
reveal a limited developmentally regulated expression pattern,
potentially overlapping with CaMKIV expression in the cerebellum
(35-37), thymus, testis, and ovaries (38). Further exploration of
possible roles for CaMKIV in HSP25 regulation may provide new insight
into CaMK functions, but should include comparisons between CaMKI and
CaMKIV to determine whether the differences we have observed in
biochemical assays have any biological correlation.
More importantly than identifying specific substrates, our results
emphasize the distinction between CaMKI and CaMKIV substrate preferences. These differences have been downplayed in many papers examining calcium-dependent signaling, and, to the extent
that it has been addressed in biological systems, differences between CaMKI and CaMKIV have been attributed primarily to localization. Although many similarities exist, these kinases must be considered independently and should not be confused. Furthermore, given the close
relationship in both the primary sequences and activation requirements
of these kinases, discrete substrate preferences provide an opportunity
to explore substrate determinants that may improve our ability to
predict pathway targets.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Ser276 is a
CaMKII substrate but not a CaMKIV substrate (7, 8); CaMKI
phosphorylates site 1 of synapsin I, whereas CaMKII efficiently phosphorylates sites 2 and 3 (9), and CaMKIV can phosphorylate all
three (10). Assessment of CaMK activities toward a panel of site 1 peptide variants identified various primary sequence determinants in
the vicinity of the phosphoacceptor site (P = 0). CaMKII
phosphorylates efficiently only those peptides that incorporate a
nonbasic residue in the P
2 position and a hydrophobic residue in the
P+1 position (11), whereas CaMKI prefers hydrophobic residues at the
P+4 position (12). All three kinases favor a hydrophobic residue at
P
5 and a basic residue, preferably Arg, at P
3. Thus, although
kinetic differences between the kinases have been documented in these
peptide studies, this definition of substrate discrimination indicates
similar and highly degenerate consensus sequences, and implies that
CaMKIV will phosphorylate a superset of CaMKI targets.
isoform, hsCaMKI), CaMKIV (rat
isoform, rnCaMKIV), and the CaMK
homologue from C. elegans (ceCaMKI). Our first experiments identify a novel CaMKIV phosphorylation site in the N-terminal fragment
p300-(1-117). Recognizing that additional determinants of
phosphorylation could potentially include protein folding, binding
partners, or post-translational modifications of substrates, we examine
the spectrum of proteins phosphorylated by the kinases in complex cell
extracts using methodology similar to that described for
mitogen-activated protein kinases by Knebel et al.
(16). The application of this proteomic approach reveals dramatic
differences between the sets of substrates phosphorylated by CaMKs I
and IV, and supports the classification of ceCaMKI as a CaMKI-like
enzyme. Furthermore, applying microsequencing techniques in conjunction with proteomic display, we identify a novel and differentially phosphorylated CaMKI/IV substrate, HSP25, in mouse embryo fibroblast (MEF) extracts. Using purified recombinant HSP25, we characterize it as
a CaMK substrate both to identify sites and to validate our approach
for evaluating CaMK substrates in complex mixtures.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
were provided by S. Hook (17) and K. Anderson (18). Wild type, S89A, S19A/S20A/S89A, and S19A/S20A/S82A/S89A variations of pET30-p300-(1-117) were provided by C. Kane (15). Additional mutants were generated by PCR mutagenesis using
pET30-p300-(1-117) as template. The vector for production of
His6-CREB was the gift of M. Montminy (19). Recombinant
proteins were prepared using these vectors as previously described.
-32P]ATP and substrate. Specific
radioactivity was determined for each set of reactions, generally
falling between 500 and 2500 cpm/pmol of ATP. Assays were stopped by
either addition of Laemmli buffer and boiling (for reactions to be run
on SDS-PAGE, dried, and autoradiographed), or spotting on Whatman 3MM
paper and washing (for liquid scintillation quantification of counts
incorporated) (21). To determine phosphorylation stoichiometry, 2 µM recombinant substrate was incubated in standard
reaction buffer with 100 nM kinase and 500 µM
ATP (1000 cpm/pmol) for 60 min, and specific incorporation over
background was quantified using the 3MM filter assay described above.
Recombinant CREB, which has a stoichiometry of phosphorylation by
CaMKIV (1.0 (22)), was used to normalize stoichiometry results.
80 °C until use.
80 °C.
-32P]ATP
(~35,000 cpm/pmol ATP) and 200 µg of cell extract, starting the
reaction. The reaction was stopped by precipitation with 50 µl of
100% trichloroacetic acid (for two-dimensional electrophoresis), incubated on ice for 20 min, centrifuged 20 min at 14,000 × g, washed 3 times with ether, and air dried. The protein
pellet was resuspended directly in IEF sample buffer and frozen at
20 °C. Two-dimensional gels were run using a Bio-Rad Mini-IEF
apparatus, with pH 3 to 10 isoelectric focusing as the first dimension
using a standard mixture of ampholytes (0.8% 5/7 ampholyte, 0.8% 6/8 ampholyte, and 0.4% 3/10 ampholyte) between 100 mM NaOH
and 10 mM H3PO4 overnight at 300 volts. After an additional hour at 600 volts, gels were extruded from
their tubes, equilibrated in 4× Laemmli buffer, and run on 10%
SDS-PAGE for the second dimension, fixed, silver-stained, and exposed
to film.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
although
incubation with rnCaMKK
alone failed to incorporate 32P
into 117(wt), 117(wt) is clearly an activation-dependent
substrate of the CaMKs. However, only rnCaMKIV phosphorylation retarded the electrophoretic mobility of the protein (Fig. 1B).
Phosphorylation by any of the kinases did not seem to require any
specific secondary structure, because boiling the substrate for 5 min
prior to the kinase reaction did not affect the results of the assay
(data not shown). Thus, assuming that the amino acid sequence was the primary site determinant, Ser89 was identified the likely
phosphoacceptor by analogy to CaMK peptide substrate requirements (26);
the site includes hydrophobic residues at P
5 and P+4, and Arg at
P
3. Mutation to Ala89 eliminated detectable
phosphorylation by hsCaMKI and ceCaMKI but not by rnCaMKIV.
View larger version (37K):
[in a new window]
Fig. 1.
Phosphorylation of p300-(1-117) by CaMKs and
site identification. A, deduced amino acid sequence of
recombinant, His6-tagged p300-(1-117), with
Ser24 and Ser89 indicated by
asterisks, and other Ser mutated without effect on phosphate
incorporation indicated by underlines. B, autoradiogram of
SDS-PAGE following in vitro kinase reactions under standard
conditions with the indicated components. Phosphorylation of 117 is
markedly reduced by S89A mutation for activated CaMKI and by S24A
mutation for CaMKIV. C, 117 proteins labeled with CaMKI or
CaMKIV were purified by affinity resin, digested with trypsin, and used
for radiation release by Edman degradation to determine the
32P released at each cycle.
, were nearly identical to the extract alone. Thus,
phosphorylation of the recombinant kinases by endogenous activators did
not contribute substantially to the observed patterns. Likewise,
rnCaMKK
alone did not alter significantly the extract pattern.
Because the kinases were preincubated in unlabeled ATP for 20 min, it
was not surprising to find that the control gels had minimal signal,
although the protein was readily detected by silver staining.
View larger version (99K):
[in a new window]
Fig. 2.
Autoradiograms of CaMK reactions in MEF and
C. elegans N2 extracts. The origin
(high Mr, high pH) is indicated, with
arrows in the direction of decreasing pH and
Mr. Representative sets of matched gels are
shown with added kinases indicated.
View larger version (48K):
[in a new window]
Fig. 3.
Identification HSP25 as a CaMKIV substrate
from MEF extracts. A, enlargement of the region of the
aligned silver-stained gel and autoradiogram used to obtain the sample
for MS/MS. Circles indicate two independent samples that
were identified as HSP25. B, MS/MS sequencing of HSP25 from
gel fragments pooled from eight gels. Assignment of the b
and y series peptide ions is listed with the predicted
primary sequence. amu, atomic mass units.
View larger version (27K):
[in a new window]
Fig. 4.
Kinetics of HSP25 phosphorylation by
CaMKs. A, deduced amino acid sequence of recombinant
His6-tagged mouse HSP25 used to study the initial rate
kinetics. Underlined sequences correspond to the
phosphopeptides identified by MS/MS following high performance liquid
chromatography purification. Major consensus determinants are indicated
in bold, and phosphorylated Ser residues are indicated by
asterisks. B, phosphorylation by 0.5 µM CaMKs in 2-min reactions; curves are direct
Michaelis-Menten fits to the data. Apparent Km
values for HSP25 phosphorylation were 36 ± 4 and 45 ± 5 µM for CaMKI and CaMKIV, respectively. C, high
performance liquid chromatography purification of trypsin-digested
HSP25 phosphorylated by the indicated kinases and purified by affinity
resin before digestion. Two major peaks of radiation (labeled I and II)
are identifiable in all traces corresponding to the underlined peptides
in A that include Ser85 and Ser15,
respectively.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-activated CaMKI and CaMKIV were overlaid in the indicated
color channels onto extract alone to visualize similarities and
differences (Supplemental Materials). Clear global differences between
the sets of proteins phosphorylated by CaMKs I and IV are apparent,
which are particularly useful for examining the related kinases from
other organisms. Looking more closely at ceCaMKI, it phosphorylated a
similar set of proteins to hsCaMKI, with high specific incorporation
into a cluster of acidic, high molecular weight proteins. This is in
contrast to high incorporation into more basic, lower molecular weight
proteins of rnCaMKIV. Overlapping substrate sets were also observed,
including a prominent cluster in the range of 50 kDa, near the middle
of the IEF range. Thus, CaMKI and CaMKIV could be easily distinguished
by this approach, and the biochemical similarity between ceCaMKI and
mammalian CaMKI over CaMKIV was reinforced.
![]() |
FOOTNOTES |
---|
* This work was supported in part by a Natural Sciences and Engineering Research Council of Canada postdoctoral fellowship (to J. A. M.) and National Institutes of Health Grants GM33976-19 (to A. R. M.), HD07503-24 (to A. R. M.), and DK52378-06 (to T. A. J. H.).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.
The on-line version of this article (available at
http://www.jbc.org) contains Fig. 1.
Both authors contributed equally to the results of this article.
§ Present address: Dept. of Medicine, University of California, San Francisco, CA.
¶ Present address: Pharmacia Corporation, Chesterfield, MI.
To whom correspondence should be addressed: Dept. of
Pharmacology and Cancer Biology, Duke University Medical Center, P.O. Box 3813, Durham, NC 27710. Tel.: 919-681-6209; Fax: 919-681-7767; E-mail: means001@mc.duke.edu.
Published, JBC Papers in Press, January 21, 2003, DOI 10.1074/jbc.M210642200
2 E. E. Corcoran, unpublished data.
3 C. D. Kane, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
CaM, calmodulin;
CaMKI, calcium/calmodulin-dependent protein kinase I;
CaMKII, calcium/calmodulin-dependent protein kinase II;
CaMKIV, calcium/calmodulin-dependent protein kinase IV;
CaMKK, calcium/calmodulin-dependent protein kinase
kinase
;
ceCaMKK, C. elegans
calcium/calmodulin-dependent protein kinase kinase;
CREB, cAMP response element-binding protein;
HSP25, mouse 25-kDa heat shock
protein;
MEF, mouse embryonic fibroblast;
CBP, CREB-binding protein;
hsCaMKI, human
isoform;
rnCaMKIV, rat
isoform.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Means, A. R. (2000) in Principles of Molecular Regulation (Conn, P. M. , and Means, A. R., eds) , pp. 187-204, Humana Press, Totowa, NJ |
2. | Chin, D., and Means, A. R. (2000) Trends Cell Biol. 10, 322-328[CrossRef][Medline] [Order article via Infotrieve] |
3. | Ahmed, B. Y., Yamaguchi, F., Tsumura, T., Gotoh, T., Sugimoto, K., Tai, Y., Konishi, R., Kobayashi, R., and Tokuda, M. (2000) Neurosci. Lett. 290, 149-153[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Eto, K.,
Takahashi, N.,
Kimura, Y.,
Masuho, Y.,
Arai, K.,
Muramatsu, M. A.,
and Tokumitsu, H.
(1999)
J. Biol. Chem.
274,
22556-22562 |
5. |
Kimura, Y.,
Corcoran, E. E.,
Eto, K.,
Gengyo-Ando, K.,
Muramatsu, M. A.,
Kobayashi, R.,
Freedman, J. H.,
Mitani, S.,
Hagiwara, M.,
Means, A. R.,
and Tokumitsu, H.
(2002)
EMBO Rep.
3,
962-966 |
6. | Sun, P., Enslen, H., Myung, P. S., and Maurer, R. A. (1994) Genes Dev. 8, 2527-2539[Abstract] |
7. | Wegner, M., Cao, Z., and Rosenfeld, M. G. (1992) Science 256, 370-373[Medline] [Order article via Infotrieve] |
8. |
Cruzalegui, F. H.,
and Means, A. R.
(1993)
J. Biol. Chem.
268,
26171-26178 |
9. | Czernik, A. J., Pang, D. T., and Greengard, P. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 7518-7522[Abstract] |
10. |
Ohmstede, C. A.,
Jensen, K. F.,
and Sahyoun, N. E.
(1989)
J. Biol. Chem.
264,
5866-5875 |
11. |
White, R. R.,
Kwon, Y. G.,
Taing, M.,
Lawrence, D. S.,
and Edelman, A. M.
(1998)
J. Biol. Chem.
273,
3166-3172 |
12. | Dale, S., Wilson, W. A., Edelman, A. M., and Hardie, D. G. (1995) FEBS Lett. 361, 191-195[CrossRef][Medline] [Order article via Infotrieve] |
13. |
Yuan, L. W.,
and Gambee, J. E.
(2000)
J. Biol. Chem.
275,
40946-40951 |
14. |
Yang, W.,
Hong, Y. H.,
Shen, X. Q.,
Frankowski, C.,
Camp, H. S.,
and Leff, T.
(2001)
J. Biol. Chem.
276,
38341-38344 |
15. |
Kane, C. D.,
and Means, A. R.
(2000)
EMBO J.
19,
691-701 |
16. |
Knebel, A.,
Morrice, N.,
and Cohen, P.
(2001)
EMBO J.
20,
4360-4369 |
17. | Haribabu, B., Hook, S. S., Selbert, M. A., Goldstein, E. G., Tomhave, E. D., Edelman, A. M., Snyderman, R., and Means, A. R. (1995) EMBO J. 14, 3679-3686[Abstract] |
18. |
Anderson, K. A.,
Means, R. L.,
Huang, Q. H.,
Kemp, B. E.,
Goldstein, E. G.,
Selbert, M. A.,
Edelman, A. M.,
Fremeau, R. T.,
and Means, A. R.
(1998)
J. Biol. Chem.
273,
31880-31889 |
19. |
Alberts, A. S.,
Arias, J.,
Hagiwara, M.,
Montminy, M. R.,
and Feramisco, J. R.
(1994)
J. Biol. Chem.
269,
7623-7630 |
20. | Guo, Z., and Cooper, L. F. (2000) Biochem. Biophys. Res. Commun. 270, 183-189[CrossRef][Medline] [Order article via Infotrieve] |
21. |
Chin, D.,
and Means, A. R.
(1996)
J. Biol. Chem.
271,
30465-30471 |
22. | Matthews, R. P., Guthrie, C. R., Wailes, L. M., Zhao, X., Means, A. R., and McKnight, G. S. (1994) Mol. Cell. Biol. 14, 6107-6116[Abstract] |
23. |
Russo, G. L.,
Vandenberg, M. T., Yu, I. J.,
Bae, Y. S.,
Franza, B. R., Jr.,
and Marshak, D. R.
(1992)
J. Biol. Chem.
267,
20317-20325 |
24. |
MacDonald, J. A.,
Mackey, A. J.,
Pearson, W. R.,
and Haystead, T. A.
(2002)
Mol. Cell. Proteomics
1,
314-322 |
25. | Bruning, J. C., Winnay, J., Cheatham, B., and Kahn, C. R. (1997) Mol. Cell. Biol. 17, 1513-1521[Abstract] |
26. | Lee, J. C., Kwon, Y. G., Lawrence, D. S., and Edelman, A. M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 6413-6417[Abstract] |
27. |
Mackey, A. J.,
Haystead, T. A.,
and Pearson, W. R.
(2002)
Mol. Cell. Proteomics
1,
139-147 |
28. | Geier, A., Hemi, R., Haimsohn, M., Beery, R., and Karasik, A. (1997) In Vitro Cell Dev. Biol. Anim. 33, 129-136[Medline] [Order article via Infotrieve] |
29. | Impey, S., Fong, A. L., Wang, Y., Cardinaux, J. R., Fass, D. M., Obrietan, K., Wayman, G. A., Storm, D. R., Soderling, T. R., and Goodman, R. H. (2002) Neuron 34, 235-244[Medline] [Order article via Infotrieve] |
30. | MacRae, T. H. (2000) Cell. Mol. Life Sci. 57, 899-913[Medline] [Order article via Infotrieve] |
31. | Stokoe, D., Engel, K., Campbell, D. G., Cohen, P., and Gaestel, M. (1992) FEBS Lett. 313, 307-313[CrossRef][Medline] [Order article via Infotrieve] |
32. | Rouse, J., Cohen, P., Trigon, S., Morange, M., Alonso-Llamazares, A., Zamanillo, D., Hunt, T., and Nebreda, A. R. (1994) Cell 78, 1027-1037[Medline] [Order article via Infotrieve] |
33. | Engel, K., Ahlers, A., Brach, M. A., Herrmann, F., and Gaestel, M. (1995) J. Cell. Biochem. 57, 321-330[Medline] [Order article via Infotrieve] |
34. | Melander Gradin, H., Marklund, U., Larsson, N., Chatila, T. A., and Gullberg, M. (1997) Mol. Cell. Biol. 17, 3459-3467[Abstract] |
35. | Loones, M. T., Chang, Y., and Morange, M. (2000) Cell Stress Chaperones 5, 291-305[Medline] [Order article via Infotrieve] |
36. | Armstrong, C. L., Krueger-Naug, A. M., Currie, R. W., and Hawkes, R. (2000) J. Comp. Neurol. 416, 383-397[CrossRef][Medline] [Order article via Infotrieve] |
37. | Armstrong, C. L., Krueger-Naug, A. M., Currie, R. W., and Hawkes, R. (2001) J. Comp. Neurol. 434, 262-274[CrossRef][Medline] [Order article via Infotrieve] |
38. | Wakayama, T., and Iseki, S. (1998) Cell Biol. Int. 22, 295-304[CrossRef][Medline] [Order article via Infotrieve] |