From the Helix Research Institute, Inc., 1532-3 Yana,
Kisarazu-shi, Chiba 292-0812, Japan, the ¶ Vollum Institute,
Oregon Health Sciences University, Portland, Oregon 97201, and the
Department of Biological Cybergenetics, Medical Research
Institute, Tokyo Medical Dental University, Bunkyo-ku,
Tokyo 113-8510, Japan
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ABSTRACT |
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Mammalian
Ca2+/CaM-dependent protein kinase kinase
(CaM-KK) has been identified and cloned as an activator for two
kinases, CaM kinase I (CaM-KI) and CaM kinase IV (CaM-KIV), and a
recent report (Yano, S., Tokumitsu, H., and Soderling, T. R. (1998) Nature 396, 584-587) demonstrates that CaM-KK can
also activate and phosphorylate protein kinase B (PKB). In this study,
we identify a CaM-KK from Caenorhabditis
elegans, and comparison of its sequence with the mammalian
CaM-KK Ca2+/calmodulin-dependent protein kinases
(CaM-Ks)1 constitute a
diverse group of enzymes which are involved in many aspects of calcium
signaling such as neurotransmitter release, excitation-contraction coupling in muscle, and gene expression (1-4). Recent studies have
demonstrated that two CaM kinases, CaM-KI and -IV, are activated through phosphorylation by an upstream CaM kinase kinase (CaM-KK) (5-12), analogous to other kinase cascades such as PKA/phosphorylase kinase (13), MAP kinase (14), and AMP kinase (15). CaM-KK is a recently
cloned protein kinase that phosphorylates and activates CaM-KI and
CaM-KIV, constituting the CaM-K cascade (11, 39, 47). Like other
CaM-Ks, CaM-KK is negatively regulated by an intrasteric mechanism
through its autoinhibitory domain (residue 436-441) and activated by
the Ca2+·CaM complex (16). Ca2+/CaM binding
to both CaM-KK and its downstream target CaM-Ks are required to
activate the CaM kinase cascade (6, 7, 12). This CaM-K cascade has been
functionally demonstrated for CaM-KIV activation in response to
Ca2+ mobilization using transfected COS-7 cells (12),
Jurkat cells (17), and cultured hippocampal neurons (18) and for CaM-KI activation in PC-12 cells upon membrane depolarization (19). The
CaM-KK/CaM-KIV cascade can stimulate gene transcription through phosphorylation of Ser133 in cAMP response element-binding
protein, and this may play an important physiological role in learning
and memory (11, 18, 20-23). It has also been demonstrated that the MAP
kinase pathways, especially JNK and p38, may be indirectly activated by
the CaM-KK/CaM-KIV cascade (24). Recently, evidence has been provided
that CaM-KK may mediate the anti-apoptotic effect of modest elevations
of Ca2+ through phosphorylation and activation of protein
kinase B (PKB) (25). This result also indicates that multiple protein
kinases might be phosphorylated and activated by CaM-KK, resulting in regulation of a wide variety of functions.
The activation sites in CaM-KI (Thr177) (7), CaM-KIV
(Thr196) (6, 12), and PKB (Thr308) (25) which
are phosphorylated by CaM-KK are located in their "activation
loops" analogous to Thr197 in PKA (26) and
Thr160 in cdk2 (27). For most protein kinases the substrate
recognition determinants are located just NH2- and
COOH-terminal of the phosphorylated Ser/Thr. For example, many
substrates of PKA have basic residues at position 2 and 3 NH2-terminal of the phosphorylated Ser, and these basic
residues are recognized by Glu127 and Glu170 in
PKA (33-37). However, there is little sequence similarities in the
NH2-terminal sequences of the activation sites of CaM-KI, CaM-KIV, or PKB. There is considerable COOH-terminal sequence identity,
but these sequences are also conserved in many other kinases that have
activation loops but are not phosphorylated by CaM-KK. This suggests
that CaM-KK has specific mechanisms to recognize its target kinases
other than the primary sequence surrounding the phosphorylated Thr.
Although CaM-KK can phosphorylate a synthetic peptide corresponding to
the sequence in the CaM-KIV activation loop
(KKKEHQVLMKT196VCGTPGY), very high concentrations of the
peptide are required (28). In this report, we explored the mechanisms
of selective substrate recognition of CaM-KK by identifying a unique
kinase insert domain which is involved in its interaction and
activation of CaM-KI and IV but not PKB.
Materials--
CaM-KK cDNA (GenBank accession number L42810)
was from a rat brain cDNA library (11). Recombinant CaM-KIV was
expressed in Sf9 cells and purified as described previously
(21). Recombinant CaM-KKs were expressed in Escherichia coli
BL-21 (DE3) pLys(S) with pET16b vector and purified by CaM-Sepharose as
described previously (12). CaM was purified from bovine brain (29). CaM-KIV peptide (KKKEHQVLMKT196VCGTPGY (28)) was
synthesized by the Bio-Synthesis, Inc. Caenorhabditis elegans CaM-KK cDNA was cloned by RT-PCR by using a sense
oligonucleotide (5'-TTACTCGAGATGTACACATTTCAGTCGGTCTCACAGCAG-3'), and an
antisense oligonucleotide
(5'-AGACTAGTTTACCTGAATGGATTGCCAAACCGCTTGCGA-3') based on
the sequence of C. elegans cosmid (C05H8.1) and reverse transcribed DNA from mRNA of N2 stage C. elegans as a
template, which was kindly provided from Dr. Shouhei Mitani (Tokyo
Woman's Medical College). A 1.1-kilobase pair of PCR fragment was
digested with XhoI and SpeI and then inserted
into pME18s vector. Nucleotide sequence of cloned C. elegans
CaM-KK cDNA (GenBank accession number AB016838) was confirmed
and the deduced amino acid sequence (357 amino acids) was completely
matched with predicted coding sequence using the program Genefinder
from C05H8.1 (30). The 5'-untranslated region of C. elegans
CaM-KK cDNA was also cloned by RT-PCR using a sense primer
(5'-AAACTTCTGTAGTATTTACA-3') and antisense primer
(5'-AAATAGTTGTCATTTGGATCG-3') based on the cosmid sequence and reverse
transcribed DNA from mRNA of N2 stage C. elegans as a
template (see Fig. 1A) and completely sequenced. Rat CaM-KI
cDNA was cloned by RT-PCR using a sense oligonucleotide (5'-ATGCCAGGGGCAGTGGAAGG-3') and an antisense-oligonucleotide (5'-TCAGTCCATGGCCCTAGAGC-3') based on the published sequence (31) and
reverse transcribed cDNA from rat brain mRNA
(CLONTECH) as a template. The PCR product (1.1 kilobase pairs) was subcloned into pT7Blue plasmid and then sequenced.
CaM-KI cDNA was digested with EcoRI and SalI,
and then subcloned into pGEX-4T3 and transformed into E. coli JM109. Expressed GST-CaM-KI was purified on
glutathione-Sepharose followed by CaM-Sepharose. GST-CaM-KIV (17-469,
T196A) was constructed into pGEX-KG as follows: His-tagged CaM-KIV
(T196A) cDNA in pME18s vector (12) was digested with
BstEII at the position of Val17 and then filled
in and digested with XbaI. CaM-KIV fragment was ligated into
SmaI-XbaI digested pGEX-KG and then transformed
into E. coli BL-21 (DE3). Nucleotide sequence of this
construct was confirmed. Expressed GST-CaM-KIV was purified by
CaM-Sepharose. All other chemicals were from standard commercial sources.
Construction of Plasmids--
Mutagenesis of CaM-KK using
pME-CaM-KK (wild-type) plasmid as a template was carried out by using
either the GeneEditorTM in vitro Site-Directed Mutagenesis
System (Promega Co.) or QUANT-ESSENTIALTM (QUANTUM Biotechnologies
Inc.) and mutagenic oligonucleotides as follows; P237E,
5'-CTCCTGAGAAAGGGAGAAGTCATGGAAGTGCCC-3'; P237V, 5'-CTCCTGAGAAAGGGAGTAGTCATGGAAGTGCCC-3'; P237A,
5'-CTCCTGAGAAAGGGAGCAGTCATGGAAGTGCCC-3'; S279E,
5'-AGGGACATCAAGCCGGAGAATCTGCTCCTTGGG-3'; S279D,
5'-AGGGACATCAAGCCGGACAATCTGCTCCTTGGG-3'; 279A,
5'-AGGGACATCAAGCCGGCCAATCTGCTCCTTGGG-3'; RP-domain
deletion, 5'-CCAAAAAGAAGTTACTGAAGCTGCTGCCCCTGGAGCGTGT-3'; R172.177E,
5'-CAGTATGGCTTTCCTGAACGTCCTCCCCCGGAAGGGTCCCAAGCTCCT-3'; R172.177A,
5'-CTGTATGGCTTTCCTGCCCGTCCTCCCCCGGCAGGGTCCCAAGCCTCCT-3'; R172E, 5'-CAGTATGGCTTTCCTGAACGTCCTCCCCCGAGA-3'; R177E,
5'-CGCCGTCCTCCCCCGGAAGGGTCCCAAGCTCCT-3'; R173A,
5'-TATGGCTTTCCTCGCGCTCCTCCCCCGAGAGGG-3'; R173E,
5'-TATGGCTTTCCTCGCGAGCCTCCCCCGAGAGGG-3'. Nucleotide
sequences of each mutant were confirmed by automated sequencing using
an Applied Biosystems 377 DNA sequencer.
Transient Expression of CaM-KK Mutants--
COS-7 cells were
maintained in Dulbeco's modified Eagle's medium containing 10% fetal
calf serum. Cells were subcultured in 10-cm dishes 12 h before
transfection. The cells were then transferred to serum-free medium and
treated with a mixture of either 10 µg of pME18s plasmid DNA (DNAX
Research Institute, Inc.) or CaM-KK cDNA containing plasmid DNAs
and 60 µg of LipofectAMINE Reagent (Life Technologies, Inc.) in 6.8 ml of medium. After 32-48 h incubation, the cells were collected and
homogenized with 1 ml of lysis buffer (150 mM NaCl, 20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1% Nonidet
P-40, 10% glycerol, 0.2 mM phenylemethylsulfonyl fluroide,
10 mg/liter leupeptin, 10 mg/liter pepstatin A, 10 mg/liter trypsin
inhibitor) at 4 °C. After centrifugation at 15,000 × g for 15 min, the supernatant was used for CaM-KIV
activation assay and quantitated by Western blotting.
In Vitro Assay of CaM Kinase Activation by CaM-KK--
Either
COS-7 cell extract (0.6 µg) transfected with CaM-KK or E. coli expressed CaM-KK was incubated with either recombinant CaM-KIV (3.4 µM) or GST-CaM-KI (1 µg) at 30 °C in 50 mM HEPES (pH 7.5), 10 mM Mg(Ac)2, 1 mM DTT, 400 µM ATP, and 2 mM
CaCl2, 8 µM CaM. The reaction was terminated
at the indicated time points by a 20-fold dilution at 4 °C with 50 mM HEPES (pH 7.5), 2 mg/ml bovine serum albumin, 10%
ethylene glycol, and 2 mM EDTA. CaM-KIV (34 nM)
activity was measured at 30 °C for 5 min in a 25-µl assay containing 50 mM HEPES (pH 7.5), 10 mM
Mg(Ac)2, 1 mM DTT, 400 µM
[ PKB Activation by CaM-KK--
GST-rat PKB ( CaM-KIV Peptide Phosphorylation Assay--
CaM-KIV peptide
phosphorylation activity of CaM-KK was measured at 30 °C for 10 min
in a 25-µl assay containing 50 mM HEPES (pH 7.5), 10 mM Mg(Ac)2, 1 mM DTT, 400 µM [ Autophosphorylation of CaM-KK--
Autophosphorylation reaction
was essentially the same as CaM-KIV peptide phosphorylation assay as
described above except for 100 µM
[ Binding of CaM-KK with GST-CaM-KIV (T196A)--
After 25 µl of
glutathione-Sepharose (50 µl of 50% slurry) was loaded with either
buffer or 0.5 µg of GST-CaM-KIV (T196A) which was purified by
CaM-Sepharose, the resin was washed three times with 1 ml of
phosphate-buffered saline and then washed three times with 1 ml of 50 mM HEPES (pH 7.5), 2 mM CaCl2, and
10 mM Mg(Ac)2. Equal amounts (approximately 200 ng) of either wild-type or mutant CaM-KK were applied to the resin in a
solution (100 µl) containing 50 mM HEPES (pH 7.5), 2 mM CaCl2, 10 mM
Mg(Ac)2, 5 µM CaM, and 1 mM ATP
and then incubated for 1 h at room temperature. These resins were
washed three times with 500 µl of 50 mM HEPES (pH 7.5), 2 mM CaCl2, 10 mM
Mg(Ac)2, 1 µM CaM at 4 °C and then GST-CaM-KIV was eluted with 80 µl of 50 mM Tris-HCl (pH
8.0) and 10 mM glutathione. Equal volumes of samples were
subjected to SDS-7.5% PAGE followed by Western blotting by using both
anti-CaM-KIV antibody or anti-CaM-KK antibody.
Others--
Western blotting was carried out using antiserum
(1/1000 dilution) against a peptide corresponding to a conserved
protein kinase motif (residues 132-146 of CaM-KII), anti-CaM-KIV
antibody (Transduction Laboratories), or anti-CaM-KK antibody (Santa
Cruz Biotechnology, Transduction Laboratories), and the
biotinylated-CaM overlay was done as described previously (10).
Detection was performed by using chemiluminesence reagent (NEN Life
Science Products Inc.). Protein concentration was estimated by
Coomassie dye binding (Bio-Rad) using bovine serum albumin as a standard.
Identification and Characterization of C. elegans CaM-KK--
A
protein kinase gene was recently identified in the C. elegans genome data base that is highly homologous to rat CaM-KK
(30, 47). To determine whether this clone encodes CaM-KK, cDNA of 1074 bp was cloned by RT-PCR from C. elegans mRNA (Fig.
1A). In order to ensure that
the methionine at position 1 is the point of translation initiation, we
also cloned the 5'-untranslated region by RT-PCR using two nucleotide
sequences, 200 bp upstream in C. elegans genome sequence and
353 bp downstream from the first Met (ATG), as PCR primers (Fig.
1A, underlined). The nucleotide sequence of the PCR product
(553 bp) includes 200 bp of the 5'-untranslated region containing three
in-frame stop codons and the remaining 353-bp region is completely
matched with its open reading frame from the first ATG. Since the
antisense primer is located in the third exon, this PCR product is
derived from C. elegans mRNA and the Met at position 1 is likely the translation initiation. The full-length clone encoded a
protein of 357 amino acids with a calculated molecular mass of 40,701 (Fig. 1A). This protein is likely the C. elegans
homologue of CaM-KK as it contains the unique RP-rich insert (residues
60-81) in the catalytic domain highly homologous to similar inserts in
the Acidic Residues Are Not Essential for CaM-KK Substrate
Recognition--
The mechanism of substrate recognition by CaM-KK has
not been studied. To determine critical residues or domains for
substrate recognition in CaM-KK, we first aligned and compared the
amino acid sequences of the catalytic domains of mammalian The RP-domain Is Essential for CaM-KIV Activation by
CaM-KK--
The function of the unique RP-domain insert in all cloned
CaM-KKs (Fig. 2A) is completely unknown. To analyze its
function, the RP-domain (residues 167-189) was deleted from
Next we tested whether the RP-deletion mutant could physically interact
with CaM-KIV using GST-CaM-KIV (T196A). The rational for using the
T196A mutant, which cannot be activated by CaM-KK, is that it may
capture the intermediate complex form between CaM-KK and CaM-KIV.
GST-CaM-KIV (T196A) loaded glutathione-Sepharose was mixed with either
the wild-type or RP-deletion mutant of CaM-KK, and a pull-down
experiment was performed in essentially the same condition as an
activation reaction with low ionic strength. As shown in Fig.
3D, association of wild-type CaM-KK with GST-CaM-KIV (T196A)
was readily detected which was approximately 10% of applied CaM-KK,
and the interaction occurred in a
Ca2+/CaM-dependent manner (data not shown).
Deletion of the RP-domain resulted in a loss of interaction to CaM-KIV
compared with the wild-type CaM-KK.
RP-domain Mutants of CaM-KK Are Catalytically Active--
To test
whether these RP-domain mutants are catalytically defective, we
measured their abilities to autophosphorylate. As shown in Fig.
4A, all of the RP-domain
mutants showed Ca2+/calmodulin-dependent
autophosphorylation similar to wild-type kinase although the
R172A,R177A mutant gave a slightly weaker activity as compared with
others. As a second test of catalysis, we determined their abilities to
phosphorylate the CaM-KIV activation domain sequence
(KKKEHQVLMKT196VCGTPGY) (28) which contains the
phosphorylation-activation Thr196 in CaM-KIV. Again, all of
the mutants including the RP-domain deletion could phosphorylate
CaM-KIV peptide in a Ca2+/CaM-dependent manner
(Fig. 4B). In these experiments, we used recombinant mutant
enzymes expressed in E. coli and partially purified by
CaM-Sepharose instead of using transfected COS-7 extract. CaM-KIV
activating and phosphorylating activity of both wild-type and RP-domain
deletion mutants of E. coli expressed CaM-KK was shown to be
essentially the same as COS-7 cell expressed enzyme (see Fig.
5B). We also checked CaM-KIV
activating activity of other E. coli-expressed CaM-KK
mutants with the same conditions as shown in Fig. 3A with
triplicate experiments and obtained essentially the same results as
that obtained with COS-7 cell-expressed enzymes (data not shown). These
results indicate that deletion and mutations on the RP-domain do not
affect catalytic activity with regard to autophosphorylation and
peptide phosphorylation. Also, the regulatory mechanism of CaM-KK, such
as autoinhibition and CaM binding, remained intact with these mutations
and deletions.
Requirements of the RP-domain for Activation of CaM-Kinases but Not
for PKB--
Although the RP-domain mutants can catalyze
Ca2+/calmodulin-dependent autophosphorylation
and phosphorylation of a synthetic peptide, we wanted to test other
physiological substrates. Two such substrates are CaM-KI and PKB.
CaM-KI is phosphorylated on Thr177 resulting in its
activation (7). As shown in Fig. 5A, recombinant GST-CaM-KI
was activated and phosphorylated by wild-type CaM-KK as described
previously (11). However, activation and phosphorylation of GST-CaM-KI
by the RP-deletion mutant of CaM-KK was strongly reduced compared with
wild-type (Fig. 5A) in a manner similar to CaM-KIV (Fig.
5B). PKB can be activated through phosphorylation of its
activation loop Thr308 by PDK1 (40) and CaM-KK (25). In
contrast to CaM-KI and CaM-KIV, PKB was activated and phosphorylated by
the RP-deletion mutant of CaM-KK in a manner comparable to that
obtained with wild-type CaM-KK (Fig. 5C) while the rate of
activation of PKB by CaM-KK is lower than that of CaM-KI or CaM-KIV
which is consistent with previous observations (25). This indicates
that the RP-domain deletion mutant is catalytically active and
recognizes PKB as its substrate.
In this study we demonstrated that the RP-domain of CaM-KK, which
is conserved in all three known CaM-KKs, is required for selective
substrate recognition. Thus, deletion of the RP-domain obviated CaM-KK
activity toward its two classical substrates CaM-KI and CaM-KIV.
Mutational analysis indicated that the three highly conserved Arg in
rat CaM-KK, Arg172, Arg173, and
Arg177 are essential in this substrate recognition. In
contrast, the RP-deletion mutants showed normal autophosphorylation,
phosphorylation of a synthetic substrate containing the activation site
of CaM-KIV, and, more importantly, in phosphorylation and activation of
PKB. Since these reactions were
Ca2+/CaM-dependent, these results indicate that
the RP-domain is involved in recognition of CaM-KI and CaM-KIV as
substrates. This conclusion is substantiated by the fact that the GST
fusion of CaM-KIV could pull down wild-type CaM-KK whereas the
RP-domain deletion mutant was not able to interact with the GST fusion
of CaM-KIV. The fact that RP-domain mutants could phosphorylate CaM-KIV
peptide but not CaM-KIV itself is perhaps consistent with the report
(28) that the CaM-KIV peptide (Km = 263 µM) is a very poor substrate for CaM-KK as compared with
CaM-KIV (Km = 0.71 µM). Therefore, the
recognition determinants of CaM-KK toward this peptide substrate are
likely to be quite different than for the full-length CaM-KIV. The
RP-domain of CaM-KK is localized between subdomains II and III in the
catalytic domain, placing it between Crystallographic studies of PKA with PKI (33, 34) and recent studies of
several protein kinases using an oriented degenerate peptide library
(41) clearly demonstrated that critical determinants for substrate
recognition by those protein kinases were found at residues in the
vicinity of the phosphorylation site. Two acidic residues in PKA
(Glu127 and Glu170) are predicted to form
interactions with the P-3 basic residue in substrates. However, there
is no obvious consensus basic residues at those positions in the
activation loops of CaM-KI, -IV, and PKB, and CaM-KK has a
Pro237 and Ser279 in positions analogous to
Glu127 and Glu170 of PKA. Not surprisingly,
mutations of Pro237 and Ser279 did not affect
the ability of CaM-KK to phosphorylate and activate CaM-KIV.
In conclusion, our data contribute to the understanding of how CaM-KK
specifically recognizes and phosphorylates its downstream CaM kinases.
CaM-KK contains a RP-domain which allows efficient targeting and
subsequent activation of CaM-KI and -IV. Based on a limited number of
mutants, we cannot rule out the possibility that other residues or
regions are involved in substrate recognition by CaM-KK. In this
regard, our results are consistent with a study showing that mutations
in the activation loop of MAP kinases has little effect on their
recognition as substrates for their upstream activating MAP kinase
kinases (46).
and
shows a unique Arg-Pro (RP)-rich insert in their
catalytic domains relative to other protein kinases. Deletion of the
RP-domain resulted in complete loss of CaM-KIV activation activity and
physical interaction of CaM-KK with glutathione S-transferase-CaM-KIV (T196A). However, CaM-KK
autophosphorylation and phosphorylation of a synthetic peptide
substrate were normal in the RP-domain mutant. Site-directed
mutagenesis of three conserved Arg in the RP- domain of CaM-KK
confirmed that these positive charges are important for CaM-KIV
activation. The RP- domain deletion mutant also failed to fully
activate and phosphorylate CaM-KI, but this mutant was
indistinguishable from wild-type CaM-KK for the phosphorylation and
activation of PKB. These results indicate that the RP-domain in CaM-KK
is critical for recognition of downstream CaM-kinases but not for its
catalytic activity (i.e. autophosphorylation) and PKB activation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (1000-2000 cpm/pmol), 40 µM
syntide-2, and 2 mM EGTA (Ca2+/CaM-independent
activity). CaM-KI activity (1 µg/ml) was measured at 30 °C for 5 min in a 25-µl assay containing 50 mM HEPES (pH 7.5), 10 mM Mg(Ac)2, 1 mM DTT, 400 µM [
-32P]ATP (1000-2000 cpm/pmol), 40 µM syntide-2, and 2 mM CaCl2, 8 µM CaM. The reaction was initiated by the addition of
CaM-KIV or CaM-KI and terminated by spotting aliquots (15 µl) onto
phosphocellulose paper (Whatman P-81) followed by washing in 75 mM phosphoric acid (32). CaM-KK activity is expressed in
terms of its ability to increase either
Ca2+/CaM-independent activity of CaM-KIV or
Ca2+/CaM dependent activity of CaM-KI under the defined
assay conditions. The data of CaM-KIV activation are expressed as a
percentage of the value of incubation of recombinant CaM-KIV with
wild-type CaM-KK for 10 min which was calculated to be approximately
0.5 µmol/min/mg. The data of CaM-KI activation are expressed as a percentage of the value of incubation of recombinant CaM-KI with wild-type CaM-KK for 5 min which was calculated to be approximately 4.0 µmol/min/mg.
isoform) was
transiently expressed in COS-7 cells with pME18s vector as described
above and purified on glutathione-Sepharose as described previously
(25). Purified GST-PKB (0.1 µg) was incubated for the indicated times
with 50 mM HEPES (pH 7.5), 10 mM
Mg(Ac)2, 1 mM DTT, 1 mM
CaCl2, 3 µM CaM, 0.2 mg/ml histone H2B, 400 µM [
-32P]ATP (1000 cpm/pmol), and
approximately 100 ng of wild-type, mutant CaM-KK, or buffer. After
terminating the reaction by addition of SDS-PAGE sample buffer,
32P incorporation into GST-PKB and histone H2B was analyzed
by SDS-15% PAGE followed by autoradiography and then quantitated by
densitometric scanning. The data of GST-PKB activity are expressed as
fold of control value of 32P incorporation into histone H2B
by recombinant GST-PKB with wild-type CaM-KK at 0 min.
-32P]ATP (1000-2000 cpm/pmol), 400 µM CaM-KIV peptide and either presence of 1 mM EGTA or 1 mM CaCl2, 4 µM CaM using 5 µl of partially purified CaM-KKs.
-32P]ATP (1000-2000 cpm/pmol) and omitting
peptide substrate in a reaction solution. After terminating the
reaction by addition of 5 µl of SDS-PAGE sample buffer, samples were
loaded onto SDS-10% PAGE and then subjected to autoradiograph.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(11) and
(39, 47) isoforms of mammalian CaM-KK. The
C. elegans clone was transiently expressed in COS-7 cells,
and a CaM overlay in the presence of Ca2+ (Fig. 1B,
inset panel) identified a 45-kDa protein which is smaller than the
mammalian
CaM-KK. COS-7 cell extracts from mock transfected or
transfections with vectors encoding mammalian
CaM-KK or C. elegans protein were tested for their abilities to activate
recombinant mammalian CaM-KIV. The C. elegans protein gave
activation of CaM-KIV which was about 2-3-fold less efficient than the
mammalian CaM-KK in the initial rate of activation (Fig.
1B). Next we tested Ca2+/CaM dependence of the
C. elegans enzyme by using both wild-type CaM-KIV (Fig.
1C, left panel) and constitutively active mutant of CaM-KIV
(316FN-DD, Fig. 1C, right panel) (10) which has
shown to be activated by the constitutively active form of rat
CaM-KK (1-434) in a Ca2+/CaM-independent manner (16).
As shown in Fig. 1C, both wild-type and mutant CaM-KIVs are
activated by the C. elegans enzyme in a complete
Ca2+/CaM-dependent manner suggesting that
C. elegans enzyme requires Ca2+/CaM for its
activity as well as the mammalian CaM-KK (11). Furthermore, the
COOH-terminal end of the C. elegans clone (residues 331-356, Fig. 1A) is 54% identical to the CaM-binding
domain of
CaM-KK (16). We have also detected
Ca2+-dependent CaM binding of the synthetic
peptide corresponding to residues 331-356 in the C. elegans
clone (data not shown) similar to that of a CaM-binding peptide of
CaM-KK (16). These biochemical criteria confirm that the protein is
the C. elegans homologue of CaM-KK.
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Fig. 1.
Cloning and characterization of C. elegans CaM-KK. A, nucleotide and deduced
amino acid sequence of C. elegans CaM-KK. C. elegans CaM-KK cDNA was cloned by RT-PCR using reverse
transcribed DNA from mRNA of N2 stage C. elegans as a
template (see "Experimental Procedures"). Nucleotide sequence of
the 5'-untranslated region of the cDNA cloned by RT-PCR is
combined. Underlines indicate the sequence of PCR primers
for amplification of the 5'-untranslated region. In-frame stop codons
are indicated with an asterisk. CaM-binding site is shown by
a box (16). B, activation of CaM-KIV by C. elegans CaM-KK. Recombinant mouse CaM-KIV (3.4 µM)
was incubated at 30 °C for the indicated times in the presence of
Ca2+/CaM (see "Experimental Procedures") with extracts
(0.6 µg) from COS-7 cells either mock-transfected ( ) or
transfected with plasmids expressing rat
CaM-KK (
) or C. elegans CaM-KK (
). After terminating the reaction,
Ca2+/CaM independent activity of CaM-KIV was measured and
is expressed as a percentage of the 10-min value for
CaM-KK. Each
extract (18 µg) was subjected to SDS-10% PAGE, transferred onto
nitrocellulose membrane (Hybond-C, Amersham), and analyzed by CaM
overlay (panel B, inset). C, requirement of
Ca2+/CaM for C. elegans CaM-KK activity. Either
wild-type (left panel) or constitutively active mutant
(316FN-DD, right panel) of mouse CaM-KIV was
activated with extracts from COS-7 cells either mock-transfected (
)
or transfected with a plasmid expressing C. elegans CaM-KK
(+) for 15 min as described in panel B in the absence (
)
or presence (+) of 2 mM CaCl2, 10 µM CaM. After terminating the reaction,
Ca2+/CaM independent activity of each CaM-KIV was measured
and is expressed as a percentages of the value in the presence of
C. elegans CaM-KK and Ca2+/CaM. Results
represent mean and S.E. of three experiments.
and
CaM-KK and C. elegans CaM-KK with mammalian CaM-KII and
cAMP-dependent protein kinase (PKA), the best protein
kinase for enzyme-substrate recognition mechanisms (Fig.
2A). According to the studies
of: 1) the crystal structure of PKA with PKI (33, 34); 2) charged to
alanine mutagenesis of the yeast homologue of PKA (35); and 3) labeling
with a water-soluble carbodiimide (36), acidic residues Glu127 and Glu170 interact with the Arg in the
P-2 and P-3 positions of PKA substrates. It has been shown that
mutation of these Glu residues in PKA affect its Km
with the peptide substrate Kemptide (35, 37). Residues equivalent to
Glu127 and Glu170 are also conserved in
Arg/Lys-directed CaM kinases (myosin light chain kinase, CaM-KI, -II,
-IV, and phosphorylase kinase). Residues Pro237 and
Ser279 in rat CaM-KK are analogous to Glu127
and Glu170 in PKA (Fig. 2A). Therefore, we
initially mutated Pro237 to Glu, Val, or Ala and
Ser279 to Glu, Asp, or Ala to test whether these residues
are involved in CaM-KK activity. Mutant rat constructs, including
wild-type
CaM-KK, were transfected into COS-7 cells, and the
expression level of CaM-KK in the cells were quantitated by
immunoblotting (Fig. 2, B and C, inset panels).
All of the Pro237 and Ser279 mutants were
expressed to an extent similar to wild-type enzyme. The same amount of
CaM-KK mutants as wild-type enzyme were used for CaM-KIV activating
assays in the presence of Ca2+/CaM. The time course of
CaM-KIV activation by all the Pro237 and Ser279
CaM-KK mutants were similar to wild-type CaM-KK
(t1/2 = 1 min) (Fig. 2, B and
C). Our results indicate that Pro237 and
Ser279 are not essential for specific substrate recognition
of CaM-KK.
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Fig. 2.
A, comparison of amino acid sequences of
catalytic domain (subdomain I-VIB) of CaM-KKs with other protein
kinases. A, residues 121-296 of rat CaM-KK (11) are
aligned with the catalytic domains of rat
CaM-KK (39), C. elegans CaM-KK (30), rat CaM-KII (43), and mouse
cAMP-dependent protein kinase (PKA (44)).
Identical residues are indicated with an asterisk. The
subdomain location in the catalytic domain was used by numbering system
of Hanks et al. (45). RP-domain of each CaM-KK (residues
167-189 in
CaM-KK, residues 203-225 in
CaM-KK, and residues
60-81 in C.elegans CaM-KK) is indicated by a shaded
box. Arrows indicate the sites which were mutated in this study.
B, activation of CaM-KIV by
CaM-KK Pro237 and
Ser279 mutants. B, recombinant CaM-KIV (3.4 µM) was incubated at 30 °C for the indicated times in
the presence of Ca2+/CaM (see "Experimental
Procedures") with extraction buffer (
) or extracts (0.6 µg) from
COS-7 cells either mock-transfected (
in panels B and
C) or transfected with plasmids expressing wild-type rat
CaM-KK (
) or the indicated CaM-KK. After terminating the
reaction, Ca2+/CaM independent activity of CaM-KIV was
measured and is expressed as a percentage of the 10-min value for
CaM-KK wild type. Each extract (18 µg) was subjected to SDS-10%
PAGE and then transferred onto polyvinylidine difluoride membrane
(Bio-Rad) and analyzed by Western blotting using protein kinase peptide
antiserum (panel B, inset) or anti-CaM-KK antibody (Santa
Cruz Biotechnology) for panel C (inset).
Arrows indicate CaM-KK expressed in COS-7 cells.
B and C,
CaM-KK mutants P237E (
), P237V
(
), P237A (
), S279E (
), S279D (
), S279A (
).
CaM-KK,
and this mutant was expressed in COS-7 cells (Fig.
3A). As expected, deletion of
the RP-domain from CaM-KK gave a slightly faster migrating species than
wild-type enzyme (68 kDa) on SDS-PAGE. Surprisingly, the RP-deletion
mutant was completely unable to activate CaM-KIV (Fig. 3A).
The critical role of the RP-domain was further analyzed by
site-directed mutation of Arg172 and Arg177,
which are conserved among all three CaM-KKs. Mutation of both Arg172 and Arg177 to Glu resulted in
significant reduction of CaM-KK activity. Ala mutation was less
defective (t1/2 = 4 min) compared with either
RP-domain deletion or Glu mutants (Fig. 3A). Single Glu
mutation of either Arg172 and Arg177 revealed
that both residues are required for the maximum activity of CaM-KK
(Fig. 3B). Although mutation of Arg172 has a
more significantly affect on the catalysis than that of Arg177, both residues seem to synergistically contribute to
CaM-KK activity. Another Arg residue at 173 is also conserved in
and
isofoms of CaM-KK, but replaced by Gln66 in
C.elegans enzyme (Fig. 2A). Since C. elegans CaM-KK is a 2-3-fold less efficient activator than rat
CaM-KK toward mammalian CaM-KIV (Fig. 1B), we tested the
possibility that Arg173 contributes to the catalytic
efficiency (Fig. 3C). Glu mutation on Arg173
gave a significant reduction of the initial rate of CaM-KIV activation. However, when we mutated Gln66 in C. elegans
CaM-KK to Arg, we could not detect a significant increase in CaM-KK
activity (data not shown). This is consistent with the mutation of
Arg173 in rat CaM-KK to a neutral amino acid residue, such
as Ala, which caused little effect on CaM-KK activity.
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Fig. 3.
Effect of deletion and mutation of RP-domain
on CaM-KK activity. A-C, CaM-KK mutants expressed in
COS-7 cells were assayed for activity at the indicated times by their
ability to activate CaM-KIV and by Western blots (insets)
for expression levels as in Fig. 2. Mock-transfected ( and
in
panels B and C), wild-type
CaM-KK (
),
RP-domain deletion (
), R172/177E (
), R172/177A (
), R172E (
in panel B), R177E (
in panel B), R173A (
in panel C), R173E (
in panel C). Results
represent duplicate experiments (panels B and C).
D, interaction of GST-CaM-KIV(T196A) with CaM-KK. Equal
amounts (approximately 200 ng) of either wild-type or RP- domain
deletion mutant CaM-KK (Western blot, upper panel) were
applied to glutathione-Sepharose which was preloaded with GST-CaM-KIV
(17-469, T196A) and incubated for 1 h at room temperature (see
"Experimental Procedures"). The resins were washed three times and
eluted with buffer containing 10 mM glutathione. Equal
volumes of eluate were analyzed by SDS-7.5% PAGE and Western blots for
CaM-KIV (middle panel) or CaM-KK (bottom
panel).
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Fig. 4.
Catalytic activity of CaM-KK RP-domain
mutants. A, autophosphorylarion of CaM-KK. Equal
amounts of E. coli-expressed wild-type or the indicated rat
CaM-KK mutants were subjected to autophosphorylation conditions in the
presence or absence of Ca2+/CaM (see "Experimental
Procedures") and subjected to SDS-10% PAGE and autoradiography.
B, E. coli-expressed rat
CaM-KK was partially
purified by CaM-Sepharose and assayed (50 ng each) for phosphorylation
of CaM-KIV peptide (400 µM) in the absence (open
bar) or presence (solid bar) of Ca2+/CaM
for 10 min (see "Experimental Procedures"). Results represent mean
and S.E. of three experiments.
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Fig. 5.
Activation and phosphorylation of CaM-KI and
PKB by CaM-KK. GST-CaM-KI (panel A, left) and
recombinant CaM-KIV (panel B) were incubated with buffer,
wild-type, or RP-domain deletion mutants of E. coli
expressed rat CaM-KK (approximately 100 ng) at 30 °C for 10 min (5 min for GST-CaM-KI) in the presence of Ca2+/CaM and
then each protein kinase activity was measured. Panel A
(right) indicates time course of GST-CaM-KI activation by
buffer (
), wild-type (
), or RP-deletion mutant (
) of CaM-KK as
shown in the left panel. Panel C indicates the activity of
GST-PKB phosphorylated by buffer (
), wild-type (
), or RP-deletion
mutant (
) of E. coli expressed rat
CaM-KK at various
time points. Activities of CaM-KI, -IV, and PKB were measured as
described under "Experimental Procedures." The results in
panels A and B indicate the mean and S.E. of
three experiments. In panel C, the results indicate the
average of duplicate experiments. 32P incorporation into
each protein kinase during the activation by CaM-KK in the same
condition as activation reaction was also analyzed by SDS-PAGE followed
by autoradiography (inset). 32P incorporation of
PKB (arrow) at 30-min reaction times is shown in duplicate
in panel C (inset) and lower bands indicate
autophosphorylated CaM-KK.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helices B and C in PKA. It is
conceivable that this domain may recruit and stabilize the downstream
CaM-KI and -IV to maintain proper orientation toward the catalytic
cleft, resulting in high affinity and specific interaction between
CaM-KK and these substrates. Interestingly, we could not detect
significant interaction between CaM-KIV and a synthetic peptide
corresponding to the RP-domain by using a direct binding assay or an
inhibition assay for CaM-KIV activation, suggesting that domain
addition to the RD-domain might be required for the interaction (data
not shown). Since the RP-domain of CaM-KK is not necessary for
phosphorylation and activation of PKB (Fig. 5C), the
RP-domain seems to be specifically utilized to operate the CaM-kinase
cascade. This is also consistent with a recent report that the PKB
activating kinase PDK1, which does not possess the RP-domain, was
unable to phosphorylate CaM-KIV (42). In vitro activation of
PKB requires about 10-fold higher concentrations of CaM-KK than does
activation of CaM-KIV or CaM-KI (25). Likewise, in NG108 cells
stimulated by NMDA agonists, activation of PKB peaks at about 1 h
(25) whereas activation of CaM-KIV is maximal at about 2-5
min.2 These results suggest
that different substrate specificity determinants may be utilized by
CaM-KK for PKB activation, consistent with the difference in activation
of PKB versus CaM-KIV/CaM-KI by the RP-deletion mutant.
Therefore, the RP-deletion mutant of CaM-KK may serve as a useful tool
for evaluating the function of Ca2+-dependent
PKB activation and regulation of apoptosis without concurrent
activation of CaM-KI and -IV.
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FOOTNOTES |
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* 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 nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB016838.
§ To whom correspondence should be addressed: Helix Research Institute, Inc., 1532-3 Yana, Kisarazu-shi, Chiba 292-0812, Japan. Tel.: 81-0438-52-3967; Fax: 81-0438-52-3952; E-mail: tokumitu{at}hri.co.jp.
2 S. Yano and T. R. Soderling, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: CaM-K, Ca2+/CaM-dependent protein kinase; CaM, calmodulin; PKA, cAMP-dependent protein kinase; RP- domain, arginine-proline-rich-domain; PKB, protein kinase B; PAGE, polyacrylamide gel electrophoresis; RT-PCR, reverse transcriptase-mediated PCR; MAP, mitogen-activated protein; DTT, dithiothreitol; GST, glutathione S-transferase; bp, base pair(s).
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REFERENCES |
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