From the
Purified pig brain Ca
Ca
A critical issue in the study of CaM kinase regulation is the
molecular mechanism by which these kinases are activated in response to
elevations in Ca
It was reported that
Ca
We report here the
phosphorylation-dependent activation of recombinant CaM kinase IV by
purified CaM kinase Ia kinase. Thus, CaM kinases I and IV appear to be
regulated by either common or highly related kinase kinases. We also
report that replacement of Thr-196 of CaM kinase IV with Ala by
site-directed mutagenesis prevents both phosphorylation and activation,
indicating that Thr-196 phosphorylation is essential for activation.
The activity of recombinant CaM kinase IV is enhanced 24-fold
to a specific activity of 1.7 µmol min
Figure 1:
Activation of CaM
kinase IV by CaM kinase Ia kinase (CaMKIa Kinase). Recombinant
CaM kinase IV, expressed in Sf9 cells and purified as described under
``Experimental Procedures'' (1 µg/ml), was preincubated
in the presence or absence of CaM kinase Ia kinase (0.1 µg/ml) as
indicated for 120 min at 30 °C. All preincubations also contained
10 mM MgCl
Figure 2:
Time
course and requirements for CaM kinase IV activation. Recombinant CaM
kinase IV, expressed in Sf9 cells and purified as described under
``Experimental Procedures'' (0.8 µg/ml), was preincubated
in the presence (closedsymbols) or absence (opensymbols) of CaM kinase Ia kinase (0.1 µg/ml) for the
indicated time periods of preincubation, at 30 °C, with the
following additions (as final concentrations; CaCl
Figure 3:
The phosphorylation of CaM kinase IV
requires CaM kinase Ia kinase, Ca
Figure 4:
Relationship between the phosphorylation
and activation states of CaM kinase IV. Recombinant CaM kinase IV,
expressed in Sf9 cells and purified as described under
``Experimental Procedures'' (2.8 or 4.9 µg/ml), and CaM
kinase Ia kinase (0.5 µg/ml) were preincubated in a total volume of
76 µl at 30 °C, in a mixture containing 50 mM Tris, pH
7.6, 1 mM CaCl
Figure 5:
Phosphoamino acid analysis of
phosphorylated CaM kinase IV. Recombinant CaM kinase IV, expressed in
Sf9 cells and purified as described under ``Experimental
Procedures'' (21 µg/ml), was incubated with CaM kinase Ia
kinase (1 µg/ml) for 15 min., at 30 °C, in a mixture containing
25 mM Tris, pH 7.6, 1 mM CaCl
Figure 6:
Replacement of Thr-196 of CaM kinase IV
with alanine prevents the phosphorylation of CaM kinase IV by CaM
kinase Ia kinase. Recombinant CaM kinase IV wild type (CaMK IV
wt) and T196A mutant, expressed in E. coli as glutathione S-transferase fusion proteins and purified as described under
``Experimental Procedures,'' were incubated (both at
concentrations of 20 µg/ml) in the presence or absence of CaM
kinase Ia kinase (0.04 µg/ml) as indicated for 10 min at 30 °C.
All incubations also contained 50 mM Hepes, pH 7.5, 1 mM CaCl
Figure 8:
Activity
of CaM kinase IV (CaMKIV) in the presence and absence of CaM
kinase Ia kinase (CaMKIa Kinase) and the presence and absence
of rat brain extract. Recombinant CaM kinase IV, expressed in Sf9 cells
and purified as described under ``Experimental Procedures''
(7.6 µg/ml), was preincubated in the presence or absence of CaM
kinase Ia kinase (0.04 µg/ml) and in the presence or absence of rat
brain extract (15 µg/ml) as indicated for 30 min at 30 °C. All
preincubations also contained 50 mM Hepes, pH 7.5, 1 mM CaCl
In a number of respects, the responses of CaM kinases Ia and
IV to CaM kinase Ia kinase are virtually identical. Both kinases are
maximally activated >20-fold in reactions requiring CaM kinase Ia
kinase, Ca
Whatever its mechanism, the
presence of multiple phosphorylation sites raises a question as to
whether phosphorylation of an amino acid residue other than Thr-196 is
ultimately responsible for induction of the
Ca
The complete inability of the T196A mutant to be both phosphorylated
and activated provides direct evidence that phosphorylation of Thr-196
is essential for the activation of CaM kinase IV ( Fig. 6and Fig. 7). Nevertheless, it is conceivable that the T196A
substitution by itself could induce a conformational change that blocks
activation. The latter possibility would, however, appear to be highly
unlikely considering that this is a single conservative replacement and
that in all of its properties we have tested other than its inability
to be activated the T196A mutant enzyme is indistinguishable from the
wild type enzyme. For example, its basal specific activity is the same
as that of the wild type enzyme (Fig. 7); it binds calmodulin in
a CaM overlay procedure; and it is of equivalent immunoreactivity as
the wild type enzyme using a CaM kinase IV antibody.
Figure 7:
Replacement of Thr-196 of CaM kinase IV
with alanine prevents the activation of CaM kinase IV by CaM kinase Ia
kinase (CaMKIa Kinase). Recombinant CaM kinase IV wild type (CaMKIVwt) and T196A mutant, expressed in E. coli as
glutathione S-transferase fusion proteins and purified as
described under ``Experimental Procedures,'' were
preincubated (both at concentrations of 16 µg/ml) in the presence
or absence of CaM kinase Ia kinase (0.04 µg/ml) as indicated for 10
min at 30 °C. All preincubations also contained 50 mM Hepes, pH 7.5, 1 mM CaCl
The prevalence of these
observations raises a question as to the ultimate physiological
importance of such phosphorylation events. In some instances, for
example in that of cAMP-dependent protein kinase, this phosphorylation
may be autocatalytic following translation and resistant to
intracellular phosphatase action(49, 57) . There are,
however, a number of lines of evidence to suggest that in some cases,
these phosphorylations represent a form of acute regulation and serve
cell-specific functions. First, although a requirement for a
phosphorylated residue (or acidic residue as a phosphoamino acid
mimetic) at this position may be common, it is by no means universal.
For example, within the CaM kinase family such residues are absent from
CaM kinase II (58, 59, 60) and myosin light
chain kinase isoforms(61, 62) . Second, the ability to
isolate CaM kinase Ia from tissue in the nonactivated, i.e. dephosphorylated state, and the ability to reverse activation with
purified phosphoprotein phosphatase 2A (6) imply that
dephosphorylation is reasonably facile and that there is, therefore,
the potential for a phosphorylation/dephosphorylation cycle in
vivo. Third, although perhaps autocatalytic in the case of
cAMP-dependent protein kinase, it is now established that in most of
the other cases, cited above, a heterologous kinase(s) is involved, and
in some of the other cases, for example mitogen-activated protein
kinase, the signal transduction pathway involved has been elucidated.
Two recent reports (36, 37) described
66-68-kDa CaM kinase IV activators from rat brain that appear
themselves to be protein kinases. And another report described a
preparation capable of activating and phosphorylating both CaM kinase I
and CaM kinase IV(42) . It will ultimately be of interest to
determine the relationship between these CaM kinase activators and the
The upstream regulatory signals that may serve to drive CaM kinase
kinase-catalyzed phosphorylation of CaM kinases I and IV is an area of
active investigation in our laboratories. An example of the form this
regulation might take was suggested by Hanissian et al.(63) , who observed that in Jurkat cells, in addition to
Ca
We are indebted to Dr. J. C. Lee for performing
valuable preliminary studies. We also thank Dr. J. Aletta for helpful
discussions and T. Atkinson for photographic illustrations.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-calmodulin
(CaM)-dependent protein kinase Ia kinase (Lee, J. C., and Edelman, A.
M.(1994) J. Biol. Chem. 269, 2158-2164) enhances, by up
to 24-fold, the activity of recombinant CaM kinase IV in a reaction
also requiring Ca
-CaM and MgATP. The addition of
brain extract, although capable of activating CaM kinase IV by itself,
provides no further activation beyond that induced by purified CaM
kinase Ia kinase, consistent with the lack of a requirement of
additional components for activation. Activation is accompanied by the
development of significant (38%) Ca
-CaM-independent
CaM kinase IV activity. In parallel fashion to its activation, CaM
kinase IV is phosphorylated in a CaM kinase Ia kinase-,
Ca
-CaM-, and MgATP-dependent manner. Phosphorylation
occurs on multiple serine and threonine residues with a Ser-P:Thr-P
ratio of
3:1. The identical requirements for phosphorylation and
activation and a linear relationship between extent of phosphorylation
of CaM kinase IV and its activation state indicate that CaM kinase IV
activation is induced by its phosphorylation. Replacement of Thr-196 of
CaM kinase IV with a nonphosphorylatable alanine by site-directed
mutagenesis abolishes both the phosphorylation and activation of CaM
kinase IV, demonstrating that Thr-196 phosphorylation is essential for
activation.
-calmodulin
(CaM)
(
)-dependent protein kinases (CaM kinases)
have long been known to mediate effects of elevated intracellular
Ca
on cellular functions such as glycogen metabolism,
muscle contraction, and neurotransmitter synthesis and release. New
observations suggest that, in addition, CaM kinases are involved in
Ca
-dependent regulation of translational rates, of
the transcription of specific genes, and of cell cycle events (for
reviews, see (1) ). The CaM kinases constitute a family of
related enzymes that includes, among its better-characterized members,
phosphorylase kinase, myosin light chain kinase, and CaM kinase II
(reviewed in (2) and (3) ). Recently, a significant
amount of information about CaM kinase structure and function has been
added with the successful purification, characterization, and/or
cloning of CaM kinases
I(4, 5, 6, 7, 8, 9, 10) ,
III(11, 12) , and
IV(13, 14, 15, 16, 17, 18, 19, 20, 21) .
concentrations. The currently
accepted paradigm is a conformational change induced by the
stoichiometric binding of Ca
-CaM, which in turn
results in decreased interaction of an autoinhibitory domain with the
active site, thereby permitting substrate accessibility. This mechanism
is ideally suited to the decoding of rapid Ca
transients provoked by extracellular signals and is well
understood at the molecular level primarily through studies with myosin
light chain kinase and CaM kinase
II(22, 23, 24, 25, 26, 27, 28) .
Evidence has now accumulated, based on studies with CaM kinase I and
CaM kinase IV, for an important mechanism of CaM kinase activation in
addition to Ca
-CaM-induced relief from
autoinhibition.
-CaM-dependent autophosphorylation of purified rat
brain CaM kinase I (a-isoform) is accompanied by a dramatic
(>10-fold) potentiation of activity and that an activator exists
that is separable from CaM kinase Ia during the final steps of the
latter's purification(5, 6) . These observations
were confirmed for CaM kinase V(29) , a CaM kinase I
isoform(9) . The successful purification of CaM kinase Ia
activator from pig brain was recently reported(30) . In the
presence of the purified activator, CaM kinase Ia is phosphorylated and
is activated up to 50-fold. Autophosphorylation-autoactivation, if it
occurs at all, does so at an extremely slow rate relative to
phosphorylation and activation by the activator(30) , an
observation consistent with the lack of significant autoactivation by
bacterially expressed CaM kinase I(8) . Since CaM kinase Ia
activator is able to phosphorylate and activate brain CaM kinase Ia
inactivated by an irreversible ATP affinity analogue or a
kinase-negative mutant of recombinant CaM kinase I, it may be concluded
that the activator is itself a protein kinase, that is, a CaM kinase I
kinase(31) .
(
)In similar fashion to
CaM kinase I, purified rat brain CaM kinase IV was reported to be
subject to Ca
-CaM-dependent autophosphorylation
resulting in activation(32, 33) . Compared with the
brain enzyme, recombinant CaM kinase IV expressed in bacterial or
insect cells has high K
values for substrates,
slowly autophosphorylates, and demonstrates minimal
activation(19, 34) . However, phosphorylation and
activation of recombinant CaM kinase IV was found to be markedly
stimulated by the addition of rat brain extract (35) or CaM
kinase IV activators that appear themselves to be protein
kinases(36, 37) .
Peptides
Syntide-2 was purchased from Life
Technologies, Inc. GS10 was generously provided by Dr. Bruce Kemp (St.
Vincent's Institute for Medical Research, Melbourne, Australia).
Porcine and chicken calmodulin were obtained from Boehringer Mannheim
or produced by bacterial expression(38) , respectively.
PKI-tide (TTYADFIASGRTGRRNAIHD) was purchased from Sigma.
Expression and Mutagenesis of CaM Kinase
IV
General molecular biology techniques were carried out
according to Sambrook et al.(39) . CaM kinase IV was
expressed in insect Sf9 cells using the baculovirus system as described
previously(19) . Alternatively, CaM kinase IV was expressed in E. coli as follows. The cDNA encoding rat brain CaM kinase IV
was subcloned as a pGCaMKIV (15) SacI/XbaI
fragment into the SacI/XbaI sites of pGEX-3XMCS
(pGEX-3X from Pharmacia Biotech Inc., modified so as to contain the
multiple cloning site of pUC19), to generate pGEXCaMKIV. Recombinant
protein was then expressed from pGEXCaMKIV in E. coli strain
BL21 (DE3) as a glutathione S-transferase fusion protein and
purified using glutathione-Sepharose. T196A mutant cDNA was constructed
as follows. A 659-base pair cDNA fragment was prepared from a 30-cycle
polymerase chain reaction using pGCaMKIV as template, an
oligonucleotide primer (sense) overlapping a SacI site (5` CAC
TAT AGG GCG AAT TCG AGC TCG GAC CCG GCG AAG ATG 3`), and, an
oligonucleotide primer (antisense) overlapping a SmaI site
that introduces the point mutation indicated by boldface type (5` AAT
CTC AGG TGC ACA GTA CCC CGG GGT TCC ACA CAC CGC CTT CAT GAG 3`).
The 659-base pair polymerase chain reaction product was subcloned as a SacI/SmaI fragment into the SacI/SmaI sites of pGCaMKIV to generate
pGCaMKIVT196A. After confirming the presence of the mutated base and
the correct ligation junctions by nucleotide sequence analysis using
U.S. Biochemical Corp. Sequenase version 2.0 DNA sequencing kit and
custom-designed oligonucleotides, the mutant cDNA was subcloned into
pGEX-3XMCS and expressed and purified as described above.
CaM Kinase Ia Kinase
CaM kinase Ia kinase was
purified from pig brain by a procedure previously described in
detail(30) , modified by reversal of the order of the
CaM-Sepharose and phenyl-Sepharose chromatographic steps and by
omission of the hydroxylapatite chromatographic step.
Rat Brain Extract Preparation
Rat brain was
homogenized with a Potter-Elvehjem homogenizer in 3 volumes of a buffer
containing 5 mM Tris, pH 7.5 (at 4 °C), 0.1 mM DTT, 10 µg/ml antipain, 10 µg/ml leupeptin, 10 µg/ml
phenylmethylsulfonyl fluoride, and 10 µg/ml trypsin inhibitor.
After centrifugation of the homogenate at 100,000 g at
4 °C, the supernatant was desalted over Sephadex G-25 and stored in
small aliquots at -70 °C.
Peptide Kinase Assay
The activity of CaM kinase IV
was assayed at 30 °C using either Syntide-2 or GS10 as substrate as
indicated in the figure legends. Syntide-2 kinase activity was measured
by incubation in a mixture containing 50 mM Tris, pH 7.6, 1
mM CaCl, 1 µM CaM, 0.5 mM DTT, 0.5 mg/ml bovine serum albumin, 10 mM
MgCl
, 200 µM
[
-
P]ATP (
0.2
10
cpm/pmol), and 50 or 100 µM Syntide-2. GS10 kinase
activity was measured by incubation in a mixture containing 50 mM Hepes, pH 7.5, 1 mM CaCl
, 1 µM CaM, 30 µM PKI-tide, 10 mM MgCl
,
50 or 200 µM [
-
P]ATP
(0.1-1.2
10
cpm/pmol), and 40-50
µM GS10. Quantitation of
P incorporation into
peptides was determined by their binding to P81 phosphocellulose paper
as described previously(5, 19) .
Phosphoamino Acid Analysis
P-labeled
CaM kinase IV was subjected to SDS-polyacrylamide gel electrophoresis
and electrophoretically transferred onto polyvinylidene difluoride
membrane (Immobilon, Millipore) (100 V, 90 min). The membrane was then
rinsed in deionized water, dried, and autoradiographed. Using the
autoradiogram as template, the polyvinylidene difluoride strip
containing
P-labeled CaM kinase IV was excised from the
blot, rinsed successively in methanol and water, and hydrolyzed in 6 N HCl (110 °C, 1 h) under vacuum. The
P-labeled phosphoamino acids were dried, resuspended in
deionized water containing phosphoamino acid standards, and subjected
to two-dimensional separation by ascending chromatography in
2-propanol:HCl:H
O (7:1.5:1.5) followed by high voltage
electrophoresis (500 V, 140 min) in 7.5% acetic acid, 2.85% formic
acid, pH 1.9. The standards and
P-labeled phosphoamino
acids were identified by ninhydrin staining and autoradiography,
respectively. For relative quantitation of
P
incorporation, the cellulose corresponding to each phosphoamino acid
was scraped from the plate using the autoradiogram as a template and
counted in Ready-solve CP (Beckman) after extraction in methanol.
Other Methods
SDS-polyacrylamide gel
electrophoresis was performed as described
previously(19, 30) . The concentration of CaM kinase
IV was determined by the method of Lowry et al. (40) as modified (5) or by the method of Bradford et al.(41) . CaM kinase Ia kinase concentration was
determined by a colloidal gold staining method(30) . CaM was
quantified by spectrophotometry (E = 3300).
mg
by the presence of purified pig brain CaM
kinase Ia kinase (Fig. 1). Unlike CaM kinase Ia, which remains
fully Ca
-CaM-dependent in the activated
state(6, 30) , CaM kinase IV activation is accompanied
by the development of significant (38%)
Ca
-CaM-independent activity. The time course and
requirements for the activation of CaM kinase IV are illustrated in Fig. 2. As shown, the combined presence of CaM kinase Ia kinase,
Ca
-CaM, and MgATP is necessary for full activation to
occur. The mechanism underlying the
Ca
-CaM-requirement for activator responsiveness, a
phenomenon also observed for CaM kinase Ia(6, 30) , is
under active investigation in our laboratories.
, 0.2 mM ATP, 1 mM CaCl
, 1 µM CaM. Peptide kinase activity
was then measured using Syntide-2 as substrate, as described under
``Experimental Procedures'' for 2 min with the exception that
the kinase assay was performed in either the presence of 1 mM CaCl
and 1 µM CaM or the absence of
Ca
-CaM and presence of 2 mM EGTA as
indicated. Each bar represents the mean ± S.E. (n = 6). One unit of CaM kinase IV activity = 1
µmol of
P transferred to Syntide-2 in 1
min.
and
MgCl
are given as concentrations in excess of EGTA and EDTA
concentrations, respectively): 10 mM MgCl
, 0.2
mM ATP (squares); 1 mM CaCl
, 1
µM CaM (triangles); or 10 mM MgCl
, 0.2 mM ATP, 1 mM
CaCl
, 1 µM CaM (circles). Peptide
kinase activity was then measured using Syntide-2 as substrate in the
presence of Ca
(1 mM in excess of EGTA) and
CaM (1 µM) as described under ``Experimental
Procedures'' for 1-2 min. For background subtractions under
each condition, activity in the absence of preincubation (zero time of
preincubation) has been subtracted to control for activation occurring
during the assay itself, and where appropriate, the activity of CaM
kinase Ia kinase alone has also been subtracted. Each point represents
the mean of duplicate determinations.
Based on the MgATP
requirement for activation, we investigated whether CaM kinase IV is
phosphorylated in the presence of CaM kinase Ia kinase. As shown in Fig. 3, CaM kinase IV is phosphorylated in a reaction requiring
(in addition to MgATP), CaM kinase Ia kinase and
Ca-CaM. The identical requirements for both
activation and phosphorylation suggest that activation of CaM kinase IV
is caused by its Ca
-CaM-dependent phosphorylation by
CaM kinase Ia kinase. Consistent with this mechanism is a linear
relationship between the extent of phosphorylation and state of
activation of CaM kinase IV (Fig. 4). The maximal extent of
P incorporation achieved (6.5 ± 1.7 mol of
P/mol of CaM kinase IV) demonstrated some variability
between experiments but indicates multiple sites of phosphorylation.
Phosphoserine is the predominant phosphoamino acid detected with a
ratio of serine to threonine phosphorylation of 2.7 (± 0.6):1 (Fig. 5).
-CaM, and MgATP.
Recombinant CaM kinase IV, expressed in Sf9 cells and purified as
described under ``Experimental Procedures'' (7 µg/ml),
and CaM kinase Ia kinase (0.5 µg/ml) were incubated alone or
together for 15 min., at 30 °C, with the following additions as
indicated (final concentrations; CaCl
is given as
concentration in excess of the EGTA concentration): 1 mM
CaCl
, 1 µM CaM; or 0.6 mM EGTA (CaM
omitted). All incubations also contained the following: 50 mM Tris, pH 7.6, 0.5 mM DTT, 10 mM
MgCl
, and 20 µM
[
-
P]ATP (3
10
cpm/pmol). Reactions were terminated by boiling in
SDS-
-mercaptoethanol dissociation solution and electrophoresed in
a 10% polyacrylamide SDS-gel. The resultant autoradiogram of the
stained and destained gel is shown. The arrow indicates the
position of CaM kinase IV.
, 1 µM CaM, 0.5 mM DTT, 0.5 mg/ml bovine serum albumin, 10 mM
MgCl
, and 20 µM
[
-
P]ATP (
7
10
cpm/pmol). At five time points over a period of 2 h, replicate
aliquots of the reaction mixture were separately and simultaneously
analyzed for the states of activation and phosphorylation of CaM kinase
IV. CaM kinase IV activation state was assessed by Syntide-2 kinase
activity in the presence of Ca
-CaM, utilizing
background subtractions as described in the legend to Fig. 2.
The phosphorylation of CaM kinase IV was determined by applying
8-µl aliquots of the preincubation reaction mixture to 2
2
cm squares of 3MM filter paper (Whatman). Squares were then washed once
for 30 min at 0 °C in 10% trichloroacetic acid containing 2% sodium
pyrophosphate followed by two washes for 15 min each in 5%
trichloroacetic acid at 22 °C and dried with 95% ethanol.
P-incorporation was quantified by liquid scintillation
counting with subtraction of a CaM kinase Ia kinase alone condition as
background. Results are expressed as the linear regression of
activation versus phosphorylation after normalization to the
maximal extent of both achieved. Each point represents the mean of
duplicate determinations. Linear regression was performed using
Sigmaplot version 1.02 (Jandel Scientific).
, 1
µM CaM, 0.5 mM DTT, 10 mM MgCl
, and 20 µM [
-
P]ATP (13
10
cpm/pmol). The reaction was terminated by boiling in
SDS-
-mercaptoethanol dissociation solution and electrophoresed in
a 10% SDS-polyacrylamide gel.
P-labeled CaM kinase IV was
then subjected to phosphoamino acid analysis by two-dimensional,
ascending chromatography/high voltage electrophoresis as described
under ``Experimental Procedures.'' The positions of
phosphotyrosine (PY), phosphoserine (PS), and
phosphothreonine (PT) are
indicated.
Parallel studies in our laboratories have
established that the phosphorylation of Thr-177 of recombinant CaM
kinase I by CaM kinase Ia kinase is essential for CaM kinase I
activation. In addition, Sugita et al.(42) reported that Thr-177 of CaM kinase V is a
phosphorylation site for a CaM kinase kinase preparation. With this
information as rationale, we addressed the critical question of the
phosphorylation site(s) responsible for CaM kinase IV activation
through site-directed mutagenesis of the equivalent amino acid residue
in CaM kinase IV, Thr-196. As shown in Fig. 6and 7, replacement
of Thr-196 with a nonphosphorylatable alanine abolishes both the
phosphorylation and activation of CaM kinase IV by CaM kinase Ia
kinase. Thr-196 phosphorylation is thus absolutely required for CaM
kinase IV activation to occur.
, 1 µM CaM, 30 µM PKI-tide, 10 mM MgCl
, and 50 µM [
-
P]ATP (0.9
10
cpm/pmol). Reactions were terminated by boiling in
SDS-
-mercaptoethanol dissociation solution and electrophoresed on
a 10% polyacrylamide SDS-gel. The resultant autoradiogram of the
stained and destained gel is shown. The arrow indicates the
position of CaM kinase IV which electrophoreses with slower mobility
than as illustrated in Fig. 3due to fusion with glutathione S-transferase.
Finally, despite the maximal extent
of activation achievable (>20-fold, Fig. 1), there remained a
possibility that additional components necessary for full activation
are lost during CaM kinase Ia kinase purification. As shown in Fig. 8, although rat brain extract is capable of activating
recombinant CaM kinase IV, this effect is no longer observed after
activation by CaM kinase Ia kinase. These results indicate that
purified CaM kinase Ia kinase is sufficient for full activation of CaM
kinase IV.
, 1 µM CaM, 10 mM
MgCl
, and 200 µM ATP. Peptide kinase activity
was then measured using GS10 as substrate, as described under
``Experimental Procedures'' for 6 min. Each bar represents the mean ± S.E. (n = 2) with the
exception that the activity of the brain extract alone was calculated
as the difference between CaM kinase Ia kinase plus brain extract and
CaM kinase Ia kinase alone.
-CaM, and MgATP, and both are
phosphorylated with these same requirements. Nonetheless, we have
observed several prominent differences between the two CaM kinases. The
first is that while the activity of CaM kinase Ia is completely
Ca
-CaM-dependent after
activation(6, 30) , CaM kinase IV develops significant
(
38%) Ca
-CaM-independent activity as a result of
activation. Second, while incubation of CaM kinase Ia with CaM kinase
Ia kinase in the presence of Ca
-CaM and MgATP results
in phosphorylation almost exclusively on
threonine(5, 31) , incubation of CaM kinase IV under
the same conditions leads to the phosphorylation of multiple serine and
threonine residues with a Ser-P/Thr-P ratio of
3:1 (Fig. 5). These observations are similar to those of McDonald et al.(43) who reported multiple CaM kinase IV
autophosphorylation sites with serine the predominant phosphoamino acid
detected. Since there is no significant phosphorylation of the T196A
mutant enzyme (Fig. 6), most of the observed
P-incorporation appears to be generated in a burst of
secondary autophosphorylation reflecting the activated state of CaM
kinase IV. In this scenario, the close temporal relationship between total CaM kinase IV phosphorylation and activation, evident
from the linear relationship between the two (Fig. 4), implies
that autophosphorylation rapidly follows Thr-196 phosphorylation and
that the former is therefore, in all probability, intramolecular.
Supportive of this hypothesis is the observation that a kinase-negative
mutant of CaM kinase IV is phosphorylated in the presence of CaM kinase
Ia kinase to a considerably lesser extent than is the wild type
enzyme.
(
)
-CaM-independent activity of CaM kinase IV. That
this indeed may be the case is suggested by two additional
observations. First, as noted above, CaM kinase I does not acquire
Ca
-CaM independence after phosphorylation by CaM
kinase Ia kinase (6, 30) even though phosphorylation
occurs at a site (Thr-177)
equivalent to Thr-196 in CaM
kinase IV. Activation and Ca
-CaM independence are
therefore, for any given case, potentially dissociable phenomena.
Second, precedence for the generation of a
Ca
-CaM-independent CaM kinase activity was
established through studies of the autophosphorylation of CaM kinase
II. In this case, phosphorylation occurs in the autoinhibitory domain
itself (Thr-286 in CaM kinase II
, (44, 45, 46, 47) ), whereas in CaM
kinase IV, Thr-196 is considerably removed, at least in the linear
sequence from the autoinhibitory domain, identified as including
residues 313-323 (19) or 308-317(36) .
Thus, apart from the activation process per se, elucidation of
the mechanism of generation of Ca
-CaM-independent CaM
kinase IV activity may yield insights into the means for prolongation
of the duration of action of Ca
-mobilizing signals.
It
should also be noted that the requirement for Thr-196 phosphorylation
does not preclude the possibility that this event may induce
phosphorylation at other sites that may also participate in the
activation process.
, 1 µM CaM, 10 mM MgCl
, and 50 µM ATP.
Peptide kinase activity was then measured using GS10 as substrate as
described under ``Experimental Procedures'' for 6 min. Each bar represents the mean ± S.E. (n =
2).
Thr-196 is found in the sequence
LMKTVCGTPGYCAPE(15) , contained in the catalytic
domain in subdomain VIII, a region highly conserved among protein
kinases(48) . It has recently become apparent that for many
protein kinases, phosphorylation of a Ser or Thr residue located 9 or
10 residues amino-terminal of the nearly invariant (A/S)PE sequence is
essential for the expression of full catalytic activity. Kinases for
which this phenomenon has been demonstrated include the following:
cAMP-dependent protein kinase catalytic subunit (49) , protein
kinase C(50) , mitogen-activated protein kinase(51) ,
mitogen-activated protein kinase kinase-1 (52, 53) and
the cyclin-dependent kinases, cdc 2, cdk 2, and cdk
4(54, 55, 56) . This list has now been
expanded to include the CaM kinase family members, CaM kinase IV (this
report), and CaM kinase I.
52-kDa CaM kinase Ia kinase purified from pig brain(30) ,
which was used here. Moreover, as noted previously, CaM kinase Ia
kinase activity demonstrates chromatographic heterogeneity during
purification, suggesting the existence of isoforms (30) . Thus,
although, CaM kinase Ia kinase is capable of fully activating CaM
kinase IV (Fig. 8), it is possible that there are multiple CaM
kinase I/CaM kinase IV activating kinases and that these enzymes have
similar substrate specificities but relative preferences for the two
CaM kinase targets. It is also possible, however, that there are
species differences between rat and pig brain CaM kinase activators,
which may account for the apparent differences in reported molecular
weights. A final possibility is that there are distinct activators that
produce their effects by different mechanisms. For example, it was
proposed that phosphorylation of Ser-437 results in CaM kinase IV
activation (33) and that CaM kinase IV is phosphorylated by
the 66-kDa rat brain CaM kinase IV activator kinase exclusively on
serine residues(37) . Also Tokumitsu et al. (36) have hypothesized that a 68-kDa CaM kinase IV activator
kinase may recognize argininyl residues amino-terminal of its
phosphorylation site. It will be of importance, therefore, to evaluate
these suggestions in light of the information presented here indicating
that Thr-196 phosphorylation is essential for CaM kinase IV activation.
-influx, a signal delivered through TCR.CD3 is
required for full CaM kinase IV activation, an observation consistent
with a need for an additional signal for the activation of CaM kinase
IV kinase(s). Finally, it may be noted that whatever the precise
physiological role of these phosphorylation events for the CaM kinase
family, the existence of CaM kinase kinase(s) as well as the earlier
emergence of the concept of Ca
-CaM-independent CaM
kinase activity, suggest that a full appreciation of the complexity of
regulation of these key signal-transducing enzymes has yet to be
attained.
-CaM-dependent protein kinase; Syntide-2
and GS10, synthetic peptides PLARTLSVAGLPGKK and PLRRTLSVAA,
respectively, based on phosphorylation site 2 in glycogen synthase;
PKI-tide, synthetic peptide based on the heat-stable inhibitor of
cAMP-dependent protein kinase (amino acid residues 5-24); DTT,
dithiothreitol.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.