(Received for publication, September 18, 1995; and in revised form, December 27, 1995)
From the
When brain calmodulin-dependent protein kinase IV is incubated
with calmodulin-dependent protein kinase IV kinase under the
phosphorylation conditions in the presence of
Ca/calmodulin, rapid initial incorporation of 1 mol
of phosphate into 1 mol of the enzyme by the action of the kinase
kinase occurs, resulting in marked activation of the enzyme, and the
subsequent incorporation of more than 3 mol of phosphate by
autophosphorylation occurs, resulting in no significant change in the
activity (Okuno, S., Kitani, T., and Fujisawa, H.(1994) J. Biochem. (Tokyo) 116, 923-930; Okuno, S., Kitani, T., and
Fujisawa, H.(1995) J. Biochem. (Tokyo) 117,
686-690). After the maximal phosphorylation, the continued
incubation in the presence of excess EGTA resulted in additional
autophosphorylation of the enzyme, leading to a complete loss of the
Ca
/calmodulin-dependent activity, while causing no
significant change in the Ca
/calmodulin-independent
activity. The amino acid sequence analysis revealed that the
autophosphorylation after removal of Ca
occurred on
Ser
, Ser
, Ser
, and
Ser
. Analysis by site-directed mutagenesis clearly showed
that the autophosphorylation site responsible for the inactivation is
Ser
. Thus, calmodulin-dependent protein kinase IV
activated by the kinase kinase may lose its
Ca
/calmodulin-dependent activity by
autophosphorylation on Ser
located within the putative
calmodulin-binding domain in the absence of Ca
.
Calmodulin-dependent protein kinase IV (CaM-kinase IV) ()(also called CaM-kinase Gr) is a
Ca
/calmodulin-dependent multifunctional protein
kinase(1) , which is enriched in the brain (2, 3) and T-lymphocytes(4, 5) , and
is therefore expected to play important roles in controlling a variety
of functions in response to an increase in intracellular Ca
in the central nervous system and in the immune system. A recent
discovery of CaM-kinase IV kinase in the brain (6) suggested
the existence of a Ca
/calmodulin-dependent protein
kinase kinase cascade which is involved in the activation of CaM-kinase
IV in the central nervous system. CaM-kinase IV kinase has more
recently been purified to apparent homogeneity from rat cerebral
cortex(7) , and the activation mechanism of rat brain
CaM-kinase IV by CaM-kinase IV kinase has been studied(8) . The
present study demonstrates that CaM-kinase IV activated by CaM-kinase
IV kinase lost its Ca
/calmodulin-dependent activity
by autophosphorylation on Ser
(in CaM-kinase IV
)
located within the putative calmodulin-binding domain in the absence of
Ca
. Thus, unlike CaM-kinase II, which is activated
upon autophosphorylation on Thr
in the autoinhibitory
domain (9, 10, 11) , CaM-kinase IV is
activated upon phosphorylation by another
Ca
/calmodulin-dependent protein kinase, CaM-kinase IV
kinase, but after activation, both the CaM-kinases are inactivated by
autophosphorylation on threonine or serine residue located within their
calmodulin-binding domain in the absence of Ca
(Thr
in CaM-kinase II
(12, 13, 14, 15, 16) and
Ser
in CaM-kinase IV
).
Figure 1:
Time course
of decrease in the activity of CaM-kinase IV by phosphorylation in the
absence of Ca. A, recombinant CaM-kinase IV
(10 µg/ml) expressed in Sf9 cells was incubated with 0.2 µg/ml
CaM-kinase IV kinase in the phosphorylation mixture containing
nonradioactive ATP at 30 °C, as described under ``Experimental
Procedures'' (circles). At 60 min, EGTA was added to a
final concentration of 0.24 mM (triangles), and at 65
min, EDTA was added to a final concentration of 10 mM (squares). At the indicated times, 2-µl aliquots were
withdrawn, and the CaM-kinase IV activity was determined in the
presence of Ca
(closed symbols) or EGTA (open symbols) for 1 min, as described under
``Experimental Procedures.'' B, CaM-kinase IV was
incubated under the same conditions as described above, except that
radioactive ATP (3.7
10
cpm/nmol) was used. At the
indicated times, 4-µl aliquots were withdrawn and the incorporation
of [
P]phosphate into protein was determined as
described under ``Experimental Procedures.'' The
reproducibility of the data was confirmed by five independent
experiments.
To characterize the mechanism of the
phosphorylation causing the inactivation of the enzyme, effect of
varying the concentration of the enzyme on the phosphorylation rate was
investigated as shown in Fig. 2. The plot of the logarithm of
the phosphorylation rate versus the logarithm of the enzyme
concentration (van't Hoff plot) (34, 35) gave a
straight line with a slope of 1.2 (approximately 1), suggesting that
the phosphorylation of CaM-kinase IV in the absence of Ca causing the enzyme inactivation occurs through an intramolecular
autophosphorylation mechanism. The fact that the rate of the
phosphorylation after removal of Ca
was not affected
by increasing the concentration of CaM-kinase IV kinase (data not
shown) provides support for the autophosphorylation mechanism.
Figure 2: Effect of the enzyme concentration on autophosphorylation of CaM-kinase IV. A, CaM-kinase IV (20 µg/ml) was preincubated with 0.2 µg/ml CaM-kinase IV kinase in the phosphorylation mixture containing nonradioactive ATP for 1 h at 30 °C. The mixture was so diluted as to give the indicated concentrations of CaM-kinase IV and was incubated in the phosphorylation mixture containing radioactive ATP and excess EGTA without CaM-kinase IV kinase added. The initial velocity of the phosphorylation was calculated from the slope of the linear portion of the time course of the phosphate incorporation. The specific activity of the phosphorylation was plotted against the concentration of the enzyme. B, the results were replotted as logarithm of the initial velocity versus the logarithm of the enzyme concentration. The slope was 1.2. Data are the mean ± S.E. (n = 6).
Figure 3:
Phosphoamino acid analysis. Brain
CaM-kinase IV was incubated in the phosphorylation mixture containing
CaM-kinase IV kinase for 1 h at 30 °C with
[-
P]ATP (lane 1) or nonradioactive
ATP followed by incubation for another 4 h with
[
-
P]ATP in the presence of excess EGTA (lane 2). The samples were subjected to phosphoamino acid
analysis as described under ``Experimental Procedures.'' The
positions of phosphoserine (P-Ser), phosphothreonine (P-Thr), and phosphotyrosine (P-Tyr) are indicated by
the arrows.
Figure 4: Reverse-phase HPLC elution profiles of phosphopeptides from lysyl endopeptidase digest of phosphorylated CaM-kinase IV. A and B, brain CaM-kinase IV was incubated with CaM-kinase IV kinase in the phosphorylation mixture containing radioactive ATP for 60 min and then for another 60 min in the presence of excess EGTA. C, brain CaM-kinase IV was incubated with CaM-kinase IV kinase in the mixture containing radioactive ATP for 60 min. D, brain CaM-kinase IV was incubated with CaM-kinase IV kinase in the mixture containing nonradioactive ATP for 60 min and then for another 60 min in the mixture containing radioactive ATP in the presence of excess EGTA. After phosphorylation, the protein was digested with lysyl endopeptidase, and the resulting phosphopeptides were fractionated by HPLC in the triethylamine phosphate/acetonitrile solvent system, as described under ``Experimental Procedures.'' E and F, the radioactive fractions eluted as a big peak at about 48 min in B were subjected to the second HPLC in the trifluoroacetic acid/acetonitrile solvent system. G, the radioactive fractions eluted as a big peak at about 48 min in D were subjected to the second HPLC in the trifluoroacetic acid/acetonitrile solvent system. H, the radioactive fractions corresponding Peak E2 in B were subjected to the second HPLC in the triethylamine acetate/acetonitrile solvent system. Peptide (A, E, and inset in H) and phosphopeptide (B-D and F-H) peaks were monitored spectrophotometrically and radiometrically, respectively, as described under ``Experimental Procedures.'' The broken lines indicate the concentrations of acetonitrile. The recoveries of radioactivity upon HPLC were 80-97%.
Figure 5: Sequence analysis of the phosphopeptides. A, peptide C1 was subjected to amino acid sequence analysis. B-E, peptides C3 (B), E1 (C), E2-4 (D), and E2-6 (E) were subjected to amino acid sequence analysis after treatment with alkali ethanethiol, as described under ``Experimental Procedures.'' Recoveries of PTH-derivatives (open circles) and the relative amounts of S-ethylcysteine determined from the ratio toward the internal standard (closed circles) at each cycle are presented.
Figure 8:
Autophosphorylation sites of CaM-kinase
IV after removal of Ca. A, schematic
representation of the locations of phosphopeptides purified as
described in the legend to Fig. 5. The putative ATP-binding and
calmodulin-binding domains are represented by the solid and hatched bars, respectively. B, amino acid sequences
around the CaM-binding domains of CaM-kinase IV and CaM-kinase II. The
calmodulin-binding domain of CaM-kinase II is indicated by the solid underline. For details see
text.
Figure 6:
Time course of changes in the enzyme
activity of mutant CaM-kinase IV during the incubation under the
phosphorylation conditions. Aliquots of 2.8 µl of crude extracts of
bacteria transformed with expression vector pET11a carrying a cDNA
coding wild-type enzyme (A), mutant S332A (B), S332D (C), S333A (D), and S333D (E) were incubated
with 0.2 µg/ml CaM-kinase IV kinase in 140 µl of the
phosphorylation mixture containing nonradioactive ATP for 60 min at 30
°C and then for a further 4 h in the presence of excess EGTA. At
the indicated times, 5-µl aliquots were withdrawn, and the
CaM-kinase IV activity was determined in the presence (closed
circles) or absence (open circles) of
Ca, as described under ``Experimental
Procedures.'' Since all the bacterial crude extracts gave similar
intense protein bands corresponding to the position of the
isoform with a molecular weight of 63,000 upon SDS-polyacrylamide gel
electrophoresis, all the extracts contained similar amounts of the
enzymes. The reproducibility of the data was confirmed by three
independent experiments.
Since Ser is located within the putative
calmodulin-binding domain of CaM-kinase IV (40, 41) (Fig. 8), the loss of the
Ca
/calmodulin-dependent activity of the wild-type
enzyme autophosphorylated in the presence of EGTA and the mutant S332D
was thought to be due to loss of Ca
/calmodulin
binding. Calmodulin overlay analysis (Fig. 7) showed that the
wild-type enzyme, unphosphorylated or phosphorylated in the presence of
Ca
/calmodulin, and the mutant enzyme S333D bound
calmodulin in the presence of Ca
, but the wild-type
enzyme phosphorylated after addition of EGTA and the mutant S332D did
not significantly bind calmodulin even in the presence of
Ca
. Thus, the autophosphorylation of CaM-kinase IV on
Ser
occurring only in the absence of Ca
appears to cause loss of the ability of the enzyme to bind
calmodulin, thereby leading to loss of its
Ca
/calmodulin-dependent activity. The shift in
mobility on SDS-polyacrylamide gel electrophoresis of the enzyme upon
phosphorylation in the presence of Ca
/calmodulin
observed in Fig. 7A (lane 2) has been reported
previously(42) .
Figure 7:
Analysis of mutant CaM-kinase IV by
calmodulin overlay after SDS-polyacrylamide gel electrophoresis. A, 0.05 µg of recombinant CaM-kinase IV expressed in Sf9
cells (lanes 1 and 5), 0.05 µg of the enzyme
incubated for 1 h in the phosphorylation mixture containing 0.001
µg of CaM-kinase IV kinase in the presence of
Ca/calmodulin (lanes 2 and 6), 0.05
µg of the enzyme incubated for another 4 h in the presence of
excess EGTA after incubation for 1 h in the presence of
Ca
/calmodulin (lanes 3 and 7), and
0.001 µg of CaM-kinase IV kinase (lanes 4 and 8)
were subjected to SDS-polyacrylamide gel electrophoresis on 7.5% gel. B, 0.03-µl aliquots of crude extracts of bacteria carrying
wild-type enzyme (lanes 1 and 4), mutant S332D (lanes 2 and 5), and S333D (lanes 3 and 6) were electrophoresed as described above. After
electrophoresis, separate proteins were transferred onto polyvinylidene
difluoride membranes. The membranes were blocked with 5% non-fat milk
in 50 mM Tris-HCl (pH 7.5) containing 150 mM NaCl for
30 min at 24 °C and then incubated with 25 µg/ml biotinylated
calmodulin for 60 min, followed by incubation with 2 µg/ml avidin
conjugated with peroxidase for 2 h, in the presence of 1 mM CaCl
(lanes 1-4 in A and lanes 1-3 in B) or 5 mM EGTA (lanes 5-8 in A and lanes 4-6 in B). The positive bands which bound calmodulin were detected
with diaminobenzidine tetrahydrochloride and H
O
in the presence of CoCl
.
CaM-kinase IV is thought to play important roles in the
functioning of Ca in the central nervous system,
along with another Ca
-responsive multifunctional
protein kinase, CaM-kinase II, and therefore the regulation of its
activity is very important. Discovery of CaM-kinase IV kinase in the
brain (6) provided insight into the mechanism by which
CaM-kinase IV may be regulated, and phosphorylation of CaM-kinase IV by
CaM-kinase IV kinase has recently been demonstrated to cause a marked
activation of the enzyme(8) . Unlike CaM-kinase IV, CaM-kinase
II is activated upon Ca
/calmodulin-dependent
autophosphorylation at
Thr
(9, 10, 11) . Thus, the two
Ca
-responsive multifunctional protein kinases
occurring abundantly in the brain are activated upon phosphorylation,
by two contrasting mechanisms. After activation by
Ca
/calmodulin-dependent autophosphorylation at
Thr
, CaM-kinase II undergoes autophosphorylation at
Thr
in the absence of Ca
, resulting in
decrease in the Ca
/calmodulin-dependent activity
without decrease in the Ca
/calmodulin-independent
activity(12, 13, 14, 15, 16) .
The present study demonstrates that CaM-kinase IV loses its
Ca
/calmodulin-dependent activity by a similar
autophosphorylation mechanism.
When CaM-kinase IV was incubated with
CaM-kinase IV kinase under the phosphorylation conditions in the
presence of Ca/calmodulin until the phosphorylation
reached a maximum level (4.2 mol of phosphate/mol of enzyme), both the
Ca
/calmodulin-dependent and independent activities
were rapidly activated (Fig. 1) and phosphorylation of several
serine residues (Ser
, Ser
, Ser
,
etc.) in the segment of Val
-Lys
,
Ser
, and Ser
was observed ( Fig. 4and Fig. 5). Although threonine residues
(Thr
and Thr
) also have been reported to be
phosphorylated in the presence of
Ca
/calmodulin(42, 43) , our
phosphoamino acid analysis (Fig. 3) could not detect
phosphothreonine, probably owing to a low ratio of phosphothreonine to
phosphoserine in the phosphorylated enzyme preparation. When the
incubation was continued after the addition of EGTA to remove free
Ca
until the additional phosphorylation reached a
maximum (additionally 3 mol of phosphate/mol of enzyme), the
Ca
/calmodulin-dependent activity was decreased and
finally completely lost (Fig. 1) and phosphorylation of
Ser
, Ser
, Ser
,
Ser
, and Ser
was observed ( Fig. 4and Fig. 5). Thus, among many phosphorylation
sites, four serine residues, Ser
, Ser
,
Ser
, and Ser
, were phosphorylated only
after removal of Ca
.
Replacement of Ser with alanine by site-directed mutagenesis completely blocked the
inactivation by the incubation after removal of Ca
(Fig. 6B), probably by elimination of the
phosphorylation site responsive for the inactivation. Replacement of
Ser
with aspartic acid made the enzyme possess only very
low activity of the Ca
/calmodulin-dependent activity
even after activation by CaM-kinase IV kinase (Fig. 6C), probably by the action of Asp
mimicking phospho-Ser
. Replacement of
Ser
, Ser
, and even Ser
next
to Ser
with alanine or aspartic acid had essentially no
effect on the time course of the enzyme activity (Fig. 6, D and E). Thus, among four serine residues which were
phosphorylated in the presence of EGTA, the phosphorylation of only
Ser
appears to cause the inactivation of
Ca
/calmodulin-dependent activity, although the
possibility of a little involvement of other phosphorylation sites in
the inactivation cannot be excluded, because the mutant enzyme S332D
activated by CaM-kinase IV kinase exhibited very little but significant
Ca
/calmodulin-dependent activity, which was lost on
incubation in the presence of EGTA (Fig. 6C). The rate
of the inactivation appears to occur more slowly than that of the
phosphorylation, judging from the result of Fig. 1, suggesting
that the phosphorylation of Ser
occurs relatively slowly.
The finding that only Ser
of the four serine residues
which become phosphorylatable after removal of Ca
was
not phosphorylated in phosphopeptide E2-4 (Fig. 5E),
although all the four serine residues were phosphorylated in E2-6 (Fig. 5D), also suggests the slow phosphorylation of
Ser
. The inactivation rate of the mutant enzyme S333A was
much higher than that of the wild-type enzyme, and that of S333D was
also significantly higher than that of the wild-type enzyme, as shown
in Fig. 6, indicate that the adjacent amino acids affects the
phosphorylation rate of Ser
. As shown in Fig. 8,
alignment of the amino acid sequences of CaM-kinase IV and CaM-kinase
II showed that Ser
may be located within or near the
calmodulin-binding domain of CaM-kinase IV. The fact that replacement
of Ser
with aspartic acid strongly blocked the calmodulin
binding indicates that Ser
is located within the
calmodulin-binding domain. In contrast, replacement of
Ser
, the residue next to Ser
, with aspartic
acid did not affect the calmodulin binding, indicating that this amino
acid residue is not involved in the calmodulin binding.
A similar
regulatory mechanism by which phosphorylation of serine or threonine
residue located within a calmodulin-binding domain abolishes calmodulin
binding, thereby leading to a loss of the
Ca/calmodulin-dependent activity, is also observed
with CaM-kinase II; autophosphorylation of Thr
within the
calmodulin-binding domain in CaM-kinase II leads to a loss of the
Ca
/calmodulin-dependent activity (12, 13, 14, 15, 16) . This
together with the fact that Ser
is conserved in
rat(36, 38) , mouse(41) , and human (44, 45, 46) CaM-kinase IV suggests that the
inactivation of Ca
/calmodulin-dependent activity by
autophosphorylation at a serine or threonine residue located within a
calmodulin-binding domain in the absence of Ca
may be
a rather common and physiologically important regulatory mechanism for
Ca
/calmodulin-dependent protein kinases.