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Phosphorylation of the calmodulin-dependent protein phosphatase by protein kinase C
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 783-788
Vol. 138, No. 2,1986 July31,1986
PHOSPHORYLATION OF THE CALMODULIN-DEPENDENT PROTEIN PHOSPHATASE BY PROTEIN KINASE C H. Y. Lim Tung Clayton Foundation Biochemical Institute The University of Texas at Austin, Austin, Texas 78712 Received June 2, 1986
The c a l m o d ~ i n - d e p e n d e n t protein phosphatase was shown to be phosphorylated by the Ca , phospholipid-dependent protein kinase (protein kinase C). Analysis by sodium dodecyl sulfate-polyacrylamide gel eleetrophoresis indicated that the 61 kDa catalytic subunit was phosphorylated. Phosphorylation by protein kinase C was stimulated up to 15-fold by addition of phosphatidyl-Lserine and between 0.5 to 1.0 mole of phosphate was incorporated per mole of phosphatase. It is possible that protein kinase C is involved in the regulation of the calmodulin-dependent protein phosphatase via this novel phosphorylation of the enzyme. © 1986 AcademicPress, Inc.
The
calmodulin-dependent
protein
phosphatase
was
first
identified
in
rabbit skeletal muscle [1,2] and shown to be identical to a brain calmodulinbinding
protein
consists subunit
[3]
originally
of two subunits, of apparent
termed
calcineurin
A and B in a molar
molecular mass
61 kDa
[4,5]
Or CamBP80
ratio of 1:1
interacts with
[6] and
[7,8,9].
The A
calmodulin
in the
presence of calcium [1,7], and the B subunit is a Ca 2+ binding protein which shows
35%
binding
regions
phosphatase low
identity
containing specificity.
[i0].
activity,
molecular
with
mass
proteins
calmodulin In
with
addition
the enzyme has phospho [12,13],
to
strong
homology
possessing
around
the
Ca 2+
phosphoseryl/threonyl
recently been shown to dephosphorylate
compounds indicating
[ii]
as
that
well the
as enzyme
phosphotyrosine has
multiple
Here we demonstrate that in addition to being regulated by Ca 2+
and calmodulin,
the enzyme can also be phosphorylated by protein kinase C, a
Ca 2+, phospholipid-dependent protein kinase [14].
MATERIALS AND METHODS Histone H I , casein, phospha~yl-L-serine and cyclic AMP-dependent protein kinase ~ere from Sigma. Ca- , phospholipid-dependent protein kinase (protein kinase C) and calmodulin were purified from bovine brain essentially as described by Walsh et al. [15]. Calmodulin-dependent protein phosphatase
783
0006-291X/86 $1.50 Copyr~ht © 1986 ~ Aca~mk' Press, Inc. All r~hts 0/' r~roduction in at~ jorm reserved.
Vol. 138, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
was also purified from bovine brain essentially as described by Sharma et al. [16], except that the last chromatography on Sephadex G-200 was replaced by chromatography on Sephacryl S-300. C a l m o d ~ n - d e p e n d e n t p r o t e ~ phosphatase was assayed by determining the release of u-P-phosphate from P-labeled casein prepared by phosphorylation with cyclic AMP-dependent protein kinase. The assay (60 ~i) consisted of diluted phosphatase in 50 mM Tris-C1, pH 7.5, 0.1% 2-mercaptoethanol, 1 mg/ml bovine serum albumin, I0 mM m a g n e ~ u m chloride, 20 ~g/ml calmodulin, 0.1 mM c a l c i ~ chloride and 0.2 mg/ml of P-labeled casein. Reaction was initiated with P-labeled substrate, allowed to take place for i0 min, terminated by the addition of 200 ~i of 30% trichloroacetic acid, and the mixture was centrifuged at 15,000 x g for 3 min. An aliquot of the supernatant3~luid was taken, added to scintillation fluid (Amersham) and counted for - P-radioactivity. Phosphorylation of calmodulin-dependent protein phosphatase by protein kinase C was performed in 50 mM Tris-Cl, pH 7.5, I0 mM magnesium chloride, 0.5 •M calcium chloride, 40 ~g/ml of phosphatidyl-L-serine and 0.i mM ~ P-labeled or unlabeled ATP ~s indicated in the figure legends. The specific radioactivity of ATP was I0 ~ cpm per nmol. One unit of protein kinase C activity was that amount which incorporated 1 nmol of phosphate into hlstone HI (histone type Ill-S) in I min at 30°C in the presence of 0.5 mM calcium chloride, i0 mM magnesium chloride and 40 ~g/ml of phosphatidyl-L-serine.
RESULTS AND DISCUSSION A homogeneous preparation of calmodulin-dependent protein phosphatase was used to determine whether it could be phosphorylated by the Ca 2+, phospholipid-dependent protein kinase
(protein kinase C).
Figure i show s that the
calmodulin-dependent protein phosphatase can indeed serve as a substrate for protein kinase C.
In the absence of protein kinase C, no phosphorylation of
the phosphatase was observed
[y-32p]ATP in
the
presence
phosphorylation was present
in
phosphatase
the was
(Fig.
of
observed
IB, lane 3) when
Ca 2+,
(Fig.
intensely
lane 5) when
The
61
phosphorylated
kDa and
61
kDa
subunit,
minor
radioactive
bands
catalytic the
were
represent proteolytic fragments of the 61 kDa subunit.
In
contrast,
protein kinase C was subunit
of
phosphorylatlon
stimulated 15- to 20-fold by phospholipid (Fig. IB, lane 5). the
incubated with
Mg2+ and p h o s p h o l i p i d .
IB,
incubation mixture.
it was
the was
In addition to
observed.
These
may
The 17 kDa regulatory
calmodulin-like subunit of the phosphatase was not appreciably phosphorylated by
the protein
klnase
C.
The
amount
of
phosphate
incorporated
into
the
phosphatase was 0.5 to 1.0 mol per mol of enzyme (Fig. 2). The effect of the observed phosphorylatlon of the calmodulin-dependent phosphatase by protein kinase C on the activity of the phosphatase was studied
784
Vol. 138, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A
B
i
t M
I
2 3 4 5
l
2
3
45
Fi 8. I. Analysis of the phosphorylated products of the calmodulin-dependent protein phosphatase following phosphorylation by protein ~kinase C. The reaction mixtures (60 ~i) contained I0 mM MgCI2, 0.1 mM [y-JLP]ATP, and the following additions: lane I, i unit of protein kinase C; lane 2, I unit of protein kinase C; 0.5 mM CaClp and 50 ~g/ml of phosphatidyl-L-serine; lane 3, I mg/ml of calmodulin-dependent protein phosphatase; lane 4, 1 mg/ml of calmodulin-dependent protein phosphatase, 0.5 mM CaCI?, and 1 unit of protein kinase C; lane 5, i mg/ml of calmodulin-dependent prolein phosphatase, 0.5 mM CaClp, 50 ~g/ml ~f phosphatidyl-L-serine and 1 unit of protein kinase C. ReacEion was at 30 C and after 30 min, the mixtures were heated at 100=C for 5 min in sodium dodecyl sulfate solubilizing buffer and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Migration is from top to bottom. The arrows indicate the position of marker proteins (M) phosphorylase b (97.4 kDa), bovine serum albumin (68 kDa), ovalhumin (43 kDa), carbonic snhydrase (29.5 kDa) and myoglobin (17 kDa). Panel A shows the proteins as stained by Coomassie blue. Panel B shows the phosphorylated products as visualized by autoradiography.
using
32p-labeled
casein
as
significant
difference
and phospho
forms of the enzyme
the phosphatase and V
max
reaction factor with latter
to calmodulin
no dramatic
phosphorylation must
not
be
is required the
enzyme
enzyme
undergone
observed
Under
between
(Fig. 3). activation
values were also not significantly
Although following
was
substrate.
change
in order
tyrosine
apparently
phosphorylation.
It
hydroxylase
assay
The sensitivity was
also
the
that
adrenal
The calmodulin-dependent 785
dephospho
(Fig.
4).
The K
m
of the phosphatase significance
of this
an additional
protein
in enzyme
to an activator
the
no
(data not shown).
properties
a change
of
used,
of the two forms of
similar
different
is possible
of
conditions
activities
the physiological
to observe
responds
the
in the kinetic
was observed,
overlooked.
the
activity,
medulla
protein
only
phosphatase
as found
[17,18]. after
The it has
is the second
Vol. 1 38, NO. 2, 1 986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
t.0 hi
~-0.8 hi J O "S
w'~,0.6 "r {3O3
o 0.4 O.
"s
0.2 _._-a-----e----
0.0
'
1~
'
2b
i
3~
l
4b
TIME, min
Fi~. 2. Time course of phosphorylation of purified ealmodulin-dependent protein phosphatase by protein kinase C. Incubations were carried out at 1 unit of protein kinase C and I mg/ml of calmodulin-dependent protein phosphatase as described in Materials and Methods in the presence ( O ) or absence (Z~) of phosphatidyl-L-serine. Phosphoryla~%on in the absence of added protein kinase C was not affected by Caplus phosphatidyl-L-serine. Radioactivity due to autophosphorylation of the protein kinase C preparation was subtracted from the total radioactivity.
14000" 12000 10000 a w
8ooo
/
6000 4000 2000-
O~ o
lb
'
2b
J
TIME, min
3b
i
go
Fi~.3. Time course of activation of phospho ( O ) and dephospho ( ~ ) forms of calmodulin-dependent protein phosphatase. Calmodulln-dependent protein phosphatase was phosphorylated b~2 protein kinase C using unlabeled ATP and then assayed for activity using P-labeled casein as described in Materials and Methods. 786
Vol. 138, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
10000
8000-
,~'
a w J W
6000. 4000-
2000-
.......
1'0 ...... 1'~2 CALMODUUN, #g/ml
......
~'
Fi~. 4. Activation of phospho (O) and dephospho (~) forms of calmodulindependent protein phosphatase by calmodulln. Calmodulin-dependent protein phosphatase was phosphorylated bY3~roteln kinase C using unlabeled ATP and then assayed for activity using P-labeled casein at different calmodulin concentrations as described in Materials and Methods.
protein phosphatase known to be phosphorylated;
the other protein phosphatase
being the ATP-Mg-dependent protein phosphatase [18-21]. Recently Patel et al. [22] reported briefly that the calmodulin-dependent protein phosphatase could be phosphorylated by protein kinase C. Singh and Wang
[23] reported that the phosphatase could be phosphorylated by
casein kinase i and by cyclic AMP-dependent protein kinase. established
whether
phosphorylated
In addition,
the
in vivo
in
calmodulin-dependent response
protein
to physiological
It remains to be
phosphatase activators
of
can
be
protein
kinase C and cyclic AMP-dependent protein kinase.
ACKNOWLEDGMENTS The author wishes
to thank Dr. Lester J. Reed for his support and Dr. Zahi
Damuni for his useful comments.
REFERENCES I.
Stewart, A. A., Ingebritsen, T. S., Manalan, Cohen, P. (1982) FEBS Lett. 137, 80-84.
787
A.
S., Klee,
C. B., and
Vol. 138, No. 2, 1986
2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13 14 15 16 17. 18. 19. 20.
21. 22. 23.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Stewart, A. A., Ingebritsen, T. S., and Cohen, P. (1983) Eur. J. Biochem. 132, 297-298. Wang, J. H., and Desai, R. (1976) Biochem. Biophys. Res. Commun. 72, 926-937. Wang, J. H., and Desai, R. (1977) J. Biol. Chem. 252, 4175-4184. Klee, C. B., Crouch, T. H., and Krinks, M. H. (1979) Proc. Natl. Acad. Sci. USA 76, 6270-6273. Wallace, R. W., Tallant, E. A., and Cheung, W. Y. (1980) Biochemistry 19, 1831-1837. Klee, C. B., and Krinks, M. A. (1978) Biochemistry 17, 120-126. Sharma, R. K., Desai, R., Waismann, D. M., and Wang, T. H. (1979) J. Biol. Chem. 254, 4276-4282. Yang, S. D., Tallant, E. A., and Cheung, W. Y. (1982) Biochem. Biophys. Res. Commun. 106, 1419-1425. Aitken, A., Klee, C. B., and Cohen, P. (1984) Eur. J. Biochem. 139, 663-671. Pallen, C. J., and Wang, J. H. (1983) J. Biol. Chem. 258, 8550-8553. Chernoff, J., Sells, M. A., and Li, H. C. (1984) Biochem. Biophys. Res. Commun. 121, 5577-5584. Pallen, C. J., Valentine, K. A., Wang, J. H., and Hollenberg, M. D. (1985) 24, 4727-4730. Kikkawa, V., Takai, Y., Minakuchi, R., Inohara, S., and Nishizuka, Y. (1982) J. Biol. Chem. 257, 13341-13348. Walsh, M. P., Valentine, K. A., Ngai, P. K., Carruthers, C. A., and Hollenberg, M. D. (1984) Biochem. J. 224, 117-127. Sharma, R. K., Taylor, W. A., and Wang, J. H. (1983) Methods Enzymol. 102, 210-219. Yamauchi, T., Nakata, H., and Fujisawa, M. (1981) J. Biol. Chem. 256, 5405-5409. Hemmings, B. A., Resink, T. J., and Cohen, P. (1982) FEBS Lett. 150, 319-324. Ballou, L. M., Brautigan, D. L., and Fisher, E. H. (1983) Biochemistry 22, 3393-3399. Jurgensen, S., Schacter, E., Huang, C. Y., Chock, P. B., Yang, S. D., Vandenheede, J. R., and Merlevede, W. (1984) J. Biol. Chem. 259, 5864-5870. Tung, H. Y. L., and Cohen, P. (1984) Eur. J. Biochem. 145, 57-64. Patel, J., Lanciotti, M., and Huang, C. Y. (1986) Fed. Proc. 45, 1803, abstr 1884. Singh, T. J., and Wang, J. H. (1986) Fed. Proc. 45, 1803, abstr 1886.
788
Vol. 138, No. 2,1986 July31,1986
PHOSPHORYLATION OF THE CALMODULIN-DEPENDENT PROTEIN PHOSPHATASE BY PROTEIN KINASE C H. Y. Lim Tung Clayton Foundation Biochemical Institute The University of Texas at Austin, Austin, Texas 78712 Received June 2, 1986
The c a l m o d ~ i n - d e p e n d e n t protein phosphatase was shown to be phosphorylated by the Ca , phospholipid-dependent protein kinase (protein kinase C). Analysis by sodium dodecyl sulfate-polyacrylamide gel eleetrophoresis indicated that the 61 kDa catalytic subunit was phosphorylated. Phosphorylation by protein kinase C was stimulated up to 15-fold by addition of phosphatidyl-Lserine and between 0.5 to 1.0 mole of phosphate was incorporated per mole of phosphatase. It is possible that protein kinase C is involved in the regulation of the calmodulin-dependent protein phosphatase via this novel phosphorylation of the enzyme. © 1986 AcademicPress, Inc.
The
calmodulin-dependent
protein
phosphatase
was
first
identified
in
rabbit skeletal muscle [1,2] and shown to be identical to a brain calmodulinbinding
protein
consists subunit
[3]
originally
of two subunits, of apparent
termed
calcineurin
A and B in a molar
molecular mass
61 kDa
[4,5]
Or CamBP80
ratio of 1:1
interacts with
[6] and
[7,8,9].
The A
calmodulin
in the
presence of calcium [1,7], and the B subunit is a Ca 2+ binding protein which shows
35%
binding
regions
phosphatase low
identity
containing specificity.
[i0].
activity,
molecular
with
mass
proteins
calmodulin In
with
addition
the enzyme has phospho [12,13],
to
strong
homology
possessing
around
the
Ca 2+
phosphoseryl/threonyl
recently been shown to dephosphorylate
compounds indicating
[ii]
as
that
well the
as enzyme
phosphotyrosine has
multiple
Here we demonstrate that in addition to being regulated by Ca 2+
and calmodulin,
the enzyme can also be phosphorylated by protein kinase C, a
Ca 2+, phospholipid-dependent protein kinase [14].
MATERIALS AND METHODS Histone H I , casein, phospha~yl-L-serine and cyclic AMP-dependent protein kinase ~ere from Sigma. Ca- , phospholipid-dependent protein kinase (protein kinase C) and calmodulin were purified from bovine brain essentially as described by Walsh et al. [15]. Calmodulin-dependent protein phosphatase
783
0006-291X/86 $1.50 Copyr~ht © 1986 ~ Aca~mk' Press, Inc. All r~hts 0/' r~roduction in at~ jorm reserved.
Vol. 138, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
was also purified from bovine brain essentially as described by Sharma et al. [16], except that the last chromatography on Sephadex G-200 was replaced by chromatography on Sephacryl S-300. C a l m o d ~ n - d e p e n d e n t p r o t e ~ phosphatase was assayed by determining the release of u-P-phosphate from P-labeled casein prepared by phosphorylation with cyclic AMP-dependent protein kinase. The assay (60 ~i) consisted of diluted phosphatase in 50 mM Tris-C1, pH 7.5, 0.1% 2-mercaptoethanol, 1 mg/ml bovine serum albumin, I0 mM m a g n e ~ u m chloride, 20 ~g/ml calmodulin, 0.1 mM c a l c i ~ chloride and 0.2 mg/ml of P-labeled casein. Reaction was initiated with P-labeled substrate, allowed to take place for i0 min, terminated by the addition of 200 ~i of 30% trichloroacetic acid, and the mixture was centrifuged at 15,000 x g for 3 min. An aliquot of the supernatant3~luid was taken, added to scintillation fluid (Amersham) and counted for - P-radioactivity. Phosphorylation of calmodulin-dependent protein phosphatase by protein kinase C was performed in 50 mM Tris-Cl, pH 7.5, I0 mM magnesium chloride, 0.5 •M calcium chloride, 40 ~g/ml of phosphatidyl-L-serine and 0.i mM ~ P-labeled or unlabeled ATP ~s indicated in the figure legends. The specific radioactivity of ATP was I0 ~ cpm per nmol. One unit of protein kinase C activity was that amount which incorporated 1 nmol of phosphate into hlstone HI (histone type Ill-S) in I min at 30°C in the presence of 0.5 mM calcium chloride, i0 mM magnesium chloride and 40 ~g/ml of phosphatidyl-L-serine.
RESULTS AND DISCUSSION A homogeneous preparation of calmodulin-dependent protein phosphatase was used to determine whether it could be phosphorylated by the Ca 2+, phospholipid-dependent protein kinase
(protein kinase C).
Figure i show s that the
calmodulin-dependent protein phosphatase can indeed serve as a substrate for protein kinase C.
In the absence of protein kinase C, no phosphorylation of
the phosphatase was observed
[y-32p]ATP in
the
presence
phosphorylation was present
in
phosphatase
the was
(Fig.
of
observed
IB, lane 3) when
Ca 2+,
(Fig.
intensely
lane 5) when
The
61
phosphorylated
kDa and
61
kDa
subunit,
minor
radioactive
bands
catalytic the
were
represent proteolytic fragments of the 61 kDa subunit.
In
contrast,
protein kinase C was subunit
of
phosphorylatlon
stimulated 15- to 20-fold by phospholipid (Fig. IB, lane 5). the
incubated with
Mg2+ and p h o s p h o l i p i d .
IB,
incubation mixture.
it was
the was
In addition to
observed.
These
may
The 17 kDa regulatory
calmodulin-like subunit of the phosphatase was not appreciably phosphorylated by
the protein
klnase
C.
The
amount
of
phosphate
incorporated
into
the
phosphatase was 0.5 to 1.0 mol per mol of enzyme (Fig. 2). The effect of the observed phosphorylatlon of the calmodulin-dependent phosphatase by protein kinase C on the activity of the phosphatase was studied
784
Vol. 138, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A
B
i
t M
I
2 3 4 5
l
2
3
45
Fi 8. I. Analysis of the phosphorylated products of the calmodulin-dependent protein phosphatase following phosphorylation by protein ~kinase C. The reaction mixtures (60 ~i) contained I0 mM MgCI2, 0.1 mM [y-JLP]ATP, and the following additions: lane I, i unit of protein kinase C; lane 2, I unit of protein kinase C; 0.5 mM CaClp and 50 ~g/ml of phosphatidyl-L-serine; lane 3, I mg/ml of calmodulin-dependent protein phosphatase; lane 4, 1 mg/ml of calmodulin-dependent protein phosphatase, 0.5 mM CaCI?, and 1 unit of protein kinase C; lane 5, i mg/ml of calmodulin-dependent prolein phosphatase, 0.5 mM CaClp, 50 ~g/ml ~f phosphatidyl-L-serine and 1 unit of protein kinase C. ReacEion was at 30 C and after 30 min, the mixtures were heated at 100=C for 5 min in sodium dodecyl sulfate solubilizing buffer and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Migration is from top to bottom. The arrows indicate the position of marker proteins (M) phosphorylase b (97.4 kDa), bovine serum albumin (68 kDa), ovalhumin (43 kDa), carbonic snhydrase (29.5 kDa) and myoglobin (17 kDa). Panel A shows the proteins as stained by Coomassie blue. Panel B shows the phosphorylated products as visualized by autoradiography.
using
32p-labeled
casein
as
significant
difference
and phospho
forms of the enzyme
the phosphatase and V
max
reaction factor with latter
to calmodulin
no dramatic
phosphorylation must
not
be
is required the
enzyme
enzyme
undergone
observed
Under
between
(Fig. 3). activation
values were also not significantly
Although following
was
substrate.
change
in order
tyrosine
apparently
phosphorylation.
It
hydroxylase
assay
The sensitivity was
also
the
that
adrenal
The calmodulin-dependent 785
dephospho
(Fig.
4).
The K
m
of the phosphatase significance
of this
an additional
protein
in enzyme
to an activator
the
no
(data not shown).
properties
a change
of
used,
of the two forms of
similar
different
is possible
of
conditions
activities
the physiological
to observe
responds
the
in the kinetic
was observed,
overlooked.
the
activity,
medulla
protein
only
phosphatase
as found
[17,18]. after
The it has
is the second
Vol. 1 38, NO. 2, 1 986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
t.0 hi
~-0.8 hi J O "S
w'~,0.6 "r {3O3
o 0.4 O.
"s
0.2 _._-a-----e----
0.0
'
1~
'
2b
i
3~
l
4b
TIME, min
Fi~. 2. Time course of phosphorylation of purified ealmodulin-dependent protein phosphatase by protein kinase C. Incubations were carried out at 1 unit of protein kinase C and I mg/ml of calmodulin-dependent protein phosphatase as described in Materials and Methods in the presence ( O ) or absence (Z~) of phosphatidyl-L-serine. Phosphoryla~%on in the absence of added protein kinase C was not affected by Caplus phosphatidyl-L-serine. Radioactivity due to autophosphorylation of the protein kinase C preparation was subtracted from the total radioactivity.
14000" 12000 10000 a w
8ooo
/
6000 4000 2000-
O~ o
lb
'
2b
J
TIME, min
3b
i
go
Fi~.3. Time course of activation of phospho ( O ) and dephospho ( ~ ) forms of calmodulin-dependent protein phosphatase. Calmodulln-dependent protein phosphatase was phosphorylated b~2 protein kinase C using unlabeled ATP and then assayed for activity using P-labeled casein as described in Materials and Methods. 786
Vol. 138, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
10000
8000-
,~'
a w J W
6000. 4000-
2000-
.......
1'0 ...... 1'~2 CALMODUUN, #g/ml
......
~'
Fi~. 4. Activation of phospho (O) and dephospho (~) forms of calmodulindependent protein phosphatase by calmodulln. Calmodulin-dependent protein phosphatase was phosphorylated bY3~roteln kinase C using unlabeled ATP and then assayed for activity using P-labeled casein at different calmodulin concentrations as described in Materials and Methods.
protein phosphatase known to be phosphorylated;
the other protein phosphatase
being the ATP-Mg-dependent protein phosphatase [18-21]. Recently Patel et al. [22] reported briefly that the calmodulin-dependent protein phosphatase could be phosphorylated by protein kinase C. Singh and Wang
[23] reported that the phosphatase could be phosphorylated by
casein kinase i and by cyclic AMP-dependent protein kinase. established
whether
phosphorylated
In addition,
the
in vivo
in
calmodulin-dependent response
protein
to physiological
It remains to be
phosphatase activators
of
can
be
protein
kinase C and cyclic AMP-dependent protein kinase.
ACKNOWLEDGMENTS The author wishes
to thank Dr. Lester J. Reed for his support and Dr. Zahi
Damuni for his useful comments.
REFERENCES I.
Stewart, A. A., Ingebritsen, T. S., Manalan, Cohen, P. (1982) FEBS Lett. 137, 80-84.
787
A.
S., Klee,
C. B., and
Vol. 138, No. 2, 1986
2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13 14 15 16 17. 18. 19. 20.
21. 22. 23.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
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