Vol.
166,
January
No.
1, 1990
AND
BIOCHEMICAL
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
15, 1990
Inactivation
of Bovine Kidney Cytosolic Subunit of Protein Zahi
Department
of
Received
November
Protamine Phosphatase
Kinase 2A
by the
449-456
Catalytic
Damuni
Biological Sciences, CLS 511, University of South at Columbia, Columbia, South Carolina 29208
Carolina
1, 1989
Incubation of highly purified preparations of the bovine kidney cytosolic protamine kinase in the presence of near homogeneous preparations of the catalytic subunit of protein phosphatase 2A (PrP2A ) from bovine kidney resulted in time-dependent inactivation of the p&tamine kinase. By contrast, incubation of bovine kidney cytosolic casein kinase II with PrP2Ac had no effect on the activity of this casein kinase II. In the presence of 10 mM sodium fluoride, 10 mM inorganic orthophosphate, 1 mM pyrophosphate or 0.1 mM ATP, the inactivation of the protamine kinase by PrP2A was completely inhibited. Half-maximal inhibition by ATP occurred atCabout 20 PM. The rate of inactivation of the protamine kinase by PrP2A was unaffected by Mg2+, Mn2+, Ca2+, EDTA or EGTA at l&l. The resulss strongly indicate that the activity of the cytosolic protamine kinase is regulated by phosphorylation/dephosphorylation. o 1990 Academic Press, Inc. The bovine polypeptide sulfate
kidney
of
cytosolic
apparent
Mr
polyacrylamide
graphy
This
(1).
its
unique
The
physiological
has
not
gel
enzyme
substrate
yet
regulators
been
and
that
identical
to the bovine
ing
treatment
for
this
cellular
kidney rat
by
kidney to
other
hepatocytes
responses
protamine
However,
with
449
insulin hormone
protein EDTA,
by
(1,2). kinase relevant
the
and chromatographic
to this
The abbreviations used are: PrP2A, protein phosphatase 2A catalytic subunit; tic acid; EGTA, ethyleneglycol-bis-(B-aminoethyl tetraacetic acid.
kinases
properties
enzyme was stimulated
dodecyl chromato-
physiologically
identified.
catalytic
sodium
protein
cytosolic
date,
of a single
permeation
chromatographic
been
exhibits
composed
determined
from
bovine
enzyme had not
ties
in
the
is
and gel
and
elucidated,
isolated
as
electrophoresis
of
kinase
enzyme
45,000
kinase
was differentiated
of a protamine
of
-
specificity role
of this
protamine
rapidly indicating
activity properfollowa role
(2).
phosphatase 2A; PrP2Ac, ethylenediaminetetraaceether) N, N, N', N'
0006-291x/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
166, No. 1, 1990 In kinase
this of
communication, bovine
homogeneous 2A
BIOCHEMICAL
(PrP2Ac).
bovine
The
kidney
phorylation. which isolated
the
The
rat
is of
results
protamine
activity
we report
kidney
preparations
hepatocytes
that
inactivated the
kinase
is
protamine is
purified
subunit
of protein
that
the
results
to
was stimulated
protamine with
the
near
phosphatase
activity
by phosphorylation/
the
kinase
highly
incubation
regulated of
RESEARCH COMMUNICATIONS
following
indicate
relevance
the
the
catalytic
strongly
possible of
AND BIOPHYSICAL
of
the
dephosmechanism by insulin
by in
discussed.
Materials
and Methods
Affigel Blue gel was obtained from Biorad. Phosphorylase b, phosphorylase kinase and casein were obtained from Sigma Chemical Company. All other materials are given in references 1 and 2. Purification of the bovine kidney cytosolic protamine kinase to apparent homogeneity (1) and partial purification of the rat hepatocyte protamine kinase (2) were as described. Determination of protamine kinase activity was as documented (2). One unit of protamine kinase activity was the amount of enzyme which catalyzed the incorporation of 1 nmol of phosphoryl groups into protamine. Protein was determined by the procedure of Bradford (3). Polyacrylamide gel electrophoresis was performed in slab gels (12% acrylamide) with 0.1% sodium dodecyl sulfate and Tris/glycine buffer, pH 8.3 (4). Protein bands were detected by staining with Coomassie Blue or silver (5). 3'P-labeled phosphorylase a was prepared from phosphorylase b using phosphorylase kinase, and phosphorylase phosphatase activity was determined as described (6,7). One unit of phosphorylase phosphatase activity was the amount of enzyme which catalyzed the release of 1 run01 inorganic phosphate/min. The catalytic subunit of PrP2A was purified to apparent homogeneity from bovine kidney cytosol by a procedure similar to the one employed to purify PrP2A from rabbit skeletal muscle and liver (8,9). The final preparations cznsisted of a single Coomassie Blue or silver staining polypeptide of apparent M - 36,000 as determined by sodium dodecyl sulfate polyacrylamide gel efectrophoresis. The criteria employed to identify the purified phosphatase preparations as type 2A (10,ll) were that the enzyme specifically dephosphorylthat the enzyme was divalent ated the 01 subunit of phosphorylase kinase, cation-independent and that ATP at 0.1 mM completely inhibited its activity with 32P-labeled phosphorylase. Half-maximal inhibition with ATP occurred at about 20 FM ATP (see Fig. 3 below). 32P-labeled phosphorylase kinase was prepared by incubation with the catalytic subunit of CAMP-dependent protein kinase as described (11,12). Dephosphorylation of the ci subunit by PrP2Ac was analyzed by autoradiography following resolution of 32P-labeled phosphorylase kinase LY and S subunits by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Casein kinase II was purified to apparent homogeneity from bovine kidney cytosol by a procedure similar to the one employed to purify the mitochondrial casein kinase II except that chromatography of the preparaThese casein kinase II tions on heparin-agarose was omitted (13). preparations consisted of two Coomassie Blue or silver staining subunits of apparent M - 36,000 and 28,000 as estimated by sodium dodecyl sulfate polyacrylamid: gel electrophoresis. Casein kinase II activity was determined as described (13) except that Mgzf at 10 mM instead of 1.5 mM 4.50
Vol.
166, No. 1, 1990
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
was employed in the incubations. One unit was the amount of the enzyme which catalyzed of phosphoryl groups into casein (13). Results Incubation cytosolic
of
near
protamine
casein kinase the incorporation
II
activity of 1 nmol
and Discussion
homogeneous
kinase
of
with
preparations
of
resulted
PrP2A
the
in
bovine
kidney
time-dependent
in-
C
activation the II
activity were
employed PrP2Ac
01
of
the
protamine
of purified unaffected (not
was
by
due
20
preparations
preparations
shown). not
kinase
incubation
of the bovine with
The inactivation to
(Fig.
any
of
of the
contaminating
the
1). kidney
casein
PrP2Ac
kidney
protease(
By contrast,
protamine if
any,
kinase
preparations kinase because
w
30
Time (min)
Fig. 1. Inactivation of bovine kidney cytosolic p rotamine kinase by PrP2A - Highly purified protamine kinase (170 units/ml) was incubated at 30°C %n the absence (o---o) and presence (o---s) of PrP2A~ (7 unit/ml) in 50 mM imidazole chloride, pH 7.3, 10% glycerol, 0.1 &l pbenylmethylsulfonyl fluoride, 1 mM benzamidine, 0.1 mM EGTA, and 14 mM B-mercaptoethanol in a final volume of 0.1 ml. At the indicated times, a 0.01 ml aliquot of the incubations was used to determine protamine kinase activiof 10 min assays were ty as described (1) except that 3 q in instead performed. Fig. 2. Incubation of the protamine kinase in the absence (A) and presence (B) of PrP2A - The conditions were as described in the legend to Fin. 1 except that following incubation of the protamine kinase with PrP2A for i h, 50 pl of sample buffer was added (7). The solutions were then teated at 100% for 5 min and sodium dodecyl sulfate polyacrylamide gel electrophoresis was carried out (7). The gel, 12% acrylamide was stained with silver (5). The arrow denotes the position of the protamine kinase. 451
by the
Vol. 166, No. 1, 1990
apparent
Mr
dodecyl
sulfate
following of
the
presence
BIOCHEMICAL
(-
45,000)
of
the
incubation protamine
with
general
gel
PrP2Ac
kinase
of the
by protein
at
1 mM, or
a specific
PrP2A
inhibited
the
inactivation
of
the
of
PrP2A c with
substrates
occurred
concentration
(Fig.
of PrP2Ac
inhibited
inhibitors
sodium
the
orthophosphate at
0.1
kinase
to
at
by
This
observe
half-maximal
3 and Refs.
11,12)
altered
in
the
fluoride
at
10 mM (not completely
PrP2Ac.
3).
sodium
inactivation
mM (11,12),
20 PM ATP (Fig.
a (Fig.
not
completely
protamine
of ATP required phosphorylase
was
was
inhibitor
at about
by
In addition,
inorganic
the
as determined
2).
phosphatase
ATP,
inhibition
kinase
RESEARCH COMMUNICATIONS
electrophoresis
PrP2A c
shown).
to
kidney
polyacrylamide
5 mM, pyrophosphate
maximal
AND BIOPHYSKAL
is
Halfsimilar
inhibition and
with
other
(11,12).
I
I
I
I
I
20
0.04
0.06
ATP (mM) Fig. 3. Inhibition of PrP2A- by ATP - Incubation of the protamine kinase with PrP2Ac for 10 min in the presence of ATP and assay of the protamine kinase for 3 min were performed (o---o) as described in the legend to Fig. 1. The 100% control value refers to the maximal inactivation of the protamine kinase observed in the absence of ATP. The effect of ATP on the phosphorylase phosphatase activity of PrP2A (o---o) was determined as described (11). The 100% control value for p%osphorylase phosphatase was the activity determined in the absence of ATP.
452
Vol.
166,
No.
1, 1990
The
rate
BIOCHEMICAL
of protamine
was unaffected The PrP2Ac
was
by NaCl
may
the
completely
of
mine
kinase
the
most
in
in
kidney
enzyme contain
because
extracts
(1).
In
does addition,
1 mM EDTA (1).
report
from
sodium
kinases
by
occurred
of
increase
all
the
Therefore,
protamlne
kinase
for
purified inhibition
indicate
kidney
either
during
the
buffers during
the
in
isolation
3 min
453
were
as
described
protacourse
appears
the
of
this
purification
of the
the
to be
determined
purification
employed
enzyme
the
kinase
the
inacti-
This
during
possibility protamine
is kidney
groups.
the
that
cytosol
NaCl (mM) Inhibition of PrPZA by NaCl Incubation of Fig. 4. kinase with PrP2Ac -for 10 min in the absence and presence assay of the to Fig. 1.
vanadate.
the
Half-maximal
the purified
The latter
activity
not
preparations
EGTA or
phosphoryl
have
--in vivo. the
that
regulatory could
PrP2Ac
strongly
isolated
and therefore bound
COMMUNICATIONS
4).
this
kinase
and/or
likely
EDTA,
by 200 mM NaCl.
phosphorylation
of the purification
RESEARCH
by the
protamine
50 mM (Fig.
protamine
covalently
Ca2+,
cytosolic
by dephosphorylation, contain
Mn2+,
presented
the
BIOPHYSICAL
inactivation
inhibited
at about
results
activity vated
of
occurred
The
kinase
by 1 mM Mg2+,
inactivation
AND
protamine
protamine
of NaCl, and in the legend
Vol.
166, No. 1, 1990 kinase,
BIOCHEMICAL
divalent-cation
dependent
expected
to be inhibited.
tion
performed
in
the
purification,
is
steps
of
is(are)
likely
stages
of
the
containing PrP2A
AND BIOPHYSICAL
It
phosphorylation
is
also
a phosphate
buffer
protamine inhibited.
purification
protamine
the
at a concentration
catalyzed
of
pertinent
to be completely
NaCl
RESEARCH COMMUNICATIONS
of
enzyme
to note
that
(1).
Therefore,
kinase
protein
kinase
the
is
Under
the
first
phosphatase(s) at all
present these
protamine
can be
homogenizaat
In addition,
> 200 mM.
inactivation
this
the
in
later
solutions
conditions,
kinase
is
the
completely
C
inhibited
(Fig.
cannot
4).
account
protein
for
any loss
phosphatases
protamine
kinase,
Protein
residues
(14,15).
exhibit
protamine
composition of
residues,
the
is
detectable it
is
the
PrP2A Whether
activity
for
of
the
proteins,
the
activity
towards
conceivable
phosphoseryl enzyme
that
on
preliminary
and
not
of
kinase
phosphoserine
that
inactivates
the
which
phosphoamino indicated
nor PrP2A
residues
been
of phosphotyrosyl
phosphothreonine
certain
has
PrP2Ac
analysis
protamine
and
phosphotyrosine
by dephosphorylation
almost
possible
may be
that
involved
idea
was First,
protein
phosphatase of
insulin
insulin-stimulated chromatography
a covalent in
in
it
was
the
the
phosphotyrosyl catalyzes
the
C
present
on
phosphorylation
mechanism
response
suggested
tions.
effect
purification.
specific
some
cytosolic
seems
enzyme was stimulated This
by
the
purified
kinase.
It kinase
but
based
of
the
regulate
relatively
With
phosphoseryl
dephosphorylation
PrP2A
preparations
of
it
protamine
2A is
Therefore,
However,
during
catalyzed
to be determined.
low
kinase
residues.
presence
than
residues.
to
acid
other
phosphatase
reported
the
of activity
remains
phosphothreonyl
dephosphorylation
Therefore,
necessary
inhibitor on the increase
(2) to
in rat
the
based
sodium
on protamine-agarose,
protamine kinase
DEAF-cellulose, 454
the
protamine
activity rat
on the
homogenization
protamine
the
in isolated
include
hepatocyte in
by which
to insulin
previously
of
of
this
hepatocytes.
following
observa-
fluoride,
a general
buffers
to detect
the
kinase.
Second,
the
activity and
was
stable
Sephacryl
to S-200
Vol.
166,
gel
No.
1, 1990
BIOCHEMICAL
filtration
indicating
involved.
Third,
control
cells on
kinase
activity
tion
of
the
kinase
synthesis
increase
in the
of
that
supported
by
experiments
not
pool
the
kinase
of
the
phosphorylation
results
presented
partially were
of
in
purified
also
cyclohexiimide,
the
insulin-stimulated
that
insulin
the
associa-
Clearly, protamine
protamine
the kinase
kinase
communication.
following
a
had stimu-
kinase.
preparations
inactivated
stimulated
Fourth,
hepatocyte
this
not
of the
the
of
chromato-
modulation
indicating
was and
permeation had
prevent
activity
a pre-existing
increased
utilizing
protamine
did
kinase
insulin-treated
insulin
factor.
insulin-stimulation
via
from
or via
COMMUNICATIONS
of the
by gel
that
a regulatory
inhibitor,
activity
occur
indicating
kinase
kinase
as estimated
proteolysis
with
protamine
possibility may
S-200
RESEARCH
modification
Mr of the
45,000
by limited
the
BIOPHYSICAL
a covalent
apparent
Sephacryl
protein
lated
that
was about
graphy
AND
is
Moreover,
of
the
incubation
hepatocyte with
PrP2A C
(not
shown).
In
preparations and
this
of
an
partially
regard,
purified
inactivated
following and
inactivated
the
has
been
in
(16)
kinase
that
the
together
with
purified
S6 kinase
(16).
also
been
shown
In
the
been
ribosomal
cascade
kinase(s)
the
protamine
of sequential
the
of
which
would
the
PrP2Ac
identified. protein
of
protein-2
reactivation
yet
to be
addition,
30% of maximum)
The
(16)
insulin-stimulated
PrP2Ac.
kinase,
an insulin-stimulated
that
microtubule-associated
not
protein-2
phosphorylation/activation
have
concomitant
has
note
an
(about
reported and
microtubule-associated
participate
of
with
by
phosphorylation
however,
protein
reactivation
kinase
protamine
possible,
ribosomal
incubation
S6
preparations the
to
kinase
partial
protein
catalyze
interesting
preparations protein-2
phosphorylation
kinase
is
insulin-stimulated
microtubule-associated
ribosomal
it
It
is
S6 kinase
and
kinase
may
protein
kinase
reactions.
Acknowledgments I Lisa
am grateful
Ball
for
typing
to
Clint this
Cook for report.
the
I also 455
art thank
work Dr.
and Debra Michael
Williams Felder
and and my
Vol.
166, No. 1, 1990 graduate useful
students comments.
and by the Weight
BIOCHEMICAL Mr. This
Shrikanth work
Watcher's
AND BlOPHYSICAL A.G.
was
Reddy
supported
RESEARCH COMMUNICATIONS
and Mr.
Grayson
D. Amick
for
by the
Carolina
Venture
Fund
Foundation. References
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
Damuni, Z., Amick, G.D., and Sneed, T.R. (1989) J. Biol. Chem. 264, 6412-6416. Reddy, S.A.G., Amick, G.D., Cooper, R.H., and Damuni, Z. (1989) J. Biol. Chem. in press. Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. Laemmli, U.K. (1970) Nature 227, 680-685. Merril, C.R., Goldman, D., Sedman, S.A., and Ebert, M.M. (1981) Science 211, 1437-1438. Tung, H.Y.L., Pelech, S., Fisher, M.J., Pogson, M.J., and Cohen, P. (1985) Eur. J. Biochem. 149, 305-313. Stewart, A.A., Hemmings, B.A., Cohen, P., Goris, J., and Merlevede, W. (1981) Eur. J. Biochem. 115, 197-205. H.Y.L., Resink, T.J., Hemmings, B.A., Shenolikar, S., and Tmz, Cohen, P. (1984) Eur. J. Biochem. 145, 51-56. Lee, E.Y.C., Silberman, S.R., Ganapathi, M.R., Paris, H., and Petrovic, S. (1981) Cold Spring Harb. Conf. Cell. Prolif. 8, 425-439. Ingebritsen, T.S., and Cohen, P. (1983) Science 221, 331-338. Ingebritsen, T.S., Foulkes, J.G., and Cohen, P. (1980) FEBS Lett. 119, 9-15. Ingebritsen, T.S., Foulkes, J.G., and Cohen, P. (1983) Eur. J. Biochem. 132, 263-274. Damuni, Z., and Reed, L.J. (1988) Arch. Biochem. Biophys. 262, 574-584. Chernoff, J., Li, H.-C., Cheng, Y.-S.E., and Chen, L.B. (1983) J. Biol. Chem. 258, 7852-7857. Foulkes, J.G., Erickson, E., and Erickson, R.L. (1983) J. Biol. Chem. 258, 431-438. Sturgill, T.W., Ray, B.L., Erickson, R.E., and Maller, J.L. (1988) Nature 334, 715-718.
456
166,
January
No.
1, 1990
AND
BIOCHEMICAL
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
15, 1990
Inactivation
of Bovine Kidney Cytosolic Subunit of Protein Zahi
Department
of
Received
November
Protamine Phosphatase
Kinase 2A
by the
449-456
Catalytic
Damuni
Biological Sciences, CLS 511, University of South at Columbia, Columbia, South Carolina 29208
Carolina
1, 1989
Incubation of highly purified preparations of the bovine kidney cytosolic protamine kinase in the presence of near homogeneous preparations of the catalytic subunit of protein phosphatase 2A (PrP2A ) from bovine kidney resulted in time-dependent inactivation of the p&tamine kinase. By contrast, incubation of bovine kidney cytosolic casein kinase II with PrP2Ac had no effect on the activity of this casein kinase II. In the presence of 10 mM sodium fluoride, 10 mM inorganic orthophosphate, 1 mM pyrophosphate or 0.1 mM ATP, the inactivation of the protamine kinase by PrP2A was completely inhibited. Half-maximal inhibition by ATP occurred atCabout 20 PM. The rate of inactivation of the protamine kinase by PrP2A was unaffected by Mg2+, Mn2+, Ca2+, EDTA or EGTA at l&l. The resulss strongly indicate that the activity of the cytosolic protamine kinase is regulated by phosphorylation/dephosphorylation. o 1990 Academic Press, Inc. The bovine polypeptide sulfate
kidney
of
cytosolic
apparent
Mr
polyacrylamide
graphy
This
(1).
its
unique
The
physiological
has
not
gel
enzyme
substrate
yet
regulators
been
and
that
identical
to the bovine
ing
treatment
for
this
cellular
kidney rat
by
kidney to
other
hepatocytes
responses
protamine
However,
with
449
insulin hormone
protein EDTA,
by
(1,2). kinase relevant
the
and chromatographic
to this
The abbreviations used are: PrP2A, protein phosphatase 2A catalytic subunit; tic acid; EGTA, ethyleneglycol-bis-(B-aminoethyl tetraacetic acid.
kinases
properties
enzyme was stimulated
dodecyl chromato-
physiologically
identified.
catalytic
sodium
protein
cytosolic
date,
of a single
permeation
chromatographic
been
exhibits
composed
determined
from
bovine
enzyme had not
ties
in
the
is
and gel
and
elucidated,
isolated
as
electrophoresis
of
kinase
enzyme
45,000
kinase
was differentiated
of a protamine
of
-
specificity role
of this
protamine
rapidly indicating
activity properfollowa role
(2).
phosphatase 2A; PrP2Ac, ethylenediaminetetraaceether) N, N, N', N'
0006-291x/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
166, No. 1, 1990 In kinase
this of
communication, bovine
homogeneous 2A
BIOCHEMICAL
(PrP2Ac).
bovine
The
kidney
phorylation. which isolated
the
The
rat
is of
results
protamine
activity
we report
kidney
preparations
hepatocytes
that
inactivated the
kinase
is
protamine is
purified
subunit
of protein
that
the
results
to
was stimulated
protamine with
the
near
phosphatase
activity
by phosphorylation/
the
kinase
highly
incubation
regulated of
RESEARCH COMMUNICATIONS
following
indicate
relevance
the
the
catalytic
strongly
possible of
AND BIOPHYSICAL
of
the
dephosmechanism by insulin
by in
discussed.
Materials
and Methods
Affigel Blue gel was obtained from Biorad. Phosphorylase b, phosphorylase kinase and casein were obtained from Sigma Chemical Company. All other materials are given in references 1 and 2. Purification of the bovine kidney cytosolic protamine kinase to apparent homogeneity (1) and partial purification of the rat hepatocyte protamine kinase (2) were as described. Determination of protamine kinase activity was as documented (2). One unit of protamine kinase activity was the amount of enzyme which catalyzed the incorporation of 1 nmol of phosphoryl groups into protamine. Protein was determined by the procedure of Bradford (3). Polyacrylamide gel electrophoresis was performed in slab gels (12% acrylamide) with 0.1% sodium dodecyl sulfate and Tris/glycine buffer, pH 8.3 (4). Protein bands were detected by staining with Coomassie Blue or silver (5). 3'P-labeled phosphorylase a was prepared from phosphorylase b using phosphorylase kinase, and phosphorylase phosphatase activity was determined as described (6,7). One unit of phosphorylase phosphatase activity was the amount of enzyme which catalyzed the release of 1 run01 inorganic phosphate/min. The catalytic subunit of PrP2A was purified to apparent homogeneity from bovine kidney cytosol by a procedure similar to the one employed to purify PrP2A from rabbit skeletal muscle and liver (8,9). The final preparations cznsisted of a single Coomassie Blue or silver staining polypeptide of apparent M - 36,000 as determined by sodium dodecyl sulfate polyacrylamide gel efectrophoresis. The criteria employed to identify the purified phosphatase preparations as type 2A (10,ll) were that the enzyme specifically dephosphorylthat the enzyme was divalent ated the 01 subunit of phosphorylase kinase, cation-independent and that ATP at 0.1 mM completely inhibited its activity with 32P-labeled phosphorylase. Half-maximal inhibition with ATP occurred at about 20 FM ATP (see Fig. 3 below). 32P-labeled phosphorylase kinase was prepared by incubation with the catalytic subunit of CAMP-dependent protein kinase as described (11,12). Dephosphorylation of the ci subunit by PrP2Ac was analyzed by autoradiography following resolution of 32P-labeled phosphorylase kinase LY and S subunits by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Casein kinase II was purified to apparent homogeneity from bovine kidney cytosol by a procedure similar to the one employed to purify the mitochondrial casein kinase II except that chromatography of the preparaThese casein kinase II tions on heparin-agarose was omitted (13). preparations consisted of two Coomassie Blue or silver staining subunits of apparent M - 36,000 and 28,000 as estimated by sodium dodecyl sulfate polyacrylamid: gel electrophoresis. Casein kinase II activity was determined as described (13) except that Mgzf at 10 mM instead of 1.5 mM 4.50
Vol.
166, No. 1, 1990
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
was employed in the incubations. One unit was the amount of the enzyme which catalyzed of phosphoryl groups into casein (13). Results Incubation cytosolic
of
near
protamine
casein kinase the incorporation
II
activity of 1 nmol
and Discussion
homogeneous
kinase
of
with
preparations
of
resulted
PrP2A
the
in
bovine
kidney
time-dependent
in-
C
activation the II
activity were
employed PrP2Ac
01
of
the
protamine
of purified unaffected (not
was
by
due
20
preparations
preparations
shown). not
kinase
incubation
of the bovine with
The inactivation to
(Fig.
any
of
of the
contaminating
the
1). kidney
casein
PrP2Ac
kidney
protease(
By contrast,
protamine if
any,
kinase
preparations kinase because
w
30
Time (min)
Fig. 1. Inactivation of bovine kidney cytosolic p rotamine kinase by PrP2A - Highly purified protamine kinase (170 units/ml) was incubated at 30°C %n the absence (o---o) and presence (o---s) of PrP2A~ (7 unit/ml) in 50 mM imidazole chloride, pH 7.3, 10% glycerol, 0.1 &l pbenylmethylsulfonyl fluoride, 1 mM benzamidine, 0.1 mM EGTA, and 14 mM B-mercaptoethanol in a final volume of 0.1 ml. At the indicated times, a 0.01 ml aliquot of the incubations was used to determine protamine kinase activiof 10 min assays were ty as described (1) except that 3 q in instead performed. Fig. 2. Incubation of the protamine kinase in the absence (A) and presence (B) of PrP2A - The conditions were as described in the legend to Fin. 1 except that following incubation of the protamine kinase with PrP2A for i h, 50 pl of sample buffer was added (7). The solutions were then teated at 100% for 5 min and sodium dodecyl sulfate polyacrylamide gel electrophoresis was carried out (7). The gel, 12% acrylamide was stained with silver (5). The arrow denotes the position of the protamine kinase. 451
by the
Vol. 166, No. 1, 1990
apparent
Mr
dodecyl
sulfate
following of
the
presence
BIOCHEMICAL
(-
45,000)
of
the
incubation protamine
with
general
gel
PrP2Ac
kinase
of the
by protein
at
1 mM, or
a specific
PrP2A
inhibited
the
inactivation
of
the
of
PrP2A c with
substrates
occurred
concentration
(Fig.
of PrP2Ac
inhibited
inhibitors
sodium
the
orthophosphate at
0.1
kinase
to
at
by
This
observe
half-maximal
3 and Refs.
11,12)
altered
in
the
fluoride
at
10 mM (not completely
PrP2Ac.
3).
sodium
inactivation
mM (11,12),
20 PM ATP (Fig.
a (Fig.
not
completely
protamine
of ATP required phosphorylase
was
was
inhibitor
at about
by
In addition,
inorganic
the
as determined
2).
phosphatase
ATP,
inhibition
kinase
RESEARCH COMMUNICATIONS
electrophoresis
PrP2A c
shown).
to
kidney
polyacrylamide
5 mM, pyrophosphate
maximal
AND BIOPHYSKAL
is
Halfsimilar
inhibition and
with
other
(11,12).
I
I
I
I
I
20
0.04
0.06
ATP (mM) Fig. 3. Inhibition of PrP2A- by ATP - Incubation of the protamine kinase with PrP2Ac for 10 min in the presence of ATP and assay of the protamine kinase for 3 min were performed (o---o) as described in the legend to Fig. 1. The 100% control value refers to the maximal inactivation of the protamine kinase observed in the absence of ATP. The effect of ATP on the phosphorylase phosphatase activity of PrP2A (o---o) was determined as described (11). The 100% control value for p%osphorylase phosphatase was the activity determined in the absence of ATP.
452
Vol.
166,
No.
1, 1990
The
rate
BIOCHEMICAL
of protamine
was unaffected The PrP2Ac
was
by NaCl
may
the
completely
of
mine
kinase
the
most
in
in
kidney
enzyme contain
because
extracts
(1).
In
does addition,
1 mM EDTA (1).
report
from
sodium
kinases
by
occurred
of
increase
all
the
Therefore,
protamlne
kinase
for
purified inhibition
indicate
kidney
either
during
the
buffers during
the
in
isolation
3 min
453
were
as
described
protacourse
appears
the
of
this
purification
of the
the
to be
determined
purification
employed
enzyme
the
kinase
the
inacti-
This
during
possibility protamine
is kidney
groups.
the
that
cytosol
NaCl (mM) Inhibition of PrPZA by NaCl Incubation of Fig. 4. kinase with PrP2Ac -for 10 min in the absence and presence assay of the to Fig. 1.
vanadate.
the
Half-maximal
the purified
The latter
activity
not
preparations
EGTA or
phosphoryl
have
--in vivo. the
that
regulatory could
PrP2Ac
strongly
isolated
and therefore bound
COMMUNICATIONS
4).
this
kinase
and/or
likely
EDTA,
by 200 mM NaCl.
phosphorylation
of the purification
RESEARCH
by the
protamine
50 mM (Fig.
protamine
covalently
Ca2+,
cytosolic
by dephosphorylation, contain
Mn2+,
presented
the
BIOPHYSICAL
inactivation
inhibited
at about
results
activity vated
of
occurred
The
kinase
by 1 mM Mg2+,
inactivation
AND
protamine
protamine
of NaCl, and in the legend
Vol.
166, No. 1, 1990 kinase,
BIOCHEMICAL
divalent-cation
dependent
expected
to be inhibited.
tion
performed
in
the
purification,
is
steps
of
is(are)
likely
stages
of
the
containing PrP2A
AND BIOPHYSICAL
It
phosphorylation
is
also
a phosphate
buffer
protamine inhibited.
purification
protamine
the
at a concentration
catalyzed
of
pertinent
to be completely
NaCl
RESEARCH COMMUNICATIONS
of
enzyme
to note
that
(1).
Therefore,
kinase
protein
kinase
the
is
Under
the
first
phosphatase(s) at all
present these
protamine
can be
homogenizaat
In addition,
> 200 mM.
inactivation
this
the
in
later
solutions
conditions,
kinase
is
the
completely
C
inhibited
(Fig.
cannot
4).
account
protein
for
any loss
phosphatases
protamine
kinase,
Protein
residues
(14,15).
exhibit
protamine
composition of
residues,
the
is
detectable it
is
the
PrP2A Whether
activity
for
of
the
proteins,
the
activity
towards
conceivable
phosphoseryl enzyme
that
on
preliminary
and
not
of
kinase
phosphoserine
that
inactivates
the
which
phosphoamino indicated
nor PrP2A
residues
been
of phosphotyrosyl
phosphothreonine
certain
has
PrP2Ac
analysis
protamine
and
phosphotyrosine
by dephosphorylation
almost
possible
may be
that
involved
idea
was First,
protein
phosphatase of
insulin
insulin-stimulated chromatography
a covalent in
in
it
was
the
the
phosphotyrosyl catalyzes
the
C
present
on
phosphorylation
mechanism
response
suggested
tions.
effect
purification.
specific
some
cytosolic
seems
enzyme was stimulated This
by
the
purified
kinase.
It kinase
but
based
of
the
regulate
relatively
With
phosphoseryl
dephosphorylation
PrP2A
preparations
of
it
protamine
2A is
Therefore,
However,
during
catalyzed
to be determined.
low
kinase
residues.
presence
than
residues.
to
acid
other
phosphatase
reported
the
of activity
remains
phosphothreonyl
dephosphorylation
Therefore,
necessary
inhibitor on the increase
(2) to
in rat
the
based
sodium
on protamine-agarose,
protamine kinase
DEAF-cellulose, 454
the
protamine
activity rat
on the
homogenization
protamine
the
in isolated
include
hepatocyte in
by which
to insulin
previously
of
of
this
hepatocytes.
following
observa-
fluoride,
a general
buffers
to detect
the
kinase.
Second,
the
activity and
was
stable
Sephacryl
to S-200
Vol.
166,
gel
No.
1, 1990
BIOCHEMICAL
filtration
indicating
involved.
Third,
control
cells on
kinase
activity
tion
of
the
kinase
synthesis
increase
in the
of
that
supported
by
experiments
not
pool
the
kinase
of
the
phosphorylation
results
presented
partially were
of
in
purified
also
cyclohexiimide,
the
insulin-stimulated
that
insulin
the
associa-
Clearly, protamine
protamine
the kinase
kinase
communication.
following
a
had stimu-
kinase.
preparations
inactivated
stimulated
Fourth,
hepatocyte
this
not
of the
the
of
chromato-
modulation
indicating
was and
permeation had
prevent
activity
a pre-existing
increased
utilizing
protamine
did
kinase
insulin-treated
insulin
factor.
insulin-stimulation
via
from
or via
COMMUNICATIONS
of the
by gel
that
a regulatory
inhibitor,
activity
occur
indicating
kinase
kinase
as estimated
proteolysis
with
protamine
possibility may
S-200
RESEARCH
modification
Mr of the
45,000
by limited
the
BIOPHYSICAL
a covalent
apparent
Sephacryl
protein
lated
that
was about
graphy
AND
is
Moreover,
of
the
incubation
hepatocyte with
PrP2A C
(not
shown).
In
preparations and
this
of
an
partially
regard,
purified
inactivated
following and
inactivated
the
has
been
in
(16)
kinase
that
the
together
with
purified
S6 kinase
(16).
also
been
shown
In
the
been
ribosomal
cascade
kinase(s)
the
protamine
of sequential
the
of
which
would
the
PrP2Ac
identified. protein
of
protein-2
reactivation
yet
to be
addition,
30% of maximum)
The
(16)
insulin-stimulated
PrP2Ac.
kinase,
an insulin-stimulated
that
microtubule-associated
not
protein-2
phosphorylation/activation
have
concomitant
has
note
an
(about
reported and
microtubule-associated
participate
of
with
by
phosphorylation
however,
protein
reactivation
kinase
protamine
possible,
ribosomal
incubation
S6
preparations the
to
kinase
partial
protein
catalyze
interesting
preparations protein-2
phosphorylation
kinase
is
insulin-stimulated
microtubule-associated
ribosomal
it
It
is
S6 kinase
and
kinase
may
protein
kinase
reactions.
Acknowledgments I Lisa
am grateful
Ball
for
typing
to
Clint this
Cook for report.
the
I also 455
art thank
work Dr.
and Debra Michael
Williams Felder
and and my
Vol.
166, No. 1, 1990 graduate useful
students comments.
and by the Weight
BIOCHEMICAL Mr. This
Shrikanth work
Watcher's
AND BlOPHYSICAL A.G.
was
Reddy
supported
RESEARCH COMMUNICATIONS
and Mr.
Grayson
D. Amick
for
by the
Carolina
Venture
Fund
Foundation. References
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
Damuni, Z., Amick, G.D., and Sneed, T.R. (1989) J. Biol. Chem. 264, 6412-6416. Reddy, S.A.G., Amick, G.D., Cooper, R.H., and Damuni, Z. (1989) J. Biol. Chem. in press. Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. Laemmli, U.K. (1970) Nature 227, 680-685. Merril, C.R., Goldman, D., Sedman, S.A., and Ebert, M.M. (1981) Science 211, 1437-1438. Tung, H.Y.L., Pelech, S., Fisher, M.J., Pogson, M.J., and Cohen, P. (1985) Eur. J. Biochem. 149, 305-313. Stewart, A.A., Hemmings, B.A., Cohen, P., Goris, J., and Merlevede, W. (1981) Eur. J. Biochem. 115, 197-205. H.Y.L., Resink, T.J., Hemmings, B.A., Shenolikar, S., and Tmz, Cohen, P. (1984) Eur. J. Biochem. 145, 51-56. Lee, E.Y.C., Silberman, S.R., Ganapathi, M.R., Paris, H., and Petrovic, S. (1981) Cold Spring Harb. Conf. Cell. Prolif. 8, 425-439. Ingebritsen, T.S., and Cohen, P. (1983) Science 221, 331-338. Ingebritsen, T.S., Foulkes, J.G., and Cohen, P. (1980) FEBS Lett. 119, 9-15. Ingebritsen, T.S., Foulkes, J.G., and Cohen, P. (1983) Eur. J. Biochem. 132, 263-274. Damuni, Z., and Reed, L.J. (1988) Arch. Biochem. Biophys. 262, 574-584. Chernoff, J., Li, H.-C., Cheng, Y.-S.E., and Chen, L.B. (1983) J. Biol. Chem. 258, 7852-7857. Foulkes, J.G., Erickson, E., and Erickson, R.L. (1983) J. Biol. Chem. 258, 431-438. Sturgill, T.W., Ray, B.L., Erickson, R.E., and Maller, J.L. (1988) Nature 334, 715-718.
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