(Received for publication, February 9, 1995; and in revised form, May 19, 1995)
From the
Inhibitor-2 (I-2) is the regulatory subunit of the cytosolic
ATP-Mg-dependent form of type 1 serine/threonine protein phosphatase
and its phosphorylation at Thr-72 by glycogen synthase kinase-3 results
in phosphatase activation. Activation of cytosolic type 1 phosphatase
has been observed in cells treated with growth factors. Reported here
is the phosphorylation and activation of the ATP-Mg-dependent
phosphatase by mitogen-activated protein kinase (MAPK). Recombinant I-2
was phosphorylated by activated MAPK to an extent (
Much recent work on growth factor-mediated signal transduction
pathways has centered on the role of protein kinase cascades activated
by initial stimulation of the receptors (for reviews see Posada and
Cooper(1992), Fantl et al.(1993), Crews and Erickson (1993),
Nishida and Gotoh(1993), and Neiman(1993)). Although tyrosine
phosphorylation has a special significance for the events proximal to
the receptor, much of the intracellular signaling is mediated by
serine/threonine phosphorylation. The protein Ser/Thr phosphatases are
therefore important candidates to have major regulatory roles in the
control of the transmission of the signals. The type 1 phosphatases
exist in cells as holoenzymes composed of a common 37-kDa catalytic
subunit and a specific regulatory subunit, which influences both
substrate specificity and subcellular localization of the enzyme. These
holoenzymes include the ATP-Mg-dependent phosphatase, the
glycogen/sarcoplasmic reticulum-associated phosphatase, the
myosin-associated phosphatase (for reviews see Hubbard and Cohen(1993)
and DePaoli-Roach et al.(1994)) and the nuclear phosphatase
containing NIPP1 (Beullens et al., 1992). Among these
holoenzymes, the widely distributed cytosolic form of type 1
phosphatase, the ATP-Mg-dependent phosphatase, has been studied most
extensively (Yang et al., 1981; Hemmings et al.,
1982; Ballou et al., 1983; DePaoli-Roach, 1984; Park and
DePaoli-Roach, 1994). The activity of this phosphatase is regulated
through reversible phosphorylation of its regulatory subunit,
inhibitor-2 (I-2). ( There is evidence that the ATP-Mg-dependent phosphatase activity in
cells is regulated by extracellular signals. Chan et al.(1988)
initially reported that treatment of 3T3 cells with insulin and other
growth factors caused a rapid and transient activation of a cytosolic
type 1 protein phosphatase. Subsequently, Yang et al.(1989)
and Villa-Moruzzi(1989) also observed activation of the
ATP-Mg-dependent phosphatase and the activating factor F GSK-3, as mentioned above, has generally
been considered to be responsible for activation of the
ATP-Mg-dependent phosphatase. However, recent observations raise
questions as to whether this protein kinase functions as an activator
of the phosphatase in vivo under growth factor-stimulated
conditions. Ramakrishna and Benjamin(1988) initially reported
inactivation of GSK-3 in insulin-treated adipocytes. More recently,
Welsh and Proud (1993) have described an insulin-induced inactivation
of the kinase in Chinese hamster ovary cells, apparently mediated by
activation of the p90 Mitogen-activated protein -kinases (MAPK) or extracellular
signal-regulated protein kinases (ERK) are components of the signal
transduction pathways triggered by numerous extracellular signals
including mitogens and growth factors (for reviews, see Cobb et
al.(1991), Sturgill and Wu(1991), Blenis(1993), and Davis(1993)).
Their activation proceeds via a cascade that involves two upstream
protein kinases, a MAPK kinase and a MAPK kinase kinase. The downstream
targets of MAPK may include the S6 ribosomal protein kinase
p90 The MAPK signaling cascade has already been
implicated in the regulation of protein serine/threonine phosphatase
activity. It has been postulated, from in vitro experiments,
that p90
Figure 1:
Phosphorylation of recombinant I-2 by
ERK1. Purified recombinant GST-ERK1 (31 µg/ml) was incubated with
or without the GST-MEK2 (12.5 µg/ml) for 10 min at 30 °C in a
14-µl reaction containing [
Figure 2:
Identification of Thr-72 of I-2 as the
site modified by ERK1. Panel A, phosphorylation of wild type
I-2 (I-2 WT) and I-2Ala72 mutant by ERK1. ERK1 (10 µg/ml),
after preactivation in the presence of cold ATP as described under
``Experimental Procedures,'' was incubated with equal amounts
(20 µg/ml) of I-2 proteins in a 20-µl reaction for 30 min at 30
°C. Samples corresponding to 0.12 µg of protein were then
subjected to SDS-PAGE and an autoradiogram is shown. The arrowhead indicates a lane in which a reaction mixture without substrate was
loaded. Panel B, phosphoamino acid analysis of wild type I-2
phosphorylated by ERK1 (lane1) and GSK-3
Phosphate was introduced into I-2 by
ERK1 up to a stoichiometry of 0.3 mol of phosphate/mol of I-2 (Fig. 3), similar to the extent of phosphorylation catalyzed by
GSK-3
Figure 3:
Time course of I-2 phosphorylation by
activated ERK1. Phosphorylation of recombinant I-2 (20 µg/ml) was
performed with activated ERK1 (5 µg/ml). Aliquots were removed at
different times and the phosphoproteins were separated by SDS-PAGE. Gel
regions containing
Figure 4:
Synergistic phosphorylation of I-2 by
GSK-3
Kinetic analyses of I-2 phosphorylation by
ERK1 gave a K
Figure 5:
Phosphorylation of I-2 by
p90
The effect of the phosphorylation of I-2
in the inactive holoenzyme complex was then determined with different
mitogen-regulated protein kinases. The activity of the kinases was
normalized so that approximately equal I-2 phosphorylation activities
were used. The action of GSK-3 and the activated ERK1 resulted in
activation of the phosphatase; however, no significant stimulation of
the phosphatase activity was observed with p90
Figure 6:
Time course of activation of
ATP-Mg-dependent protein phosphatase by different kinases. The purified
inactive ATP-Mg-dependent phosphatase (1 µg/ml) was incubated with
GSK-3
The ATP-Mg-dependent phosphatase represents the major
cytosolic form of type 1 phosphatase. Activation in vitro is
triggered by phosphorylation of Thr-72 of its regulatory subunit, I-2,
by GSK-3/F Cohen and co-workers, based on in vitro studies, have
proposed that the glycogen-associated phosphatase is responsible for
the control of glycogen metabolizing enzymes and that another
mitogen-regulated kinase, p90 Phosphorylation and activation of the type 1 protein phosphatases by
the growth factor-activated kinase cascade is of great interest. In
mammals, extracellular stimuli trigger numerous signal transduction
pathways leading to both increased and decreased phosphorylation of a
large number of proteins located in different cellular compartments
(Davis, 1993). Moreover, growth factor stimulation results in a
transient phosphorylation and activation of several components of the
MAPK cascade. As a controlled system, a mechanism for dephosphorylation
of these transiently activated protein kinases must exist in cells.
Activation of a cytosolic phosphatase by MAPK could be responsible for
control of proteins that undergo dephosphorylation following growth
factor stimulation, but could also be involved in a feedback
down-regulation of the kinase cascade itself. It is interesting in this
respect that the Raf-1 (Kovacina et al., 1990) and S6 protein
(Oliver et al., 1988) phosphatases appear to be of type 1. In conclusion, the present investigation demonstrates for the first
time that the ATP-Mg-dependent protein phosphatase is a substrate for
MAPK and, furthermore, that phosphorylation of the regulatory subunit
of the holoenzyme leads to activation of the phosphatase. These results
may underlie the mitogen-induced activation of the cytosolic type 1
protein phosphatase and suggest that MAPK may be a physiological kinase
activating the phosphatase in cells stimulated by insulin, growth
factors, or other mitogens.
0.3 mol of
phosphate/mol of polypeptide) similar to that reported for
phosphorylation by the
isoform of glycogen synthase kinase-3. The
phosphorylation of I-2 by MAPK was exclusively at Thr-72, the site
involved in the activation of phosphatase. Incubation of MAPK with
purified ATP-Mg-dependent phosphatase resulted in phosphorylation of
the I-2 component and activation of the phosphatase. Ribosomal S6
protein kinase II (p90
) was also able to
phosphorylate the recombinant I-2; however, this phosphorylation
occurred on serines and had no effect on phosphatase activation. Our
data may explain growth factor-induced activation of the
ATP-Mg-dependent phosphatase and suggest that MAPK may be the
physiological kinase responsible for the activation of cytosolic type 1
phosphatase in response to insulin and/or other growth factors.
)Several protein kinases such as
glycogen synthase kinase-3 (GSK-3; Hemmings et al., 1982),
casein kinase II (CK II; DePaoli-Roach, 1984) and casein kinase I
(Agostinis et al., 1992) have been identified as I-2 kinases.
Phosphorylation of I-2 at Thr-72 by GSK-3, also called F
for phosphatase activating factor, results in activation of the
phosphatase (Hemmings et al., 1982; Ballou et al.,
1983). Modification of I-2 at serine residues by CK II does not affect
the phosphatase activity directly but enhances Thr-72 phosphorylation
catalyzed by GSK-3
and thus results in a synergistic activation of
the phosphatase (DePaoli-Roach, 1984; Park et al., 1994).
.
Growth factor stimulation of CK II, which potentially could affect the
phosphatase activity, has also been reported (Sommercorn et
al., 1987; Klarlund and Czech, 1988), and an insulin-induced
increase in serine phosphorylation of I-2 has been shown (Lawrence et al., 1988).
(Welsh et al.,
1994; Cross et al., 1994). These observations indicate that
GSK-3 may not be the factor that activates the ATP-Mg-dependent
phosphatase following insulin and growth factor stimulation and,
therefore, other activating protein kinases may exist.
(Sturgill et al., 1988), the
mitogen-activated protein kinase-activated protein kinase-2 (Stokoe et al., 1992), phospholipase A2 (Davis, 1993), PHAS-1
(Haystead et al., 1994), and a number of nuclear transcription
factors encoded by proto-oncogenes (Seth et al., 1992; Davis,
1993). Investigation of the proteins and peptides phosphorylated by
MAPK indicated that MAPK recognizes a serine or threonine in a
preferred motif -Pro-Xaa-Ser/Thr-Pro- (Davis, 1993). Thus, the
substrate specificity of MAPK is somewhat similar to protein kinases
such as cyclin-dependent protein kinases and GSK-3. In addition, we
have shown recently (Wang et al., 1994a) that, similar to MAPK
(Wu et al., 1991), GSK-3 is a dual specificity protein kinase
in the sense that it autophosphorylates on serine/threonine and
tyrosine residues.
phosphorylates the 124-kDa regulatory
subunit (R
; Tang et al., 1991; Dent et
al., 1990) of glycogen-associated type 1 protein phosphatase. This
phosphorylation of R
would increase phosphatase activity
relevant to controlling glycogen metabolism. From an investigation of
the actions of several protein kinases regulated by insulin and growth
factors, we found that MAPK phosphorylates and activates the
ATP-Mg-dependent protein phosphatase. Thus, the same signaling pathway
may cause activation of another type 1 phosphatase holoenzyme. These
findings may underlie the insulin- and other growth factor-induced
activation of cytosolic type 1 phosphatase and suggest that MAPK may be
one of the in vivo activating kinases for the ATP-Mg-dependent
protein phosphatase.
Protein Kinases
The GSK-3 was purified from
rabbit skeletal muscle as reported previously (Fiol et al.,
1990). Expression and purification of the recombinant rabbit skeletal
muscle GSK-3
was as described (Wang et al., 1994a).
Recombinant human MEK2 and ERK1 (Charest et al., 1993) were
prepared as reported previously (Zheng and Guan, 1993a, 1993b). Both
ERK1 and MEK2 were expressed as fusion proteins with glutathione S-transferase (GST) and purified as previously reported (Zheng
and Guan, 1993a). The ERK1 used for this study was either a GST fusion
protein or else a non-fusion protein generated by cleavage of the GST
portion by thrombin. Homogeneous bovine testis CK II (Litchfield et
al., 1990) and recombinant Xenopus ribosomal S6 kinase
expressed in baculovirus (p90
, Vik et
al., 1990) were kindly provided by Dr. D. Litchfield (Manitoba
Institute of Cell Biology) and Dr. T. A. Vik (Indiana University),
respectively. One unit of CK II activity is defined as the amount of
enzyme that transfers 1 nmol of phosphate/min into the peptide
substrate RRRDDDSDDD at 30 °C. One unit of GSK-3 or MAPK catalyzes
the incorporation of 1 nmol of phosphate/min into the recombinant I-2
protein at 30 °C under the conditions described below.
In Vitro Activation of Recombinant GST-MEK2 and
ERK1
Recombinant GST-MEK2 was activated in vitro by a
mitogen-stimulated cell lysate as reported previously (Zheng and Guan,
1993a, 1993b). The activated GST-MEK2 was then purified by a
glutathione-agarose affinity column and used for the activation of
recombinant ERK1. The purified recombinant ERK1 was incubated with
preactivated GST-MEK2 at 30 °C for the indicated times, in a
phosphorylation reaction described below. To prepare larger amounts of
the activated ERK1, the non-fusion protein (10-20 µg) was
phosphorylated for 2 h by the preactivated GST-MEK2 with
non-radiolabeled ATP (0.2 mM). The activated ERK1 was
separated from the GST-MEK2 by a glutathione-agarose affinity column
(Zhang and Guan, 1993b). The pass-through containing the activated
kinase was collected, concentrated by Centriprep-30 (Amicon), and
stored at -20 °C. Activation of ERK1 by MEK was evaluated by
both myelin basic protein (Zheng and Guan, 1993a) and I-2
phosphorylation assays.Phosphorylation of Inhibitor-2
Proteins
Recombinant I-2 wild type, I-2Ala72, in which the
Thr-72 is mutated to alanine, the NH-terminal truncation
(
N) lacking the first 35 amino acids, and the COOH-terminal
deletion (
C) lacking the last 58 amino acids were prepared and
purified as described previously (Park et al., 1994; Park and
DePaoli-Roach, 1994). Wild type and mutant I-2 proteins, at the
indicated concentrations, were incubated with different protein kinases
at 30 °C in a phosphorylation reaction (20 µl) that contained
30 mM Tris-HCl, pH 7.5, 5% glycerol, 10 mM MgCl
, and 100 µM [
-
P]ATP (2,000-10,000 cpm/pmol).
Reactions were initiated by addition of
[
-
P]ATP/Mg
and terminated
by addition of 5
SDS-sample buffer (65 mM Tris
phosphate, pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, and
0.25% bromphenol blue) followed by 5 min of treatment at 100 °C.
Phosphoproteins were then separated by SDS-PAGE on 12% polyacrylamide
gels and detected by autoradiography. For quantitation, the regions of
the gel corresponding to the
P-labeled proteins were
excised and the Cerenkov radiation was counted.
Purification of the ATP-Mg-dependent
Phosphatase
Approximately 1200 g of hind limb and back rabbit
muscle were homogenized in a Waring Blendor (30 s at low speed followed
by an additional 30 s at medium speed) with three volumes of buffer,
containing 20 mM Tris-HCl, pH 7.5, 2 mM EGTA, 5
mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.1
mM tosyl-L-lysine chloromethyl ketone, 0.5
mM tosyl-L-phenylalanine chloromethyl ketone, 2
mM benzamidine, 2 mg/liter each leupeptin, antipain, and
pepstatin, and 0.1% (v/v) 2-mercaptoethanol. All procedures were
performed at 4 °C. After centrifugation for 30 min at 10,000
g, the supernatant was adjusted to pH 7.3 and made 55%
saturated with ammonium sulfate. The suspension was centrifuged for 20
min at 10,000
g and the pellet resuspended in 350 ml
of homogenization buffer, except that only 0.1 mM EDTA was
included (buffer A). Five volumes of room temperature 95% ethanol were
added, and, after rapid stirring, the sample was immediately
centrifuged for 15 min at 10,000
g. The pellet was
extracted with 450 ml of buffer A, centrifuged for 25 min at 10,000
g and the supernatant filtered through glass wool. The
pellet was reextracted with an additional 300 ml of buffer A and
centrifuged as before. The supernatants of the two extractions were
combined and taken to 65% ammonium sulfate saturation. After
centrifugation for 20 min at 10,000
g, the pellet was
resuspended in buffer B (buffer A plus 10% (v/v) glycerol) and
extensively dialyzed overnight against the same buffer. Following
clarification by centrifugation for 10 min at 10,000
g, the supernatant was made 0.1 M NaCl (buffer C) and
applied to a 4 cm
21-cm column of DEAE Sepharose CL-6B
equilibrated in the same buffer, except that benzamidine was omitted.
The column was washed with 300 ml of buffer C and developed with a
1200-ml salt gradient from 0.1 to 0.4 M NaCl. Fractions of 10
ml were collected and assayed for phosphorylase phosphatase activity in
the presence and absence of I-2. Two peaks of phosphorylase phosphatase
activity were eluted, one inhibited by I-2 at
175 mM NaCl
and the other insensitive to I-2 at
220 mM. Fractions
from the latter peak, containing the catalytic subunit of protein
phosphatase 2A, were pooled, dialyzed against buffer A plus 50% (v/v)
glycerol, and stored at -80 °C. The sample was then diluted
with an equal volume of buffer A and applied to a 10-ml
polylysine-agarose column. The enzyme activity did not bind to the
column and was recovered in the unbound material as a near homogeneous
preparation of protein phosphatase 2A catalytic subunit, which was
stored at -80 °C. The fractions from the first peak of the
DEAE-Sepharose column containing I-2-sensitive phosphatase were pooled,
diluted with an equal volume of buffer A and applied to a
polylysine-agarose column (1.5 cm
10 cm). The column was washed
with 250 ml of buffer and the phosphatase activity eluted with buffer B
containing 0.25 M NaCl. The fractions containing phosphatase
that could be inhibited by I-2 were dialyzed overnight against buffer A
containing 0.2 M NaCl and 70% (v/v) glycerol to reduce the
volume. The sample was then chromatographed on a 2.5 cm
70-cm
column of Sephadex G-100 superfine equilibrated in buffer B plus 0.2 M NaCl. Fractions were assayed for
GSK-3/F
-dependent and spontaneously active phosphatase.
Fractions containing the latter were dialyzed against buffer A
containing 60% (v/v) glycerol and stored at -80 °C. Fractions
containing the ATP-Mg-dependent phosphatase were diluted with buffer B
and rechromatographed on a 0.6 cm
5-cm polylysine affinity
column equilibrated with buffer B. After washing with 80 ml of the same
buffer, the column was developed by stepwise increasing the salt
concentration; each step was 30 ml of buffer B containing first 0.2 M then 0.25 M and finally 0.5 M NaCl. Minor
contaminants eluted at 0.2 M salt, and the ATP-Mg-dependent
phosphatase eluted at 0.25 M NaCl. The most active fractions
were frozen directly at -80 °C. The side fractions were
pooled, concentrated by dialysis against buffer A plus 60% (v/v)
glycerol, and then stored at -80 °C.
Activation of ATP-Mg-dependent Phosphatase
The
inactive form of ATP-Mg-dependent protein phosphatase purified from
rabbit skeletal muscle was activated at 30 °C in an incubation
mixture containing 50 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 0.2 mM ATP, 5 mM MgCl, 0.2%
2-mercaptoethanol in the presence of the indicated kinases. At the
indicated times, aliquots (10 µl) were used to determine
phosphorylase phosphatase activity. The phosphatase assay was for 10
min at 30 °C in a 50-µl reaction containing 50 mM Tris-HCl, pH 7.2, 0.2% 2-mercaptoethanol, 0.2 mg/ml bovine serum
albumin, 5 mM caffeine, and 2 mg/ml
[
P]phosphorylase a (200-3000
cpm/pmol) as described previously (DePaoli-Roach, 1984).
Phosphoamino Acid Analysis
Protein substrates
phosphorylated by the indicated protein kinases were separated by
SDS-PAGE and transferred to Immobilon-P membranes (Millipore). Membrane
regions that contained the P-labeled proteins were excised
and hydrolyzed in 5.7 N HCl at 110 °C for 90 min.
Phosphoamino acids were then separated by one-dimensional thin layer
electrophoresis on cellulose-coated sheets (Kodak) and detected by
autoradiography as described (Wang et al., 1994a).
Other Materials and Methods
Reagents and chemicals
for SDS-PAGE gel electrophoresis and phosphorylation studies were
obtained from Bio-Rad and Sigma. Materials for protein purification
were purchased from Whatman, Pharmacia Biotech Inc., and Sigma.
Glutathione-agarose resin and glutathione were from Sigma.
[-
P]ATP was from DuPont NEN. Restriction
enzymes and reagents for molecular biology were obtained from Life
Technologies and New England Biolabs. Protein concentration was
determined by both the method of Bradford(1976) and densitometric
scanning using bovine serum albumin as standard.
Purification of ATP-Mg-dependent Protein
Phosphatase
The ATP-Mg-dependent phosphatase was purified
through a procedure that involved treatment with 80% ethanol at room
temperature. Although such procedures have more commonly been used to
isolate the catalytic subunit of type 1 and 2A phosphatases (Brandt et al., 1975), the inclusion of the mixture of protease
inhibitors and the speed of the procedure at an early stage of the
preparation prevents degradation of I-2, thus allowing purification of
the I-2CS1 complex. Two peaks of phosphorylase phosphatase
activity were resolved on DEAE Sepharose CL-6B. Peak 1 eluted at 175
mM NaCl and was completely inhibited by I-2; peak 2 eluted at
220 mM NaCl and was not inhibited by I-2. Peak 1, which
contained type 1 phosphatase, was further processed through a
polylysine-agarose column. The enzyme activity eluted in a single peak
that again was completely inhibited by I-2. Analysis on SDS-PAGE of the
active fractions revealed the presence of two major polypeptides of M
38,000 and 31,000, respectively. The ratio
of the two polypeptides was in favor of the 38,000-dalton species. The
unexpected presence of the 31,000-dalton species, the same apparent M
as I-2, suggested that some ATP-Mg-dependent
phosphatase might be present. In fact, when the fractions containing
the enzyme activity were pooled and chromatographed on Sephadex G-100,
two distinct peaks of activity were detected. One corresponded to an
apparent M
of 140,000, dependent on ATP-Mg and
GSK-3 for activity, and the other corresponded to an apparent M
of 40,000 and was spontaneously active. SDS-PAGE
across the peaks of activity demonstrated that the enzyme with M
140,000 contained two polypeptides of 38,000 and
31,000 daltons in an approximately 1:1 ratio, whereas the spontaneously
active enzyme was composed only of a 38,000 dalton polypeptide. The
ATP-Mg-dependent phosphatase was further purified through a second
polylysine-agarose column to remove minor contaminants. The specific
activity of the purified ATP-Mg-dependent phosphatase was 24,000
units/mg and that of the type 1 catalytic subunit 23,000 units/mg of
protein.
Phosphorylation of Recombinant I-2 by Activated
MAPK
The recent reports that GSK-3 is inactivated in growth
factor-stimulated cells (Ramakrishna and Benjamin, 1988; Welsh and
Proud, 1993; Welsh et al., 1994; Cross et al., 1994)
prompted us to investigate the ability of other protein kinases to
phosphorylate I-2. Since both GSK-3 (Wang et al., 1994) and
MAPK (Wu et al., 1991) are dual specificity kinases and the
primary sequence surrounding Thr-72 in I-2 conforms to the MAPK
recognition motif, this kinase was considered as a potential candidate.
When purified recombinant wild type I-2 was used as a substrate,
non-activated GST-ERK1 was able to phosphorylate the protein, albeit to
a very low extent (Fig. 1, lane2), but
treatment of GST-ERK1 with GST-MEK2 led to a much higher level of
phosphorylation (Fig. 1, lane3). Activation
of ERK1 by MEK resulted in a similar, up to 15-fold, increase in
phosphorylation of both myelin basic protein (specific activity
2
nmol/min/mg) and wild type I-2; neither of the two proteins was a
substrate for GST-MEK2 (Fig. 1). Under similar conditions no
detectable phosphorylation of I-2Ala72, in which Thr-72 was mutated to
alanine, was observed (Fig. 2A), indicating that the
site modified was Thr-72. Phosphorylation of I-2 by both GSK-3 and ERK1
caused a reduction in electrophoretic mobility on SDS-PAGE gels of the
31-kDa polypeptide. Phosphoamino acid analysis of I-2 phosphorylated by
ERK1 confirmed that threonine was the only phosphorylated residue (Fig. 2B, lane1). These data
strongly suggest that MAPK phosphorylates I-2 solely on Thr-72, the
same site recognized by GSK-3.
-
P]ATP, as
described under ``Experimental Procedures,'' before addition
of purified recombinant wild type I-2 protein (24 µg/ml) or myelin
basic protein (MBP, 30 µg/ml). The reaction was then
continued for an additional 30 min. Aliquots corresponding to 0.13
µg of I-2 and 0.17 µg of myelin basic protein were separated on
SDS-PAGE, and an autoradiogram is shown.
(lane2). The reactions were performed under the
conditions described in panelA using either ERK1 or
GSK-3
(4 µg/ml). Phosphoamino acid analysis was performed as
described under ``Experimental Procedures.'' The migration of
phosphoserine (P-Ser), phosphothreonine (P-Thr), and
phosphotyrosine (P-Tyr) is
indicated.
(DePaoli-Roach, 1984; Park et al., 1994). Since the
action of GSK-3
is enhanced by prior phosphorylation of I-2 by CK
II (DePaoli-Roach, 1984; Park et al., 1994), we tested whether
Thr-72 phosphorylation by MAPK was similarly potentiated. No
synergistic phosphorylation by ERK1 and CK II was observed (Fig. 4), although synergism did occur when GSK-3
and CK II
were used (Fig. 4). These results demonstrate that Thr-72
phosphorylation by MAPK was independent of prior phosphorylation,
similar to the phosphorylation by the
-isoform of GSK-3 (Wang et al., 1994b).
P-labeled I-2 protein were excised and
the associated radioactivity quantitated by Cerenkov counting. The inset shows an autoradiogram of the region of the gel
corresponding to the I-2 phosphorylated at the same time points shown
in the graph.
but not ERK1. Phosphorylation of I-2 (24 µg/ml) was
performed with the indicated protein kinases as described under
``Experimental Procedures.'' The reaction was for 20 min and
contained 4.5 milliunits of CK II, 6 milliunits of GSK-3
, or 10
µg/ml activated ERK1. After SDS-PAGE, the regions of the gel
corresponding to the
P-labeled I-2 protein were excised
and the associated radioactivity quantitated by Cerenkov counting. The
stoichiometry of phosphorylation was then
determined.
value of 160
µM, 5-fold and 11-fold higher than the K
values determined for GSK-3
and
GSK-3
, respectively (Table 1). However, the V
for MAPK was
5-fold higher than that for
GSK-3
(Wang et al., 1994b). Thus, the V
/K
value for MAPK
is only
2-fold lower than that for GSK-3
. Under the
conditions used, ERK1 was activated
20% as compared with other
studies of a similar enzyme preparation (Zheng and Guan, 1993a, 1993b).
This submaximal activation does not change the sense of the results but
does imply that the V
of ERK1 for I-2
phosphorylation may have been significantly underestimated.
Phosphorylation of Recombinant I-2 by
p90
In an attempt to identify other
potential mitogen-stimulated protein kinases phosphorylating and
activating the ATP-Mg-dependent phosphatase in cells, we have also
examined the effect of p90. Xenopus p90
expressed in baculovirus was also found
to phosphorylate I-2 (Fig. 5A). Preactivation of
p90
by MAPK stimulated the activity toward I-2
by
3-fold (data not shown). While phosphorylation by either GSK-3
or MAPK resulted in a decreased electrophoretic mobility of I-2,
phosphorylation by p90
, under all conditions
used, did not affect I-2 mobility, suggesting that Thr-72 was not the
major target site for p90
. This prediction was
confirmed by the phosphorylation of the I-2 Ala72 mutant. With wild
type and Ala72 mutant I-2, a similar degree of phosphorylation by
p90
was observed (Fig. 5A). In
addition, phosphoamino acid analysis demonstrated that only
phosphoserine was present in both I-2 proteins (Fig. 5B). Based on the consensus recognition sequence
for p90
(Erickson and Maller, 1988), several
potential phosphorylation sites are present in I-2. These sites are
serines 21, 28, and 203. In order to define the sites phosphorylated by
p90
, I-2 truncation mutants lacking the first 35
(
N) or the last 58 (
C) residues (Park and DePaoli-Roach,
1994) were tested as substrates. Both I-2
N and
C were
phosphorylated by the kinase (data not shown). These results suggest
that p90
phosphorylates multiple serines of I-2.
. Panel A, phosphorylation of I-2 by
p90
. Wild type I-2 (I-2 WT) and
I-2Ala72 (24 µg/ml) were phosphorylated by p90
(10 µg/ml) for 20 min as described under
``Experimental Procedures.'' Samples corresponding to 0.13
µg of I-2 proteins were separated on SDS-PAGE, and an autoradiogram
is shown. Panel B, phosphoamino acid analysis of
P-labeled I-2 proteins. Aliquots of the same samples
prepared in panelA were subjected to phosphoamino
acid analysis as described in the legend of Fig. 2B; an
autoradiogram is shown. Lane 1 corresponds to wild type I-2
phosphorylated by GSK-3
; lanes2 and 3 correspond to wild type I-2 and I-2Ala72, respectively,
phosphorylated by
p90
.
Phosphorylation and Activation of the ATP-Mg-dependent
Phosphatase by MAPK
The activated ERK1 was able to phosphorylate
the 31-kDa I-2 subunit in the purified rabbit skeletal muscle
ATP-Mg-dependent phosphatase but no phosphorylation of the catalytic
subunit (36 kDa) in the phosphatase complex was detected (data not
shown). In a parallel experiment, we tested both the ATP-Mg-dependent
phosphatase preactivated by GSK-3 and the free active catalytic subunit
for their ability to dephosphorylate ERK1 phosphorylated by MEK2. Under
the conditions used, no significant dephosphorylation of ERK1 was
observed (data not shown).
,
MEK2, or the inactive form of MAPK (Table 2). The level of
activation of the phosphatase by MAPK was slightly lower than that
elicited by GSK-3
and GSK-3
(Table 2). Maximal
activation of the phosphatase by ERK1 and GSK-3 was observed at
10-15 min of incubation under the conditions used (Fig. 6). When the kinase concentration dependence was analyzed,
the reaction was still linear at 225 milliunits of MAPK, and activation
of the phosphatase was 70% of that elicited by 150 milliunits of
GSK-3
(data not shown).
(circles), GSK-3
(triangles), or
activated ERK1 (squares) at 30 °C for the time indicated.
Equal amounts of kinases were used based on their I-2 phosphorylation
activity. At each time point, a 10-µl aliquot was tested for
phosphorylase phosphatase activity using
P-labeled
phosphorylase a as a substrate as described under
``Experimental Procedures.''
. Previous studies have indicated that the
activity of the phosphatase and its activator protein can be
transiently stimulated in cells by growth factors (Yang et
al., 1989; Villa-Moruzzi, 1989). However, the activating factor
had been characterized based only on its ability to activate the
phosphatase and rigorous identification as GSK-3 had not been
performed. Nevertheless, it had been assumed that GSK-3 was the
activating factor. This is inconsistent with earlier (Ramakrishna and
Benjamin, 1988) and more recent (Welsh and Proud, 1993; Welsh et
al., 1994; Cross et al., 1994) reports of an
insulin-induced inactivation of GSK-3. Thus, it is likely that
the growth factor-stimulated phosphatase activator was not GSK-3 but
another kinase able to regulate the activity of the ATP-Mg-dependent
phosphatase. In this study, evidence is presented that the
ATP-Mg-dependent phosphatase can be phosphorylated and activated in
vitro by MAPK. MAPK phosphorylates the free I-2 protein
exclusively on threonine 72 (Fig. 2, A and B),
to an extent similar to that reported for GSK-3
(DePaoli-Roach,
1984). Furthermore, phosphorylation of the I-2 component in the
ATP-Mg-dependent phosphatase holoenzyme correlates with activation of
the enzyme (Fig. 6). These results suggest that MAPK may be the
phosphatase activating kinase previously detected that is responsible
for the growth factor-induced activation of the cytosolic phosphatase.
Further supporting evidence for this hypothesis comes from the
observation that the activation of the phosphatase and the activating
protein by growth factors is transient and parallels the stimulation of
MAPK, reaching a maximum at 5-10 min and returning to basal
levels at 20-30 min (Chan et al., 1988; Yang et
al., 1989; Villa-Moruzzi, 1989). The extent of activation of the
ATP-Mg-dependent phosphatase in vitro by MAPK is similar to
that reported for GSK-3. Moreover, the relatively high concentration of
MAPK in the cell (Cobb et al., 1994) and its wide tissue
distribution render this kinase a potential candidate to activate the
phosphatase. It is possible that in non-stimulated cells GSK-3 might be
involved in the control of the ATP-Mg-dependent phosphatase, whereas in
stimulated cells the MAPK becomes the predominant activating kinase.
The possibility that yet other protein kinases may regulate the
ATP-Mg-dependent phosphatase cannot be ruled out. However, it is
unlikely that p90
is an activator of this
phosphatases since this kinase, activated by MAPK, phosphorylates
exclusively serine residues in I-2 and is unable to activate the
inactive phosphatase in vitro (Table 2). It is possible,
though, that the increased serine phosphorylation observed in I-2
following insulin stimulation of adipocytes (Lawrence et al.,
1988) might have been due to the action of p90
.
The significance of this phosphorylation is not clear at present.
, phosphorylates
site 1 on R
resulting in activation of the phosphatase
(Dent et al., 1990). The latter mechanism has recently been
challenged by the observation that under conditions where both MAPK and
p90
are activated, glycogen synthase activity is
not always affected (Robinson et al., 1993; Lin and Lawrence,
1994). These results question both the proposed mechanisms for the
control of GSK-3 and the glycogen-associated phosphatase and/or their
role in the regulation of glycogen synthase activity.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.