©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Phosphorylation and Activation of the ATP-Mg-dependent Protein Phosphatase by the Mitogen-activated Protein Kinase (*)

(Received for publication, February 9, 1995; and in revised form, May 19, 1995)

Q. May Wang Kun-Liang Guan (1) Peter J. Roach Anna A. DePaoli-Roach (§)

From the Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202-5122 and the Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0606

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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 (0.3 mol of phosphate/mol of polypeptide) similar to that reported for phosphorylation by the alpha 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.


INTRODUCTION

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). (^1)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(A) 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-3alpha and thus results in a synergistic activation of the phosphatase (DePaoli-Roach, 1984; Park et al., 1994).

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(A). 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).

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 (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.

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 (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.

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 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.


EXPERIMENTAL PROCEDURES

Protein Kinases

The GSK-3alpha was purified from rabbit skeletal muscle as reported previously (Fiol et al., 1990). Expression and purification of the recombinant rabbit skeletal muscle GSK-3beta 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(2)-terminal truncation (DeltaN) lacking the first 35 amino acids, and the COOH-terminal deletion (DeltaC) 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(2), 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(A)-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(2), 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.


RESULTS

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-2bulletCS1 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(r) 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(r) 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(r) of 140,000, dependent on ATP-Mg and GSK-3 for activity, and the other corresponded to an apparent M(r) of 40,000 and was spontaneously active. SDS-PAGE across the peaks of activity demonstrated that the enzyme with M(r) 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.


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 [-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.




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-3beta (lane2). The reactions were performed under the conditions described in panelA using either ERK1 or GSK-3beta (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.



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-3alpha (DePaoli-Roach, 1984; Park et al., 1994). Since the action of GSK-3alpha 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-3alpha 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 beta-isoform of GSK-3 (Wang et al., 1994b).


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 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.




Figure 4: Synergistic phosphorylation of I-2 by GSK-3alpha 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-3alpha, 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.



Kinetic analyses of I-2 phosphorylation by ERK1 gave a K value of 160 µM, 5-fold and 11-fold higher than the K values determined for GSK-3alpha and GSK-3beta, respectively (Table 1). However, the V(max) for MAPK was 5-fold higher than that for GSK-3beta (Wang et al., 1994b). Thus, the V(max)/K value for MAPK is only 2-fold lower than that for GSK-3beta. 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(max) 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 (DeltaN) or the last 58 (DeltaC) residues (Park and DePaoli-Roach, 1994) were tested as substrates. Both I-2DeltaN and DeltaC were phosphorylated by the kinase (data not shown). These results suggest that p90 phosphorylates multiple serines of I-2.


Figure 5: Phosphorylation of I-2 by p90. 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-3alpha; 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).

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, 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-3alpha and GSK-3beta (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-3beta (data not shown).




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-3alpha (circles), GSK-3beta (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.''




DISCUSSION

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(A). 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-3alpha (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.

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, 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.

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.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants DK36569 (to A. D.-R.) and DK27221 (to P. J. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Indiana University School of Medicine, 635 Barnhill Dr., Indianapolis, IN 46202-5122. Tel.: 317-274-1585; Fax: 317-274-4686.

^1
The abbreviations used are: I-2, inhibitor-2; CK II, casein kinase II; GSK-3, glycogen synthase kinase 3; PAGE, polyacrylamide gel electrophoresis; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated protein kinase; MEK, MAPK or ERK kinase; rsk, ribosomal protein S6 kinase; GST, glutathione S-transferase.


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