THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 36, Issue of December 25, pp. 22460-22466,lWO Printed in U.S.A.

Three Forms of Phosphatase Type 1 in Swiss 3T3 Fibroblasts FREE CATALYTIC SUBUNIT APPEARS TO MEDIATE S6 DEPHOSPHORYLATION*

(Received for publication, June 11, 1990)

Andrhe R. OlivierS and George Thomas5 From the Friedrich Miescher-Institut, P. 0. Box 2543, CH-4002 Easel. Switzerland

The major 40 S ribosomal protein S6 phosphatase in Swiss mouse 3T3 fibroblasts is a type 1 enzyme (Oli- vier, A. R., Ballou, L. M., and Thomas, G. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 4720-4724). Polyclonal antibodies were raised against a synthetic peptide con- taining the carboxyl-terminal 14 amino acids of the catalytic subunit of phosphatase 1 (PP-1C). Results from Western blot analysis and immunoprecipitation show that the peptide antiserum specifically recognizes PP-1C in cell extracts. Anion-exchange chromatogra- phy of cell extracts and Western blot analysis revealed three peaks of PP-1C termed A, B, and C. Peaks A and C are associated with the major type 1 S6 phosphatase activities, but peak B exhibits little activity. The phos- phatase in peak A (J& 39,000) appears to represent the free catalytic subunit, whereas the enzymes in peaks B and C display sizes of 68,000-140,000. Peak B contains two additional proteins of M, 26,000 and 48,000 that co-immunoprecipitate with PP-lC, while peak C has a single additional protein of M, 100,000. Fifteen min after serum withdrawal there is a 2-fold stimulation of S6 phosphatase activity in peak A that can be accounted for by an increase in the amount of PP-1C. The amount of PP-1C in the inactive peak B fraction also increases during this time and this in- crease is associated with changes in the phosphoryla- tion state of the M, 26,000 and 48,000 proteins. The results are discussed in relation to regulatory mecha- nisms which are thought to modulate the activity of type 1 phosphatase.

The multiple phosphorylation of 40 S ribosomal protein S6 is thought to play a key role in the activation of protein synthesis following stimulation of quiescent cells to prolifer- ate (l-7). The overall level of S6 phosphorylation is directly controlled by two enzymes, a kinase and a phosphatase, whose activities are tightly regulated by the growth state of the cell (8). The S6 kinase is a M, 70,000 enzyme (9-11) that is activated by phosphorylation (12, 13), possibly through pro- tein kinase cascades which can be triggered either by growth factor receptors or oncogenes (14). Furthermore, at least two intracellular signalling pathways, one of them protein kinase C-dependent, are involved in regulating the activation of S6 kinase (15).

In contrast to the S6 kinase, little is known about the

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact.

$ Current address: Imperial Cancer Research Fund Laboratories, P. 0. Box 123, Lincoln’s Inn Fields, London WCZA 3PX, United Kingdom.

3 To whom correspondence should he addressed.

regulation of S6 phosphatases. The major serine/threonine- specific protein phosphatases have been classified as either type 1 or type 2 enzymes (16-18). The type 1 phosphatases preferentially dephosphorylate the p subunit of phosphorylase kinase and are inhibited by nanomolar concentrations of two heat-stable proteins termed inhibitors-l and -2 (18). In con- trast, the type 2 enzymes, termed 2A, 2B, and 2C, specifically dephosphorylate the cy subunit of phosphorylase kinase and are unaffected by the two inhibitor proteins. Employing these criteria, it has been shown that the major S6 phosphatase in both Xenopus eggs (19) and Swiss mouse 3T3 cells (8, 20, 21) is a type 1 enzyme. Furthermore, insulin activation of S6 phosphorylation in Swiss mouse 3T3 cells is transient and the dephosphorylation stage correlates with increased activity of a type 1 phosphatase in fractionated cell extracts (8, 20, 21). Earlier it had been shown that in serum-stimulated cells the rate of S6 dephosphorylation was greatly enhanced follow- ing removal of the mitogen (1). Pulse-chase experiments further revealed that the phosphate in S6 is metabolically stable in the presence of serum, but that its removal leads to immediate phosphate turnover, presumably through the ac- tivation of a phosphatase (1, 21). Whether this event is also mediated by a type 1 enzyme has not been examined.

The activation of phosphatase 1 could be controlled at a number of levels (for reviews, see Refs. 17 and 18). The first could be through increased synthesis of the enzyme. Recent experiments carried out with cycloheximide to address this question were inconclusive since the drug itself increased total phosphatase activity (23). A second level of regulation could be through post-translational modification. It has been shown in vitro that the activities of the two inhibitor proteins as well as the catalytic subunit of phosphatase 1 (PP-1C)’ can be modulated by phosphorylation (24, 27). Furthermore, the amount of phosphate in inhibitor-l has been shown to de- crease in response to insulin, resulting in an increase in phosphatase 1 activity (26). Finally, the enzyme can be regu- lated by differential compartmentalization. PP-1C has been shown to associate with targeting proteins such as the G subunit which binds the enzyme to glycogen. Phosphorylation of this subunit in vitro and in vivo by CAMP-dependent protein kinase releases PP-1C into the cytosol (28-32). Anal- ogous subunits are thought to direct phosphatase 1 to the sarcoplasmic reticulum (33, 34), myofibrils (34), and ribo- somes (35).

Here we used anion-exchange chromatography and anti- bodies against PP-1C to identify different forms of the enzyme in Swiss mouse 3T3 cells. Next we examined the S6 phospha-

1 The abbreviations used are: PP-lC, catalytic subunit of protein phosphatase 1; PBS, phosphate-buffered saline; PP-PAC, catalytic subunit of nrotein nhosuhatase 2A: EGTA, lethylenebis(oxyethy- 1enenitrilo)jtetraacetic acid; SDS-PAGE, sodium- dodecyl sulfate- polyacrylamide gel electrophoresis; Mops, 4-morpholinepropanesul- fonic acid.

22460

S6 Dephosphorylation 22461

tase activities in serum-stimulated and serum-deprived cells, determining which form of phosphatase 1 is being modulated. Finally, we attempted to identify the mechanism that me- diates this response.

EXPERIMENTAL PROCEDURES

Materials-[r-“YP]ATP (3000 Ci/mmol), ““I-labeled anti-rabbit Ig (5-20 &i/pg antibody protein), [““S]methionine (>lOOO Ci/mmol), and ‘rrPi (50 mCi/ml) were purchased from Amersham Corp. 40 S ribosomal subunits were prepared from rat liver (36). The catalytic subunits of phosphatase 1 and 2A were purified from rabbit skeletal muscle (37, 38). The 14-amino acid carboxyl-terminal peptide (RPITPPRNSAKAKK) of PP-IC was synthesized by A. Wallace, Friedrich-Miescher Institut.

Cell Culture and Labeling-Swiss mouse 3T3 fibroblasts were main- tained as previously described (12). For ““S-labeling the cells were refed with 15 ml of medium and 100 +Ci of [““Slmethionine was added to each 15-cm plate. For “YP-1abe1ing the medium was reduced to 10 ml and “lpi (3 mCi/plate) was added 16-18 h before harvesting. Extract supernatants were prepared as previously described (12, 20) in extraction buffer containing 20 mM triethanolamine, pH 7.6, 1 mM dithiothreitol, 0.5 mM EGTA, 1 mM benzamidine, 0.1 mM phenyl- methanesulfonyl fluoride, 40 mM /3-glycerolphosphate, 50 mM NaCl unless stated otherwise.

Phosphatose Assays-S6 phosphatase assays were carried out as previously described (20). One unit of S6 phosphatase activity is the amount of enzyme that releases 1 pmol of ‘rPPi from S6 per min.

Antipeptide Arztiboclies-The peptide was coupled to keyhole limpet hemocyanin using either glutaraldehyde or 1-ethyl-3-(3-dimethylam- inopropyl)carbodiimide as previously described (39). Both reactions proceeded overnight and the peptide conjugates were dialyzed against phosphate-buffered saline (PBS) to remove excess coupling agent. Approximately 0.2 mg of mixed peptide conjugate in Freund’s com- plete adjuvant (1:l) was used to inject New Zealand white rabbits subcutaneously at four sites along the back. At 4- to 5-week intervals the rabbits were boosted with 0.1 mg of peptide conjugate in Freund’s incomplete adjuvant. The rabbits were bled 1 week after each injection starting with the second boost.

Purification of IgGs-Six ml of serum was precipitated with 40% ammonium sulfate at room temperature. The precipitate was collected by centrifugation at 10,000 x g for 5 min and the pellet was dissolved in PBS and dialyzed overnight at 4 ‘C against 10 mM Tris-HCl, pH 8. The sample was then diluted 5-fold and loaded onto a lo-ml (1 x 13 cm) Fast Flow Q Sepharose (Pharmacia LKB Biotechnology Inc.) anion-exchange column. The column was washed with 30 ml of 10 mM Tris-HCI, pH 8, and the proteins were eluted with a loo-ml linear gradient from 0 to 300 mM NaCl followed by 20 ml of the same buffer containing 1 M NaCl. Fractions of 3 ml were collected and the antipeptide antibodies were detected by immunoblotting using im- mobilized PP-1C (see below). Most of the specific antibodies eluted at 1 M NaCl, yielding relatively pure IgG. The protein was concen- trated by ammonium sulfate precipitation and the pellet was dissolved in PBS and dialyzed overnight at 4 “C. The antibodies (3 mg/ml) were stored at -70 “C.

Immunoblotting-Proteins were electrophoretically transferred for 75 min onto Immobilon” uolwinvlidine difluoride membrane (Milli- pore) using a Jancos semidry electroblotter according to the manu- facturer’s instructions. The membrane was then stained with 2% Ponceau Red in 3% chloric acid to visualize proteins, destained with PBS, and then incubated in 5% milk powder in PBS for 1 h at room temperature. The blot was then washed and incubated at 4 “C over- night with preimmune serum, antipeptide serum, or purified IgG diluted 1:5000 in PBS containing 0.5% Tween 20. The membranes were washed and then incubated-for 2-3 h with ““I-anti-rabbit IgGs diluted 1:lOOO. Following incubation the blots were washed 4-5 times for 15 min with PBS, 0.5% Tween 20, dried in air, and subjected to autoradiography. For quantitation, the radioactive bands correspond- ing to PP-1C were cut out from the blot and counted with a -y-counter.

Immunoprecipitation-Cells were lysed or column fractions were diluted in lysis buffer containing 50 mM Tris-HCl, pH 8 (4 “C), 120 mM NaCl, 5 mM EDTA, 0.1% aprotinin, 1 mM phenylmethanesulfonic acid, 1 mM benzamidine, 100 pM sodium orthovanadate, 20 mM fl- glycerol phosphate, 1% Nonidet P-40. Each lysate was precleared by incubating it on ice for 1 h with 5-20 ~1 of preimmune serum diluted in lysis buffer with occasional mixing. The immunoglobulins were then precipitated by mixing with 50 ~1 of protein A-Sepharose (50%

w/v) at 4 “C for 45 min and the nonspecific immunecomplexes were collected by centrifugation (3000 rpm for 1 min). The pellet was washed as described below. Five to 20 ~1 of antipeptide serum diluted in lysis buffer was added to the supernatant and mixed for 90 min at 4 “C. The immunoglobulins were then precipitated with protein A- Sepharose as described above. After centrifugation the supernatant was discarded and the pellet was washed 3 times with wash buffer containing 50 mM Tris-HCI, pH 8 (4 “C), 120 mM NaCl, 5 mM EDTA. To measure phosphatase activity in the pellet an additional wash with assay buffer minus dithiothreitol was included. To analyze immunoprecipitated proteins the pellet was taken up in 40 ~1 of 2 times concentrated SDS samnle buffer (40) and heated at 95 “C for 5 min. The Sepharose beads were pelleted by centrifugation and the supernatant was subjected to SDS-PAGE (40). For competing PP-1C from the immunoprecipitates, 40 ~1 of wash buffer containing 5 pM peptide was added to the washed pellets and incubated for 1 h at 4 “C with mixing. The Sepharose beads were pelleted, washed with wash buffer, and the combined supernatant and wash were then either assayed for phosphatase activity or the proteins were precipitated with trichloroacetic acid and subjected to SDS-PAGE and immuno- blotting as described above.

RESULTS

Antipeptide Antibodies Specific for PP-1 C-To identify dif- ferent forms of phosphatase 1 in cell extracts, it was necessary to generate polyclonal antibodies against PP-1C that would not cross-react with the catalytic subunit of phosphatase 2A (PP-ZAC), which shares 59% homology with PP-1C (41). Because the carboxyl-terminal I4 amino acids of PP-1C should be highly antigenic and share no homology with PP- 2AC (41), polyclonal antibodies were raised against a syn- thetic peptide identical to this sequence. On Western blots the preimmune serum did not recognize purified rabbit skel- etal muscle PP-1C or PP-2AC (Fig. lA, lanes 1 and 2), although it did cross-react with some proteins in Swiss mouse 3T3 cell extracts (Fig. lA, lane 3). In contrast, the antipeptide serum and purified IgG reacted with purified PP-1C (Fig. 1, B and C, lane 1) and with a protein in Swiss mouse 3T3 cell extracts having the same molecular weight as PP-1C (Mr 38,000) (Fig. 1, B and C, lane 3). Preincubation with the synthetic peptide specifically blocked interaction with PP-1C and the M, 38,000 protein (Fig. 1, B and C, lanes 1 and 3). Under no conditions did the antibodies cross-react with PP- 2AC (Fig. 1, lane 2).

To further determine the specificity of the antiserum, we tested its ability to immunoprecipitate purified PP-1C and PP-2AC. The results show that only PP-1C was precipitated and the preimmune serum did not recognize either protein (Table I). The antibodies also precipitated phosphatase activ- ity in Swiss 3T3 fibroblast extracts. This activity was specif-

A B C

peptKi.2 - + +

1 2 3 1 2 3 123 123 123

FIG. 1. Specificity of anti-PP-1C antibodies. PP-1C (lane I ), PP-2AC (lane 2). and 20 UB of cell extract protein (lane 3) were subjected to SDS’-‘PAGE andthen transferred to polyvinylidine diflu- oride membrane as described under “Experimental Procedures.” Each membrane was then incubated as follows: Panel A, with preimmune serum; Panel B, antipeptide serum in the presence or absence of 100 nM competing peptide; Panel C, with purified antipeptide IgGs either in the presence or absence of peptide. The immunoblots were visu- alized using ““I-labeled anti-rabbit IgGs and subjected to autoradi- ography for 8 h as described under “Experimental Procedures.”

S6 Dephosphorylation

TABLE I Immunoprecipitation of SGphosphatase activity

Three and 6 units/ml, respectively, of purified rabbit skeletal muscle PP-1C and PP-2AC and 100 ~1 of cell lysate were immuno- precipitated as described under “Experimental Procedures.” The im- munoprecipitates were taken up in S6 phosphatase assay buffer and assaved for activitv as described.

PP-1c PP-2AC Cell lysate

No serum Preimmune serum Antipeptide serum Elution with peptide

’ ND, not determined.

CPm cppm CPm

12,504 20,947 12,307 30 12 ND”

1,489 40 3,156 ND ND 33,853

Fraction number

FIG. 2. Elution of phosphatase 1 from a Mono Q column. Five 15-cm plates of quiescent cells were harvested as described under “Experimental Procedures.” After homogenization and centrifuga- tion, the supernatant was applied to a l-ml anion-exchange column (Mono Q, Pharmacia) as previously described (20). Five ~1 of each fraction was used to assay for S6 phosphatase activity (A). One-tenth volume of 0.15% deoxycholate and 72% (w/v) trichloroacetic acid was added to the remaining portion of each fraction. The precipitated proteins were then washed 3 times with acetone (-20 “C) and once with ether. The dried precipitate was then taken up in SDS sample buffer and subjected to SDS-PAGE and subsequent immunoblotting as described under “Experimental Procedures.” The relative amount of PP-1C per fraction is plotted (0). Peaks A, 8, and C are described in the text.

TABLE II Specific activity of peak fractions

The specific activity is the ratio of S6 phosphatase activity to the amount of ““I-antibodies bound to PP-1C on Western blots. Fractions 11, 13, and 20 from Fig. 2 were analyzed.

S6 Phosphatase PP.1c Specific activity

units/ml cpm/d x10-”

Peak A 121.82 15,029 8.11 Peak B 39.94 19,047 2.09 Peak C 35.25 7,463 4.72

ically released from the immune complex by incubation with peptide (Table I), and was shown by Western blot analysis to be PP-1C (data not shown). Furthermore, the phosphatase activity of the eluted PP-1C was lo-fold higher than that of the complexed PP-lC, suggesting that the antibodies inhibit the enzyme. Together the results show that the peptide anti- serum recognizes PP-1C by Western blot analysis (Fig. 1) or immunoprecipitation (Table I) and that it does not cross- react with PP-2AC.

Characterization of Type 1 Phosphatases-Recently we re- ported that two peaks of type 1 S6 phosphatase activity could

0 35 45 55 65 75 65 95

Fractton number

FIG. 3. Gel filtration chromatography of type 1 phospha- tases. The proteins in fractions 11 (0), 13 (O), and 20 (A) of a Mono Q column (see Fig. 2) were adsorbed to 50 ~1 of Fast Flow Q Sepharose (Pharmacia) equilibrated in the same buffer as described in the legend to Fig. 2. The proteins were eluted with 100 ~1 of the same buffer containing 500 mM NaCl. Each eluate was loaded onto a 1 X 37-cm Sephacryl S-200 (Pharmacia) column equilibrated in a buffer con- taining 20 mM Mops, pH 7.2, 1 mM dithiothreitol, 0.05% Triton X- 100, 100 mM NaCl. Ninety-five 0.36-ml fractions were collected at a flow rate of 3.84 ml/h and assayed for S6 pbosphatase activity (data not shown). The remaining portion of each fraction was subjected to precipitation and SDS-PAGE and immunoblotting as described in the legend to Fig. 2. The band corresponding to PP-1C on the autoradiograph was quantitated by densitometry. The molecular size markers were: 1, alcohol dehydrogenase (140 kDa); 2, bovine serum albumin (68 kDa); 3, ovalbumin (43 kDa); 4, bovine carbonic anhy- drase (29.5 kDa); and 5, cytochrome c (14 kDa).

be resolved by Mono Q anion-exchange chromatography (20). The phosphatase activity in each peak was attributed to type 1 enzyme based on the ability to dephosphorylate phospho- rylase a, to selectively dephosphorylate the /3 subunit of phos- phorylase kinase, and to be inhibited by inhibitor-2. Prelimi- nary gel filtration studies indicated that the two phosphatase activities displayed distinct molecular weights, arguing that at least two forms of phosphatase 1 are present in 3T3 cell extracts. To confirm that both peaks contain PP-lC, a cell extract was fractionated on a Mono Q column and assayed for S6 phosphatase activity and PP-1C by Western blot analysis. The activity profile is similar to that previously described (compare Fig. 2 and Ref. 20), with the major peak of activity eluting in fraction 11 at 150 mM NaCl and the second peak eluting in fraction 20 at 350 mM NaCl. The small peak of activity eluting in fractions 14-17 was previously shown to contain phosphatase 2A (20). In contrast to the activity profile, three distinct peaks of PP-1C were detected on Western blots, designated as A, B, and C (Fig. 2). Peaks A and C coeluted with the major type 1 S6 phosphatase activi- ties, but peak B, which was not well resolved from peak A, contained little S6 phosphatase activity. Quantitation of this data shows that the specific activity in peak B was 4-fold lower than in peak A (Table II). Further purification studies indicated that the activity associated with peak B was largely due to contamination by peak A (data not shown).

Molecular Weight and Protein Composition of Type 1 Phos- phatases-To determine whether the PP-1C in peaks A, B, and C represents free catalytic subunit or larger protein complexes, the proteins in each peak were subjected to gel filtration and their relative position of elution was determined by Western blot analysis. The PP-1C in peak A eluted in fractions 57-59, at M, 39,000, indicating that this form prob- ably represents the free catalytic subunit (Fig. 3). Peaks B

5’6 Dephosphorylation 22463

and C eluted in fractions 46-49 and 48-51, respectively, which corresponds to molecular weights of 68,000-140,000. Thus peaks B and C appear to contain either multimers of PP-1C or PP-1C associated with one or more other proteins. To distinguish between these possibilities extracts from 3T3 cells labeled to equilibrium with [““Slmethionine were fractionated on a Mono Q column. Portions of each peak were then immunoprecipitated with either preimmune serum or peptide antiserum in the absence or presence of peptide. The only protein which was specifically precipitated and competed away with peptide in peak A was PP-1C (Mr 38,000) (Fig. 4). In peak B two additional proteins of M, 26,000 and M, 48,000 were specifically co-precipitated by the peptide antiserum, whereas in peak C a weakly labeled protein of M, 100,000 was observed. The data are consistent with peak A representing PP-1C alone and with peaks B and C representing a higher molecular weight PP-1C complex containing one copy of each of the proteins shown in Fig. 4.

Actiuation of S6 Phosphatase-As stated earlier, the rate of S6 dephosphorylation in uivo is greatly enhanced by serum withdrawal (1). It was reasoned that this effect might be

A B C

4200

4116 -98

-68

445

-3:

1 2 3 1 2 3 1 2 3

FIG. 4. Immunoprecipitation of type 1 phosphatase. Three 15-cm plates of cells were labeled to equilibrium with [ ‘“Slmethionine as described under “Experimental Procedures.” The cells were har- vested and subjected to Mono Q chromatography as described in the legend to Fig. 2 except that the buffer did not contain 1 mM dithio- threitol. The fractions were assayed for activity (data not shown) and then fractions 11 (Panel A), 13 (Panel B), and 20 (Panel C) were immuneprecipitated (see “Experimental Procedures”) with either preimmune serum (lane I ) or antipeptide serum in the absence (lane 2) or presence (lane 3) of competing peptide.

mediated through the activation of S6 phosphatases that can be measured in uitro, offering a useful model for studying the regulation of this process. To examine this possibility extracts from quiescent cells, cells stimulated for 2 h with serum, or cells first stimulated with serum and then shifted to serum- free medium were subjected to Mono Q chromatography and assayed for S6 phosphatase activity. Consistent with earlier results (1, 20), addition of serum to quiescent cells had little effect on peak C S6 phosphatase activity but caused a 25% reduction in type 1 S6 phosphatase activity in peak A (Fig. 5, A and B, Table III). Removal of serum for 15 min led to an increase in total S6 phosphatase activity which began to return to basal levels by 45 min (Fig. 5, C and D). The largest effect on activity was in peak A, which increased 2.0-fold after 15 min of serum withdrawal, and was still 1.6-fold higher at 45 min (Table III). It should also be noted that phosphatase 2A activity in fractions 14-17 is significantly increased after 15 min of serum withdrawal (Fig. 5C). These c@ta point to the phosphatase in peak A as the most likely candidate for regulating S6 dephosphorylation.

Mechanism Mediating S6 Dephosphorylation-As a first step in elucidating the mechanism by which type 1 S6 phos- phatase activity is increased, PP-1C levels in the fractions in Fig. 5 were measured by Western blot analysis. The results show that there are large changes in the amount of PP-1C in peak A and peak B but that the amount in peak C remains relatively constant. The amount of PP-1C in peak A decreased approximately 30% after 2 h with serum (Fig. 6B) and then increased almost 2-fold 15 min after serum removal before beginning to return to basal levels by 45 min (Fig. 6, C and D). The amount of PP-1C in peak B changed in a manner similar to that observed for peak A. Although it is not well resolved from peak B, the specific activity of PP-1C in peak A, based on fraction 11 alone, appears to remain relatively constant (Table III), indicating that the changes in S6 phos- phatase activity may be due to the amount of free PP-1C in the cytosol.

Phosphoproteins Associated with PP-lC-It has been ar- gued that direct phosphorylation of PP-1C on tyrosine can decrease its activity (27). This explanation could account for the lack of S6 phosphatase activity in peak B. To examine this possibility extracts from cells labeled in uiuo with “‘Pi were resolved on a Mono Q column, the PP-1C in peaks A and B was immunoprecipitated, and following SDS-PAGE the phosphoproteins were visualized by autoradiography. Un-

A 8 c D

180-

FIG. 5. S6 phosphatase activity in quiescent and serum-stimulated and 150-

-depleted cells. Quiescent cells (A) or cells treated for 2 h with 10% fetal calf serum (B) and then depleted of fetal calf

3 2 12’ I

serum for 15 (C) and 45 (D) min were harvested in the buffer described under “Experimental Procedures.” For each time point the cells from 5 15.cm plates were pooled. After homogenization and centrifugation, the supernatants were subjected to chromatography as de- scribed in the legend to Fig. 2 and as- sayed for S6 phosphatase activity. The data was normalized for the amount of protein loaded onto the column.

Fractvan number

22464 S6 Dephosphorylation

der no condition was phosphate detectable in PP-1C (Fig. 7). However, both the A4, 26,000 and M, 48,000 proteins were phosphorylated at all times examined. The phosphorylation state of these two proteins changed as a function of the growth state of the cell. In serum-stimulated cells there was an approximate 2-fold increase in the amount of 32P incorporated into both proteins compared to resting cells. Following 15 min of serum removal the level of ‘*P returned almost to basal levels. The results argue that phosphorylation of PP-1C under these conditions does not play a role in regulating its activity but suggest that phosphorylation of the M, 26,000 and M, 48,000 proteins may be important.

DISCUSSION

Using a peptide corresponding to the carboxyl-terminal sequence of PP-1C (41, 42), a polyclonal antiserum was gen- erated which specifically recognizes the protein by immuno- precipitation and on Western blots. Molecular cloning has revealed two cDNAs for PP-lC, encoding proteins termed PP-lC,, (M, 37,500) and PP-lCti (M, 35,400) (41, 42). Since the amino acid sequences of PP-lC, and PP-lCO are identical after amino acid 34 of PP-lC, (18, 41, 42), the antibody probably recognizes both forms of the protein. In examining Western blots (data not shown) and immunoprecipitates of the phosphatase in peaks A and B, we have noted a distinct doublet. These two bands are poorly resolved in Fig. 4, A and B. Only one species of PP-1C was observed in peak C (Fig. 4 and data not shown), which migrates in the position of the lower molecular weight protein seen in peaks A and B. During purification in the absence of protease inhibitors, PP-1C can be cleaved to a species of M, 33,000 (18, 38). However, the portion of PP-1C removed is the carboxyl end of the protein, so the peptide antibodies would not recognize this form of the

TABLE III Specific activities of phosphatase 1 in fraction 11 after serum

deprivation The specific activities were calculated in the same way as described

in Table II. Data are from Fig. 5.

Treatment

Resting 2-h serum 15 min deprived 45 min deprived

Activity PP.1c

units/ml cpm/ml 121.82 15,029

95.51 10,155 186.61 19,350 152.00 14,857

Specific activity

x10- 8.11 9.11 9.64

10.20

enzyme. If the two species of PP-1C seen on Western blots represent distinct polypeptides it will be of interest to deter- mine whether they are related to PP-lC, and PP-lC,+ It should be noted that we did not detect the ATP . Mg-depend- ent form of phosphatase 1 (18, 45) in Swiss 3T3 fibroblasts. This cytosolic form of phosphatase 1 is inactive and consists of a complex between PP-1C and inhibitor-2 (18, 25). A protein with the same molecular weight as inhibitor-2 (Mr 31,000) was not seen in immunoprecipitates of either whole cell lysates (data not shown) or in individual PP-1C peak fractions (Figs. 4 and 7). A possible reason for this result could be that when inhibitor-2 binds to PP-1C it prevents the antibodies from interacting with the carboxyl terminus of PP- 1c.

Polyclonal antibodies against PP-1C detected three distinct forms of the enzyme which were resolved by anion-exchange chromatography (Fig. 2). Further characterization by gel fil- tration and examination of immunoprecipitated [?S]methio- nine-labeled proteins revealed that each peak of PP-1C has a different size and protein composition. PP-1C in peak A appears to exist as the free catalytic subunit, whereas peak B consists of PP-1C associated with two proteins of M, 26,000 and 48,000 (Figs. 4 and 7). The most likely candidate for the M, 26,000 protein is inhibitor-l. Although the amino acid sequence of inhibitor-l predicts a mass of only 18.7 kDa (43), the molecular mass obtained by SDS-PAGE is closer to 26,000 (24). This difference has been attributed to a highly asym- metric structure of the protein (17, 18, 24). Inhibitor-l binds to and inhibits PP-1C in its phosphorylated form, which is consistent with the data presented in Figs. 40 and 7B. The presence of inhibitor-l in these fractions could account for the low specific activity of PP-1C in peak B (Table II). The M, 48,000 protein could be a novel subunit involved in either targeting PP-1C in the cell or regulating its activity. This protein could be identical to a recently described phosphatase inhibitor of M, 40,000 (44) that is denatured by boiling but maintains its ability to inhibit PP-1C following elution from SDS-polyacrylamide gels. It was not reported whether this protein is phosphorylated, as found for M, 48,000 protein in peak B (Fig. 7). Finally, the PP-1C present in peak C appears to be complexed with a protein of M, 100,000 (Fig. 4). This weakly labeled protein may be the G subunit which mediates binding of PP-1C to glycogen (28-32). The G subunit has a M, of 161,000 but is easily proteolyzed to a M, 103,000 species which is still functional (30, 31). This idea could be tested

FIG. 6. PP-1C content in quies- cent and serum-stimulated and -de- pleted cells. The remaining portion of

a ;;

the fractions collected in Fig. 5 were k ,5

subjected to immunoblotting as de- “. scribed under “Experimental Proce- z dures.” Quiescent cells (A), cells stimu- 8 10 lated for 2 h with 10% fetal calf serum f (E), and then deprived of fetal calf serum - for 15 min (C) and 45 (D) min, respec- tively. The data was normalized for the 5

amount of protein loaded onto each col- umn.

Fraction number

5’6 Dephosphorylation 22465

1 2 3

66k- -66k

43k- - -43k

31k- 0

-3lk

A 0 A B A B

FIG. 7. Immunoprecipitation of ““P-labeled proteins. For each time point 5 15-cm plates of cells were labeled with “‘Pi as described under “Experimental Procedures.” Throughout all manip- ulations “‘PI concentrations in the media were maintained at 3 mCi/ plate. Quiescent cells (Panel 1) or cells stimulated for 2 h with 10% fetal calf serum (Panel 2) or deprived of fetal calf serum for 15 min in the presence of “‘Pi (Panel 3) were extracted and subjected to Mono Q chromatography (20). Peaks A and B were then processed as described in the legend to Fig. 4 except that the buffers also contained 100 pM sodium orthovanadate. Autoradiographs of the immunoprecipitates with the antipeptide serum are shown. Molecular weight markers are indicated and A and E refer to peaks A and B.

with specific antibodies to the G subunit (29). The role of phosphorylation in the control of PP-1C activity

under the conditions described here is unclear. The fact that we do not detect phosphate associated with PP-1C (Fig. 7) does not exclude the possibility that the catalytic subunit itself is regulated by phosphorylation in src-transformed cells. In uitro phosphorylation of PP-1C by pp60’~“” on tyrosine inactivates the enzyme (27). However, evidence that this occurs in uiuo is still lacking. The changes in intensity of the ‘“P-labeled M, 26,000 and 48,000 proteins (Fig. 7) could be due either to changes in phosphate content or to the amounts of these proteins present in the immunocomplexes. To distin- guish between these possibilities cells could also be labeled with [““S]methionine to normalize the amount of protein in the phosphorylated bands. The M, 26,000 and 48,000 proteins could play an auxilliary role in regulating the free PP-1C released into the cytosol (see below). For example, they could act as a safety mechanism by inactivating PP-1C after it has acted on its substrate, thus preventing nonspecific dephos- phorylation of proteins. This hypothesis might account for the increase in PP-1C in both A and B after a 15-min serum withdrawal.

Withdrawal of serum from stimulated cells leads to the immediate shut-off of protein synthesis (46). This effect is paralleled by a high turnover of phosphate in S6 followed by net dephosphorylation of the protein (1). The rate of dephos- phorylation in uiuo decreases with time after serum with- drawal, being highest at 5 min and returning to basal levels by 60 min (1). These results are consistent with phosphatase activities measured in extracts of cells deprived of serum for 15 and 45 min (Fig. 5). The increase in phosphatase activity is limited to peak A and appears to be accounted for by the presence of more PP-1C in this fraction. It should be noted, however, that in the earlier studies described above (22) no turnover of phosphate in S6 was detected in uiuo at early times following addition of serum, implying that the S6 phos- phatase was inactive. However, 2 h following serum stimula- tion there is a significant amount of S6 phosphatase activity measured in peak A. This would imply that if free PP-1C is responsible for dephosphorylating S6 in uiuo, its change in activity is masked by the total pool of PP-1C in the cytosol. The large increase of PP-1C associated with peaks A and B is probably not due to de nouo synthesis of the protein; a more

likely explanation is that the protein shifts from a particulate to a cytosolic location. Quantitation of particulate and cyto- solic PP-1C should reveal if a specific population of the phosphatase is responsible for dephosphorylating S6.

Acknowle&ments-We are indebted to Drs. L. M. Ballou, Y. Khew- Goodall, S. Gal, and R. E. Layden for their critical reading of the manuscript. We also thank C. Wiedmer for secretarial assistance.

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