Autophosphorylation-Activated Protein Kinase Phosphorylates and Inactivates Protein Phosphatase 2A

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Proc. Natl. Acad. Sci. USA Vol. 90, pp. 2500-2504, March 1993 Biochemistry Autophosphorylation-activated protein kinase phosphorylates and inactivates protein phosphatase 2A HONG GUO AND ZAHI DAMUNI* Department of Biological Sciences, CLS 601, University of South Carolina, Columbia, SC 29208 Communicated by Lester J. Reed, December 9, 1992

ABSTRACT Purified preparations of a distinct autophos- and thus is a potential target for acute regulation by hormones phorylation-activated protein kinase from bovine kidney phos- and other extraceliular stimuli. phorylated and inactivated purified preparations of protein We recently purified to apparent homogeneity a distinct phosphatase 2A2 (PP2A2) by about 80% with the autophos- autophosphorylation-activated protein kinase from extracts phorylation-activated protein kinase, protamine kinase, and of bovine kidney (9). Purified preparations of this autophos- 32P-labeled myelin basic protein as substrates. Analysis of phorylation-activated protein kinase exhibited an apparent incubations performed in the presence of 0.2 mM [-32P]ATP Mr 36,000 as estimated by SDS/PAGE and by gel perme- by autoradiography following SDS/PAGE and by FPLC gel ation chromatography on Sephacryl S-200 (9). The purified permeation chromatography on Superose 12 demonstrated enzyme underwent a rapid (t1l2 = 0.5-1 min) intramolecular that the catalytic subunit of PP2A2 was phosphorylated in the autophosphorylation reaction which was accompanied by an incubation mixtures containing the kinase and phosphatase. Up =10-fold increase in enzyme activity (9). Autophosphoryla- to 0.3 mol ofphosphate groups was incorporated per mol ofthe tion and activation were reversed by purified preparations of catalytic subunit of PP2A2 following incubation with the ki- PP2A2, demonstrating that the activity of the purified kinase nase. This phosphorylation was enhanced about 5-fold in the was regulated by reversible phosphorylation (9). presence of 0.4 FM microcystin-LR. In addition, up to 1 mol In this paper we show that in the presence of Mg2+ and of phosphate groups was incorporated per mol of the PP2A2 ATP, purified preparations of the autophosphorylation- subunit of apparent Mr 60,000 when microcystin-LR was activated protein kinase phosphorylate the catalytic subunit included. Analysis by thin-layer chromatography indicated of PP2A2 and inactivate by about 80% the activity of this that PP2A2 catalyzed an autodephosphorylation reaction which phosphatase with the autophosphorylation-activated protein was inhibited by microcystin-LR. Phospho amino acid analysis kinase, purified preparations of an insulin-stimulated prot- showed that the catalytic subunit ofPP2A2 was phosphorylated amine kinase (10, 11), and 32P-labeled myelin basic protein on threonine residues by the autophosphorylation-activated (MBP) as substrates. Together with previous observations, protein kinase. Together with previous observations, the re- the results suggest that inactivation of PP2A by phosphory- sults suggest that inactivation of PP2A by phosphorylation lation could contribute to the phosphorylation of cellular catalyzed by the autophosphorylation-activated protein kinase proteins on serines and threonines in response to insulin could contribute to the marked increase in the phosphorylation and/or other mitogens. of cellular proteins in response to insulin and other mitogens. Protein phosphatase 2A (PP2A) is a protein-serine/threo- MATERIALS AND METHODS nine-phosphatase which acts on the enzymes and proteins KC2 ethyl reverse-phase TLC plates were from Whatman. that regulate the rates of glycogen metabolism, glycolysis/ Silica-gel TLC plates were from Sigma. 32P1 was from Am- gluconeogenesis, cholesterol synthesis, aromatic amino acid ersham. Protamine kinase (10) and PP2A2 (12) were purified metabolism, protein synthesis, gene transcription, and nu- to apparent homogeneity from extracts of bovine kidney as merous other processes (1, 2). Two forms, PP2A1 and PP2A2, described. Other materials are given in refs. 9-15. have been found in the cytosol of nearly all tissues examined Purififcation of Autophosphorylation-Activated Protein Ki- (1, 2). A form of PP2A2 has also been purified to apparent nase. The kinase was purified as described (9). Bovine kidney homogeneity from extracts of bovine kidney mitochondria cortex (2 kg) was homogenized in a Waring blender at high (3). PP2A1 and PP2A2 contain a catalytic subunit of apparent setting for 1 min with 2 vol ofbuffer A (25 mM Tris chloride, Mr 36,000 and a subunit of apparent Mr 60,000. PP2A1 pH 7.0/1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluo- also contains a subunit ofapparent Mr 55,000. The catalytic ride/l mM benzamidine/14 mM 2-mercaptoethanol). The subunit is highly conserved (4-8), and in some species two homogenate was centrifuged for 30 min at 10,000 rpm in a forms have been identified by cloning methods (4-8). The Beckman JA-10 rotor and the pellets were discarded. To the deduced amino acid sequences indicate that these two forms supernatant was added with stirring 0.14 vol of 50%o (wt/vol) of the catalytic subunit exhibit 97% identity, with four of the poly(ethylene glycol). After 30 min, the mixture was centri- seven amino acid substitutions located in the N terminus fuged and the pellets were discarded. The supernatant was being conservative (4-8). However, the exact physiological passed through glass wool and then applied onto a column (14 function of PP2A is uncertain because the enzyme exhibits x 10 cm) of DEAE-cellulose equilibrated in buffer B [buffer overlapping specificity with PP1 and PP2C. Furthermore, A containing 10% (vol/vol) glycerol]. The column was other than subunit-subunit interactions which modulate the washed under suction with 4 liters of buffer B/0.05 M NaCl, specificity of PP2A (1, 2), there is little information on the and the combined effluent from DEAE-cellulose was then regulation of this enzyme (1, 2). Nevertheless, PP2A is applied onto a column (5 x 6 cm) of poly(L-lysine)-agarose considered an important enzyme that is likely to occupy a equilibrated in buffer B. The column was washed with 4 liters critical position in the control of diverse metabolic pathways ofbuffer B and then developed with a 2000-ml linear gradient

The publication costs of this article were defrayed in part by page charge Abbreviations: PP2A, protein phosphatase 2A; MBP, myelin basic payment. This article must therefore be hereby marked "advertisement" protein. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 2500 Downloaded by guest on September 27, 2021 Biochemistry: Guo and Damuni Proc. Natl. Acad. Sci. USA 90 (1993) 2501 of 0-0.5 M NaCl at a flow rate of 500 ml/hr, and 6.3-ml Protamine kinase activity was determined as described (13) fractions were collected. Autophosphorylation-activated except that 0.4 ,uM microcystin-LR was included in the protein kinase was recovered at about 0.2 M NaCl. Active reaction mixtures. One unit ofprotamine kinase activity was fractions were pooled, diluted with 4 vol of buffer B, and equivalent to the amount ofenzyme that incorporated 1 nmol applied onto a column (2.5 x 6 cm) of heparin-agarose of phosphate into protamine sulfate per min. equilibrated with buffer B. The column was washed with 1 Determination of Protein Phosphatase Activity. 32P-labeled liter of buffer B and then developed with a 1000-ml linear MBP was prepared by incubation of 1 mg of MBP in 50 mM gradient of 0-0.4 M NaCl at a flow rate of 300 ml/hr, and Tris chloride, pH 7.0/10% glycerol/i mM benzamidine/0.1 6.3-ml fractions were collected. Autophosphorylation- mM phenylmethylsulfonyl fluoride, 14 mM 2-mercaptoetha- activated protein kinase was recovered at about 0.2 M NaCl. nol/0.2 mM [y-32P]ATP/10 mM MgCl2 containing 12.5 units The active fractions were pooled, mixed with 1 vol of buffer ofprotamine kinase in a final volume of0.25 ml. After 30 min B containing 1 M NaCl, and then applied onto a column (2.5 at 30°C, 1 ml of 10%6 trichloroacetic acid was added and the x 4 cm) of phenyl-Sepharose equilibrated in buffer B/0.5 M mixture was centrifuged for 2 min in a Fisher microcentri- NaCl. The column was washed with 500 ml of buffer B/0.25 fuge. The supernatant was discarded and the pellet was M NaCI and then developed with a 600-ml linear gradient washed eight times with 1-ml portions of 10% trichloroacetic from buffer B/0.25 M NaCl to buffer B/65% (vol/vol) acid followed by three i-ml portions of 95% ethanol. The ethylene glycol/0.025 M NaCl/0.1% Triton X-100 at a flow pellet was suspended in 2 ml of 50 mM Tris chloride, pH rate of 90 ml/hr, and 3.8-ml fractions were collected. Auto- 7.0/10% glycerol/i mM benzamidine/0.1 mM phenylmeth- phosphorylation-activated protein kinase was recovered at ylsulfonyl fluoride/14 mM 2-mercaptoethanol. The suspen- about 30% ethylene glycol. The active fractions were pooled, sion was stored in aliquots at -70°C. mixed with 3 vol of buffer B The phosphatase assay mixtures (50 1zl) contained 50 mM and then applied onto a column Tris chloride (pH 7.0), 10% glycerol, 1 mM benzamidine, 0.1 (2.5 x 4 cm) of CM-Sepharose equilibrated in buffer B. The mM phenylmethylsulfonyl fluoride, 14 mM 2-mercaptoetha- column was washed with 300 ml ofbuffer B and the combined nol, 0.2 mg ofbovine serum albumin, a 5-,ul sample ofPP2A2, effluent was applied onto a column (2.5 x 4 cm) ofprotamine- and 5 ,ug of 32P-labeled MBP. Reactions were initiated by the agarose equilibrated with buffer B. The column was washed addition of the 32P-labeled substrate. After a 5-min incuba- with 500 ml of buffer B and then developed with a 600-ml tion, 0.1 ml of 12% trichloroacetic acid was added and the linear gradient of 0-0.9 M NaCl at a flow rate of 300 ml/hr, mixture was centrifuged at 10,000 rpm in a Fisher microcen- and 5-ml fractions were collected. Autophosphorylation- trifuge. An aliquot of the supernatant (0.12 ml) was mixed activated protein kinase was recovered at about 0.6 M NaCl. with 1 ml of scintillant and the radioactivity was determined. The active fractions were pooled, diluted with 10 vol ofbuffer One unit of PP2A2 activity was equivalent to the amount of B, and then applied onto a small column (1.5 x 3 cm) of enzyme that released 1 nmol of 32P, per min. To ensure heparin-agarose equilibrated with buffer B. The column was linearity the extent of Pi release was limited to <10%. washed with 50 ml of this buffer, and the kinase was then SDS/12% PAGE was performed with the discontinuous eluted with buffer B/1 M NaCl. The active fractions were buffer system of Laemmli (16). Radiolabeled bands were pooled (about 2.5 ml) and applied onto a column (2.5 x 95 cm) detected with Kodax X-Omat AR-5 film. Protein was deter- of Sephacryl S-200 equilibrated and developed with buffer mined by the procedure of Bradford (17). B/0.2 M NaCl. Fractions (1.8 ml) were collected and the flow rate was 20 ml/hr. The active fractions from Sephacryl S-200 were pooled and applied onto a column (1.5 x 1.5 cm) of RESULTS phenyl-Sepharose equilibrated with buffer B/0.25 M NaCl. Autophosphorylation-Activated Protein Kinase Inactivates The column was washed with 50 ml of this buffer, and the PP2A2. During studies on the inactivation of autophosphor- kinase was eluted with buffer B/65% ethylene glycol/0.1% ylation-activated protein kinase by PP2A2, we noted that when Triton X-100. The active fractions were pooled, aliquoted, the incubation mixtures contained MgCl2 and ATP, the rate of and stored at -70°C. These preparations consisted of a single inactivation of autophosphorylation-activated protein kinase polypeptide of apparent Mr 36,000 as determined by was markedly reduced (Fig. 1). In contrast, when the incuba- SDS/PAGE (16). tion mixtures contained AMP, ADP, adenosine 5'-[3,'y Determination of Autophosphorylation-Activated Protein imido]triphosphate, or GTP instead of ATP, little or no effect Kinase Activity. A 5-,ul sample of the kinase was mixed with on the rate of inactivation of the kinase by the PP2A prepa- 50 mM Tris chloride, pH 7.0/10% glycerol/i mM benzami- rations was detected (Fig. 1). Incubation with autophosphor- dine/0.1 mM phenylmethylsulfonyl fluoride/14 mM 2-mer- ylation-activated protein kinase also inactivated the PP2A2 captoethanol in the absence or presence of 1 mM MgCl2 and preparations in a time-dependent manner with 32P-labeled 0.2 mM ATP in a final volume of 50 ,ul. After 10 min at 30°C, MBP or purified protamine kinase as substrates (Fig. 2). a 5-,lI aliquot of the incubation mixture was added to 45 ,ul of Inactivation of PP2A2 preparations depended on the rela- 50 mM Tris chloride, pH 7.0/10% glycerol/i mM benzami- tive concentrations of PP2A2 and autophosphorylation- dine/0.1 mM phenylmethylsulfonyl fluoride/14 mM 2-mer- activated protein kinase employed. Thus, after 30 min at 30°C captoethanol/10 mM MgCl2/0.2 mM [y-32P]ATP (1000 cpm/ in the presence of 1 mM Mg2+, 0.2 mM ATP, and 0.1 ,ug of pmol) containing 250 ,g of bovine serum albumin and 45 ,zg autophosphorylation-activated protein kinase, maximal in- of MBP in a microcentrifuge tube. After 10 min at 30°C, 1 ml activation (80-90%) of the PP2A2 preparations with 32p- of 12% (wt/vol) trichloroacetic acid was added and the labeled MBP as substrate was observed between 0.03 and solution was centrifuged at 10,000 rpm for 2 min in a Fisher 0.66 ,ug of PP2A2 and declined in the presence of 1.6 pLg of the microcentrifuge. The supernatant was discarded and the phosphatase. In the presence of 0.07 ,ug of PP2A2, half- pellet was washed four times with 1-ml portions of 12% maximal inactivation was observed at about 0.01 pg of the trichloroacetic acid. Aqueous counting scintillant was added kinase. The optimal concentration for Mg2+ was 1-10 mM. At to the tube and the radioactivity was determined. The kinase 1 mM Mg2+, the apparent Km for ATP was 20 ,M. The sample was omitted from controls. One unit ofautophosphor- optimal pH was about 7.0. ylation-activated protein kinase activity was equivalent to the Autophosphorylation-Activated Protein Kinase Phosphory- amount of the enzyme that catalyzed the incorporation of 1 lates PP2A2. When autophosphorylation-activated protein nmol ofphosphate into MBP per min. To ensure linearity, the kinase and PP2A2 were incubated with [y32P]ATP, SDS/ extent of incorporation was limited to <100 pmol. PAGE followed by autoradiography showed that a protein of Downloaded by guest on September 27, 2021 2502 Biochemistry: Guo and Damuni Proc. Natl. Acad. Sci. USA 90 (1993) A 100 2 3 4 5 6 7 8 9 10 11 12 ., :~ X-

80h 36- '- * 0 Co 60 F cu 5 10 15 20 5 10 15 20 c 40F Time, min

20- B

O0 AMP 0 FIG. 1. Effect of nucleotides on the PP2A-mediated inactivation x E of autophosphorylation-activated protein kinase. Autophosphoryla- 0.Q~ tion-activated protein kinase (50 ng) was activated in the presence of 0.2 mM ATP and 1 mM MgCl2 in 50 mM Tris chloride, pH 7.0/10% C- glycerol/i mM benzamidine/0.1 mM phenylmethylsulfonyl fluo- 0E.co ride/14 mM 2-mercaptoethanol in a final volume of 50 ,ul. After 15 01CS min at 30°C, a 5-,ul aliquot of the mixture was incubated (final 0 volume, 50 ,ul) in 50 mM Tris chloride, pH 7.0/10% glycerol/i mM 0 benzamidine/0.1 mM phenylmethylsulfonyl fluoride/14 mM 2-mercaptoethanol/1 mM MgC2 in the presence of 0.33 ,ug of PP2A2 with or without the indicated nucleotides (0.2 mM). After 30 min at 30°C, 5 ul of the incubation mixture was used to determine autophosphor- 12 ylation-activated protein kinase activity. AMPPNP, adenosine 5'- [8,y-imido]triphosphate. Time, min FIG. 3. Autophosphorylation-activated protein kinase phospho- apparent Mr 36,000 was phosphorylated (Fig. 3A). This rylates PP2A2. Autophosphorylation-activated protein kinase (50 ng) phosphorylation correlated closely with the inactivation of was activated in the presence of 0.2 mM ATP and 1 mM Mg2+ as the PP2A2 preparations. However, because autophosphory- described in the legend to Fig. 1. An aliquot (2.5 IL) was then incubated at 30°C (final volume, 25 ,l) in the presence of 1 mM Mg2+ lation-activated protein kinase and the catalytic subunit of and 0.2 mM [y32P]ATP in the presence (lanes 1-4) or absence (lanes 5-8) of 0.33 Mg of PP2A2. PP2A2 was also incubated in the absence of the kinase (lanes 9-12). At the indicated times, sample buffer (16) was added, the mixtures were heated at 100°C, and SDS/PAGE (16) 100 was performed. The gel was stained with Coomassie blue, washed extensively, dried, and exposed to x-ray film. (A) Autoradiogram of the dried gel. Arrow denotes the position corresponding to the 75 80 catalytic subunit of PP2A2 (Mr 36,000). (B) Incubations with the kinase and phosphatase were scaled up 4-fold to a final volume of0.1 ml and were performed as described above in the absence (e) or ~340 presence (o) of 0.4 MM microcystin-LR. After 20 min at 30TC, the mixtures were subjected separately to FPLC gel permeation chromatography on Superose 12 equilibrated and developed at a flow rate of 0.2 ml/min in 50 mM Tris chloride, pH 7.0/0.2 M NaCl/1096 glycerol/i mM benzamidine/0.1 mM phenylmethylsulfonyl fluo- 20 ride/14 mM 2-mercaptoethanol. Fractions (200 ul) were collected, and aliquots (25 Mul) of the fractions were subjected to SDS/PAGE followed by electrophoretic transfer onto Immobilon-P transfer membranes (Millipore). The bands corresponding to the catalytic 0 10 20 subunit of PP2A2 were identified by staining with Ponceau S and Time, min excised from the membrane strips for liquid scintillation counting. Arrows denote the positions at which purified preparations ofPP2A2 FIG. 2. Autophosphorylation-activated protein kinase inacti- and autophosphorylation-activated protein kinase (AK) were eluted vates PP2A2. Autophosphorylation-activated protein kinase (50 ng) from the column. Aliquots of the fractions exhibiting the highest was incubated for 15 min at 30°C in the presence of0.2 mM ATP and radioactivity from the incubations performed in the absence (lane 1) 1 mM MgCl2 in 50 mM Tris chloride, pH 7.0/10% glycerol/i mM or presence (lane 2) ofmicrocystin-LR were then electrophoretically benzamidine/0.1 mM phenylmethylsulfonyl fluoride/14 mM 2-mer- transferred onto Immobilon-P transfer membranes after SDS/ captoethanol in a final volume of 35 ,ul. A 5-,ul aliquot of PP2A2 (0.33 PAGE. The membrane strips were stained with Ponceau S, washed Mg) was then added to the incubations. At the indicated times, 5 ,ul with water, dried, and then exposed to x-ray film. Inset shows an of 32P-labeled MBP (5 Mg; e) or protamine kinase (7.5 units; A) was autoradiogram of the dried membrane strips. Arrows denote the added. After 3 min of incubation at 300C, dephosphorylation of positions corresponding to the PP2A2 subunits of apparent Mr 32P-labeled MBP was terminated with 100 Al of 12% trichloroacetic 60,000 and 36,000. acid and phosphatase activity was determined. After 10 min of incubation, protamine kinase activity was determined as described (13), except that the reaction mixtures contained 0.4 MuM microcystin- PP2A2 comigrate with an apparent Mr 36,000 in SDS/ LR. Open symbols represent control incubations in which autophos- polyacrylamide gels, the incubations were analyzed further phorylation-activated protein kinase was omitted. by FPLC gel permeation chromatography on a calibrated Downloaded by guest on September 27, 2021 Biochemistry: Guo and Damuni Proc. Natl. Acad. Sci. USA 90 (1993) 2503 subunit of apparent Mr 60,000 (Fig. 3B). Control incubations in which PP2A2 and autophosphorylation-activated protein kinase were incubated separately in the absence or y presence of microcystin-LR showed little or no phosphorylation (data not shown). There was also little or no 32P-label associated with fractions containing autophosphorylation- l.. T activated kinase (Fig. 3). We estimate that up to 0.3 mol and 1.5 mol of phosphate was incorporated per mol of the catalytic subunit of the PP2A2 preparations following incubation with autophosphorylation-activated kinase in the absence and presence of microcystin-LR, respectively. Phos- pho amino acid analysis indicated that the catalytic subunit of the PP2A2 preparations was phosphorylated on threonine residues (Fig. 4). Autodephosphorylation of PP2A2. The results suggested that in the absence of microcystin-LR, PP2A2 catalyzed an autodephosphorylation reaction. To examine this possibility, the incubation mixtures were analyzed by TLC followed by autoradiography. In the absence of microcystin-LR, phosphate was released in the incubation mixtures containing the FIG. 4. Phospho amino acid analysis. PP2A:2 was incubated with kinase and phosphatase; whereas in the presence of micro- autophosphorylation-activated protein kinase as described in the cystin-LR, this was legend to Fig. 3A. After 20 min at 30°C, 1 ml off 12% trichloroacetic dephosphorylation inhibited (Fig. 5). acid was added to the incubations. The mixture was then centrifuged for 4 min in a microcentrifuge. The supernatanit was discarded and DISCUSSION the pellets were washed eight times with 1-ml Iportions of trichloroacetic acid. After three washes with 1-ml portioins of95% ethanol, 50 The results demonstrate that PP2A2 is inactivated by phos- j,l of 6 M HCl was added to the pellets and the nnixtures were heated phorylation of its catalytic subunit following incubation with at 110°C for 1 hr. The samples were then dried with a Speed Vac purified preparations of autophosphorylation-activated pro- (Savant), suspended in water, and subjected t4o ascending TLC on tein kinase. Thus, following incubation with purified prepa- KC2 ethyl reverse-phase plates with 80% m ethanol/1.5% acetic rations of autophosphorylation-activated protein kinase, acid/0.5% formic acid as the mobile phase. T PP2A2 was inactivated by up to 80% with autophosphoryla- dried and exposed to x-ray film. Autoradiograimmhefplatesdweretthenof a dried plate is tion-activated protein kinase (Fig. 1), 32P-labeled MBP (Fig. shown. Positions of phosphoserine (S), phospl phosphotyrosine (Y) standards which were run,alongside the sample 2) and protamine kinase (Fig. 2) as substrates. Under these were identified by staining with ninhydrin. conditions, up to 0.3 mol of phosphate was incorporated per mol of the catalytic subunit ofPP2A2 (Fig. 3). Phospho amino column of Superose 12. The 32P-labeled porotein of apparent acid analysis indicated that the catalytic subunit of PP2A2 Mr 36,000 co-migrated with the PP2A2 P)reparations by gel was phosphorylated on threonine residues (Fig. 4). The permeation chromatography (Fig. 3B). In a.ddition, the extent location of the phosphorylation site(s) remains to be deter- of phosphate incorporation into the ca.talytic subunit of mined. PP2A2 was enhanced about 5-fold when the incubations were Analysis by TLC indicated that PP2A catalyzed an autode- performed in the presence of microcystirn-LR, a potent in- phosphorylation reaction during incubation with the kinase hibitor of PP2A (Fig. 3B). Under these clonditions, about 1 (Fig. 5). This dephosphorylation was inhibited when the mol ofphosphate was also incorporated pe*rmolofthePP2A2 incubations were performed in the presence of the PP2A inhibitor microcystin-LR (Fig. 5). Moreover, in the presence * I of microcystin-LR, the extent of phosphate incorporation into the catalytic subunit of PP2A2 was enhanced S5-fold (Fig. 3). In addition, under these conditions up to 1 mol of ;i11i4 phosphate was incorporated per mol of the PP2A2 subunit of apparent Mr 60,000 following incubation with the kinase (Fig. 3). Phosphorylation of this subunit was not detected when the incubations were performed in the absence of microcystin-LR (Fig. 3) and at all dilutions ofthe phosphatase f;A2'2'-',?t>',$ > (data not shown). These results indicate that phosphorylation of the catalytic subunit of the PP2A2 preparations by autophosphorylation-activated protein kinase did not inactivate 2 3 4 5 6 7 8 PP2A2 with its own subunit ofapparent Mr 60,000. Because

FIG. 5. Autodephosphorylation of PP2A2. F?P2A2 was incubated the specific activity of autodephosphorylation depended di- with 1 MM MgCl2 and 0.2 mM (yt-32P]ATP wilth (lanes 1 and 5) or rectly on the concentration of PP2A employed in the incu- without (lanes 2 and 6) autophosphorylation-,-activated protein ki- bations (data not shown), autodephosphorylation appears to nase, as described in the legend to Fig. 3, in the presence (lanes 1-4) occur via an intermolecular rather than intramolecular reac- or absence (lanes 5-8) of 0.4 uM microcystin-L.R. For lanes 3 and 7, tion mechanism. Therefore, we cannot entirely rule out the autophosphorylation-activated protein kinase w~,as incubated with 0.2 possibility that dephosphorylation was catalyzed by a con- MM [yt-32P]ATP and 1 MM MgCl2 in the absence of PP2A2. For lanes taminating phosphatase present in the incubation mixtures. 4 and 8, autophosphorylation-activated proteiiIn kinase and PP2A2 Interestingly, Serra et al. (18) have reported the purification were omitted from the incubations. After 30 min at 30gC, a 5-n.d to apparent homogeneity of a protein of apparent Mr 20,000 aliquot of each mixture was spotted onto thini-layer silica gel and which inhibited the hydroxymethylglutaryl-CoA reductase ascending chromatography was performe~ propanol/2-propanol/formic acid/water, 3:1 :1:1 (vol/vol). The phosphatase activity of purified preparations of PP2A with- plates were dried and then exposed to x-ray filmr.:The autoradiogram out affecting the phosphorylase phosphatase activity of this of the dried plates is shown. The position to whiich an aliquot of 32p;, enzyme. Whether this and/or another physiological protein run alongside the samples, migrated is denotedI by the arrow. or effector inhibits the rate of this apparent autodephosphor- Downloaded by guest on September 27, 2021 2504 Biochemistry: Guo and Damuni Proc. Natl. Acad. Sci. USA 90 (1993) ylation of PP2A2 remains to be determined. In addition, in this paper, which show that PP2A2 is inactivated by whether phosphorylation of PP2A by autophosphorylation- phosphorylation of its catalytic subunit on threonines cata- activated protein kinase affects the low but detectable pro- lyzed by purified preparations of the autophosphorylation- tein-tyrosine-phosphatase activity of PP2A (19-21) also re- activated protein kinase. It is nevertheless possible that in mains to be determined. It is pertinent that this protein- intact cells multisite phosphorylation of the catalytic subunit tyrosine-phosphatase activity of PP2A was stimulated by ofPP2A2 on threonine and tyrosine residues may, in response purified preparations of an ATP- and Mg2+-dependent pro- to extraceilular stimuli, be a mechanism for "locking for the tein factor from Xenopus oocytes (22). However, the mech- duration" the phosphorylated and inactivated form of PP2A. anism of action of this activating protein factor is uncertain, and phosphorylation of PP2A does not appear to be involved We thank Shrikanth Reddy and Grayson Amick for their help in the (22). initial stages of this work. This work was supported by Grant Our results suggest that phosphorylation and inactivation DMB-9019882 from the National Science Foundation. of PP2A may be a physiological mechanism by which auto- 1. Cohen, P. (1989) Annu. Rev. Biochem. 58, 453-508. phosphorylation and activation of autophosphorylation- 2. Shenolikar, S. & Nairn, A. C. (1991) Adv. Second Messenger activated protein kinase and phosphorylation and activation Phosphoprotein Res. 23, 1-121. of the protamine kinase may occur in cells in response to 3. Damuni, Z. & Reed, L. J. (1987) J. Biol. Chem. 262, 5133-5138. extracellular stimuli. It is important to note that the activity 4. Da Cruz e Silva, 0. B., Alemany, S., Campbell, D. G. & of the protamine kinase is stimulated rapidly following incu- Cohen, P. T. W. (1987) FEBS Lett. 221, 415-422. bation of isolated rat hepatocytes with insulin (11), appar- 5. Da Cruz e Silva, 0. B. & Cohen, P. T. W. (1987) FEBS Lett. ently 266, 176-178. via phosphorylation of the kinase (11). In addition, the 6. Stone, S. R., Hofsteenge, J. & Hemmings, B. A. (1987) Bio- evidence indicates that PP2A is a specific protamine kinase- chemistry 26, 7215-7220. inactivating phosphatase (12). Thus, in contrast to the other 7. Arino, J., Woon, C. W., Brautigan, D. L., Miller, T. B., Jr., & three major cytoplasmic protein phosphatase (PP1, PP2B, Johnson, G. L. (1988) Proc. Natl. Acad. Sci. USA 85, 4252- and PP2C), PP2A specifically inactivated purified prepara- 4256. tions of the protamine kinase (12). Interestingly, relative to 8. Snedon, M., Cohen, P. T. W. & Stark, M. J. R. (1990) EMBO PP1, PP2A preferentially inactivated mitogen-activated pro- J. 9, 4339-4345. 9. Guo, H., Reddy, S. A. G. & Damuni, Z. (1993) J. Biol. Chem., tein kinase (23), two distinct mitogen-activated ribosomal in press. protein S6 kinases (23, 24), and an insulin-stimulated Kemp- 10. Damuni, Z., Amick, G. D. & Sneed, T. R. (1989)J. Biol. Chem. tide kinase (25). Thus, it is also possible that inactivation of 264, 6412-6416. PP2A by phosphorylation may contribute to the phosphory- 11. Reddy, S. A. G., Amick, G. D., Cooper, R. H. & Damuni, Z. lation and activation of these insulin- and mitogen-activated (1990) J. Biol. Chem. 265, 7748-7752. protein kinases also and thereby contribute to the marked 12. Amick, G. D., Reddy, S. A. G. & Damuni, Z. (1992) Biochem. increase in the phosphorylation of cellular proteins in re- J. 287, 1019-1022. sponse to insulin and other mitogens. 13. Damuni, Z. (1990) Biochem. Biophys. Res. Commun. 166, However, in addition to its activity with the insulin- and 471-478. 14. Amick, G. D. & Damuni, Z. (1992) Biochem. Biophys. Res. mitogen-regulated protein kinases, PP2A exhibits activity Commun. 183, 431-437. with numerous proteins that are also phosphorylated in 15. Amick, G. D., Reddy, S. A. G. & Damuni, Z. (1992) Arch. response to insulin and other mitogens. For example, acetyl- Biochem. Biophys. 297, 80-85. CoA carboxylase (26), ATP-citrate lyase (26), and the mRNA 16. Laemmli, U. K. (1970) Nature (London) 227, 680-685. cap-binding protein, initiation factor 4E (27, 28) are phos- 17. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. phorylated in intact cells in response to insulin and other 18. Serra, D., Guillermina, A., Calvet, V. E. & Hegdart, F. G. mitogens. Acetyl-CoA carboxylase and ATP-citrate lyase (1989) J. Biol. Chem. 264, 14681-14685. are some of the best PP2A substrates identified to date (1), 19. Goris, J., Pallan, C. J., Parker, P. J., Hermann, J., Waterfield, and PP2A is an initiation factor 4E phosphatase (G. D. Amick M. D. & Merlevede, W. (1988) Biochem. J. 256, 1029-1034. 20. Goris, J., Hermann, J., Hendrix, P., Ozon, R. & Merlevede, W. and Z.D., unpublished data). Thus, phosphorylation and (1989) FEBS Lett. 245, 91-94. inactivation of PP2A in response to insulin and other mito- 21. Hermann, J., Cayla, X., Dumortier, K., Goris, J., Ozon, R. & gens may also contribute to the phosphorylation of these Merlevede, W. (1988) Eur. J. Biochem. 173, 17-25. proteins. Previous failures to observe an effect of insulin and 22. Cayla, X., Goris, J., Hermann, J., Hendrix, P., Ozon, R. & other mitogens on PP2A activity (29, 30) may well be because Merlevede, W. (1990) Biochemistry 29, 658-667. the conditions employed did not prevent the dephosphory- 23. Sturgill, T. W., Ray, L. B., Erickson, E. & Mailer, J. L. (1988) lation of PP2A either by autodephosphorylation or by other Nature (London) 334, 715-718. PP2A phosphatases present in extracts from the control and 24. Ballou, L. M., Jeno, P. & Thomas, G. (1988) J. Biol. Chem. hormone-treated 263, 1188-1194. cells. 25. Klarlund, J. K., Jaspers, S. R., Khalaf, N., Bradford, A. P., While this manuscript was in the final stages of prepara- Miller, T. B. & Czech, M. P. (1991) J. Biol. Chem. 266, tion, Brautigan and coworkers (31) reported that purified 4052-4055. preparations of the catalytic subunit of PP2A could be 26. Denton, R. M. (1986) Adv. Cyclic Nucleotide Protein Phos- phosphorylated transiently on tyrosine residues by the ty- phorylation Res. 20, 293-341. rosine-specific insulin and epidermal growth factor receptor 27. Morley, S. J. & Traugh, J. A. (1990) J. Biol. Chem. 265, kinases and by the non-receptor tyrosine-specific kinases 10611-10616. p60v-src and p561ck. The effects of the p56 ck-, insulin recep- 28. Manzella, J. M., Rychlik, W., Rhoads, R. E., Hershey, tor-, and epidermal growth factor J. W. B. & Blackshear, P. J. (1991) J. Biol. Chem. 266, 2383- receptor-catalyzed phos- 2389. phorylations on PP2A activity were not determined. How- 29. Chan, C. P., McNall, S. J., Krebs, E. G. & Fischer, E. H. ever, thiophosphorylation by p60v-src purified by immuno- (1988) Proc. Natl. Acad. Sci. USA 85, 4720-4724. precipitation from cells overexpressing the gene for this 30. Olivier, A. R., Ballou, L. M. & Thomas, G. (1988) Proc. Natl. enzyme was reported to inactivate the purified preparations Acad. Sci. USA 85, 4720-4724. of the catalytic subunit of PP2A with phosphorylase as 31. Chen, J., Martin, B. L. & Brautigan, D. L. (1992) Science 257, substrate (31). These results are in contrast to those reported 1261-1264. Downloaded by guest on September 27, 2021