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

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