phosphorylation of smg p21, a ras p21-like gtp-binding protein, by

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 264, No. 26, Issue of September 15, pp. 15688-15695,1989 Printed in U. S. A.

Phosphorylation of smg p21, a rcts p21-like GTP-binding Protein, by Cyclic AMP-dependent Protein Kinase in a Cell-free System and in Response to Prostaglandin El in Intact Human Platelets”

(Received for publication, April 21, 1989)

Masahito Kawata, Akira Kikuchi, Masahiko Hoshijima, Katsuhiko Yamamoto, Eikichi HashimotoS, Hirohei Yamamuraz, and Yoshimi Takais From the Department of Biochemistry, Kobe University School of Medicine, Kobe 650, Japan and the $Department of Biochemistry, Fukui Medical School, Matsuoka, Fukui 910-11, Japan

We have separated multiple small M, GTP-binding proteins (G proteins) from bovine brain membranes by several column chromatographies and purified to near homogeneity four of them, including a novel M, 24,000 G protein (smg p25A), a novel M, 22,000 G protein (smg p21), the rho protein (rho p20), and the c-Ki-rus protein (c-Ki-rus p21). Among these small M, G proteins, only smg p21 is phosphorylated stoichiometrically by CAMP-dependent protein kinase (protein kinase A), and c-Ki-rus p21 is phosphorylated to a small extent by protein kinase A in a cell-free system. None of smg p25A, rho p20, and other partially purified small M, G proteins is phosphorylated by protein kinase A. Neither smg p2 1 nor other small M, G proteins are phosphorylated by protein kinase C. About 1 mol of phosphate is maximally incorporated into 1 mol of smg p21 by protein kinase A. Only serine residue(s) are phosphorylated. The guanosine 5‘-3-0- (thio) triphosphate (GTP-&)-bound and GDP-bound forms of smg p21 are phosphorylated with the same reaction velocity. The phosphorylation of smg p21 affects neither its GTPyS-binding nor GTPase activity. smg p21 is found in human platelets, and this human platelet smg p21 is also phosphorylated by protein kinase A at the same site(s) as bovine brain smg p2 1 in a cell-free system. When intact human platelets are stimulated by prostaglandin El known to elevate the cAMP level, four proteins with apparent M, values of 240,000, 50,000, 24,000, and 22,000 are phosphorylated. These four proteins are also phosphorylated by the action of dibutyryl cAMP but not by the action of thrombin, Ca2+ ionophore A23187, or 12-0-tetradecanoylphorbol-13-acetate. Among the four proteins, the M, 22,000 protein is identified as smg p21. The site(s) of phosphorylation of smg p2 1 by protein kinase A in a cell-free system are identical to that phosphorylated in response to prostaglandin E1 in intact platelets. These results indicate that among many small M, G proteins, srng p21 is selectively phosphorylated by

* This investigation was supported by grants-in-aid for scientific research and cancer research from the Ministry of Education, Science, and Culture, Japan (1988), grants-in-aid for abnormalities in hor- mone receptor mechanisms and cardiovascular diseases and for cancer research from the Ministry of Health and Welfare, Japan (1988), and grants from the Yamanouchi Foundation for research on metabolic disease (1988), the research program on cell calcium signal in the cardiovascular system (1988), and the Princess Takamatsu Cancer Research Fund (1988). 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 U.S.C. Section 1734 solely to indicate this fact.

§ To whom requests for reprints should be addressed.

protein kinase A and that this G protein is also phosphorylated by this protein kinase in response to prostaglandin El in intact human platelets.

In addition to a group of G proteins’ with an subunit structure serving as transducers for membrane receptors such as G, and Gi (for reviews, see Refs. 1-4), there is another group of G proteins with M , values of about 20,000, designated here as small M , G proteins. This group includes the proteins of the three ras (Ha-, Ki-, and N-), three rho (-A, -B, and -C), ral, R-ras,yptl, four rub (-1, -2, -3, and - 4 ) , arf, rap (-lA, -lB, and -2), and Kreu-1 genes (for reviews, see Refs. 5 and 6, and 7-23). Several small M, G proteins including the arf protein have been purified and characterized (24-28). We have recently separated at least 15 small M , G proteins from bovine brain membranes by several column chromatographies and purified a novel M, 24,000 G protein (smg p25A) (29,30), a novel M, 22,000 G protein (smg p21) (31), the M , 20,000 rho protein (rho p20) (32), and the M, 21,000 c-Ki-ras protein (c-Ki-ras p21) (33). Among these small M, G proteins, the rub1 protein is identical to the yptl protein (17, 18), the rub3 protein to smg p25A (18, 19, 30), and the raplA and Kreu-1 proteins to smg p21 (22, 23, 31).

ras p21s are present in most mammalian tissues and act by regulating normal cell proliferation and differentiation in addition to the transforming action (5). The rho mRNA is expressed in many tissues (34), but its function has not been clarified. We have shown recently that rho p20 is ADP- ribosylated by an ADP-ribosyltransferase contained in botulinum toxin type C1 (35). Consistent with our observations, one small M, G protein ADP-ribosylated by an ADP-ribosyltransferase contained in botulinum toxin types C1 and D has been purified from bovine adrenal gland cytosol (27). This protein, termed Gb, has also been identified as the rho gene product (36). Moreover, a small M , G protein ADP-ribosylated by botulinum ADP-ribosyltransferase C3 has been purified from porcine brain cytosol and identified as the rho gene product (37). The yptl protein is present in many mammalian tissues and yeast (17, 38) and is suggested to be involved in secretory processes in yeast (39). The arf protein is isolated as a cofactor for the ADP-ribosylation of G, by cholera toxin and is present in many mammalian tissues (24, 26, 40), but

The abbreviations used are: G proteins, GTP-binding proteins; protein kinase A, CAMP-dependent protein kinase: PGEI, prostaglandin El; TPA, 12-0-tetradecanoylphorbol-13-acetate; TPCK, L-l-to- sylamido-2-phenylethyl chloromethyl ketone; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; GTPyS, guanosine 5’-3-O-(thio)triphosphate.

15688

Phosphorylation of smgp21 by Protein Kinase A 15689

its physiological functilon remains to be clarified. We have found that the smg p25A mRNA is present most abundantly in brain and adrenal mledulla (41). The similar results have been also reported for the rub3 (smg-25A) mRNA (34). More- over, we have shown that the smg p25A mRNA is detected in pheochromocytoma (PC-12) cells, and its level is markedly elevated in parallel with the formation of neurite-like processes in response to nerve growth factor or BtZcAMP (41). These results suggest that smg p25A is related at least to neuronal functions. On the other hand, the raplA (Kreu-1) mRNA is detected in many mammalian tissues (22, 23). We have shown that smg p2l (the raplA and Kreu-1 proteins) is present most abundantly in human platelets (42,43). smg p21 has the same putative effector domain and consensus C- terminal sequences as ras p21s (5, 6, 22, 23, 31). This unique structure of smg p21 suggests that this G protein exerts actions similar and/or antagonistic to those of ras p21s. In fact, the Kreu-1 gene has been shown to suppress the transforming action of v-Ki-ras p21 in NIH/3T3 cells (23).

Protein phosphorylation plays central roles in the regulation of various cell functions. Many protein kinases have been identified, and particulady protein kinases A and C have been shown to be involved in a wide variety of cell functions (for reviews, see Refs. 44-46). Among many small M , G proteins, c-Ki-ras p21 has been shown to be phosphorylated by both protein kinases C and A (47), v-Ha-ras p21 by protein kinase C (48), and c-Ki- and c-Ha-ras p21s by protein kinases C and A (49). The yeast RAS2 protein has been also shown to be phosphorylated by protein kinase A but not by protein kinase C (50). Moreover, the :yeast RASl and RAS2 proteins have been shown to be phosphorylated in an intact cell system (51). However, the detailed enzymatic studies including the stoichiometry of phospl~orylation of ras p21 or the RAS proteins have not been done in these reports. Moreover, it has not been examined whether other small M , G proteins are phosphorylated by these protein kinases.

In the present studies, we have examined whether bovine brain small M, G proteins are phosphorylated by protein kinases A and C. T h h paper describes that among many bovine brain small M, G proteins, only smg p21 is phosphorylated stoichiometrically by protein kinase A but not by protein kinase C and that c-Ki-ras p21 is phosphorylated to a small extent by protein kinase A but not by protein kinase C. We have also examined whether smg p21 is indeed phosphorylated by protein k:inase A in intact cells using platelets as a model. The reasom that we used platelets are (a) that platelets contain an large amount of smg p21 (42,43); ( b ) that at least four proteins with apparent M , values of 240,000, 50,000, 24,000, and 22,000 have been shown to be phosphorylated in response to CAMP-elevating PGEl and Bt,cAMP in platelets (for a review, s'ee Ref. 52); and (c) that this M , 22,000 protein has an M , value similar to that of smg p21 on SDS- PAGE. This paper describes that human platelet smg p21 is phosphorylated directly by protein kinase A in response to PGEl and Bt2cAMP in intact human platelets.

EXPERIMENTAL PROCEDURES

Materials and Chemical:;-The catalytic subunit of protein kinase A was purified to near homogeneity from rabbit skeletal muscle as described (53) with slight modifications. The holoenzyme of protein kinase A and its heat-stable protein inhibitor were highly purified from bovine heart and rabbit skeletal muscle, respectively (54, 55). Protein kinase C was purified to near homogeneity from rat brain (56). smg p21 was purified to near homogeneity from bovine brain membranes and human pl.stelet membranes (31, 43). smg p25A, rho p20, and c-Ki-ras p21 were purified to near homogeneity from bovine brain membranes (29, 32, 33) . Other small M , G proteins were partially purified from bovine brain membranes (29, 31-33). H2B

histone was prepared from calf thymus (57). Washed human platelets were obtained as described (58). PGE,, BtZcAMP, Ca2+ ionophore A23187, and TPA were from Funakoshi Pharmaceutical Co. (Tokyo, Japan), Yamasa Shoyu Co. (Chiba, Japan), Calbiochem, and CCR Inc., respectively. TPCK-treated trypsin, thrombin, and alkaline phosphatase (type VII-S) were from Sigma. Other materials and chemicals were obtained from the same sources as described (29, 31- 33, 56, 59).

Protein Phosphorylation in a Cell-free System-Phosphorylation of various small M , G proteins and H2B histone by protein kinase A was carried out in a reaction mixture (50 pl) containing 50 mM Tris/ HCI a t pH 7.5, 10 mM MgC12, 100 pg/ml bovine serum albumin, 3 mM 2-mercaptoethanol, and 25 pM [T-~'P]ATP (1600-3200 cpm/ pmol). Protein kinase A and each G protein or H2B histone were added as indicated. Where specified, 1 p~ CAMP or the heat-stable protein inhibitor of protein kinase A (750 ng of protein) was added. Phosphorylation of various G proteins by protein kinase C was carried out in a reaction mixture (50 pl) containing 50 mM Tris/HCI at pH 7.5, 5 mM Mg(CH3C00)z, a 4O-pglml mixture of bovine brain phos- pholipids, 50 nM TPA, 0.5 mM CaCl2, and 25 p M [Y-~*P]ATP (1600- 3200 cpm/pmol). The reaction mixtures of smg p21, c-Ki-ras p21, and H2B histone contained 0.2% sodium cholate and 58 mM NaCI, which were included in the preparations of smg p21 and c-Ki-ras p21. The reaction mixtures of other G proteins contained less than 100 mM NaCl. After the incubation at 30 "C for various periods of time, the reaction was stopped by the addition of 10% trichloroacetic acid. The acid-precipitable materials were collected by a Toyo-Roshi nitrocellulose filter (pore size, 0.45 pm), and the radioactivity was counted. For autoradiography of SDS-PAGE, the reaction was stopped by the addition of an SDS-stopping solution (20 pl) containing 200 mM Tris/ HCI at pH 6.7, 9% SDS, 6% 2-mercaptoethanol, 15% glycerol, and 0.01% bromphenol blue. The sample was subjected to SDS-PAGE (8-16% polyacrylamide) by the method of Laemmli (60). The gel was dried, and an autoradiograph was made. The radioactive band was excised from the gel, and the radioactivity was counted. For autoradiography of two-dimensional PAGE, the reaction was stopped by the addition of a lysis buffer (60 pl) containing 8 M urea, 2% (w/v) Nonidet P-40, 2% (v/v) ampholytes (pH range 3.5-lo), and 5% 2- mercaptoethanol. A 50-p1 aliquot was subjected to two-dimensional PAGE by the method of O'Farrell(61) with slight modifications using 2% (v/v) ampholytes (pH range 3.5-10) in the first dimension and 8- 16% polyacrylamide gel in the second dimension.

Assays for pSSIGTPyS-binding and GTPase Actiuities-The [%s] GTPyS-binding activity of smg p21 was determined by use of the nitrocellulose filter method as described (29). The GTPase activity of smg p21 was estimated by the liberation of 32P, from [y-"P]GTP as described (29).

Determination of Phosphorylated Amino Acid-smg p21 (50 ng of protein) fully phosphorylated by protein kinase A (38 ng of protein) was hydrolyzed in 6 N HCl for 3 h at 100 "C in a sealed tube. Phosphoserine, phosphothreonine, and phosphotyrosine were separated by one-dimensional high voltage electrophoresis using a cellulose thin-layer plate (62).

Protein Phosphorylation in Intact Human Platelets-Washed human platelets were labeled with 32Pi as described (63). The radioactive platelets were finally suspended at 6 X 10' cells/ml in a buffer containing 15 mM Tris/HCl at pH 7.5, 140 mM NaC1, and 5.5 mM glucose. The suspension of platelets (230 pl) prelabeled with 32Pi was incubated at 37 "C in a final volume of 250 p1 with either PGE,, BtzcAMP, thrombin, A23187, or TPA as indicated. The sample was divided into two equal volume of aliquots (125 p1 each). One aliquot was mixed with 62.5 pl of an SDS-stopping solution and boiled for 3 min. Aliquots (60 pl, 20 pg of protein) of each sample were subjected to one-dimensional SDS-PAGE (8-16% polyacrylamide). The gel was stained with Coomassie Brilliant Blue, dried, and an autoradiograph was made. The other aliquot was mixed with 187.5 p1 of a lysis buffer, and the mixture was frozen and thawed twice. Aliquots (50 pl, 10 pg of protein) of each sample were subjected to two-dimensional PAGE.

Peptide Map Analysis-Two-dimensional mapping of TPCK- treated trypsin peptides from smg p21 labeled with 32P> was made (64) with slight modifications. The SDS-polyacrylamide gel slice containing radioactive smg p21 was homogenized with 1 ml of 70% formic acid/0.05-cm2 gel (1 mm thick). The homogenate was shaken over- night at room temperature. The gel fragments were removed by centrifugation. The supernatant was carefully pooled, and the gel fragments were shaken again with the same formic acid for 2 h at room temperature. The fragments were pelleted again, and the second supernatant was pooled with the first supernatant. After the formic

15690 Phosphorylation of srng p21 by Protein Kinase A

acid was removed under reduced pressure, the solution was lyophilized. After the addition of 75 pg of bovine y-globulin as a carrier, the protein was precipitated by adding trichloroacetic acid at a final concentration of 20% followed by standing for 2 h at 4 "C. After centrifugation, the pellet was washed once with ethanol at -10 "C and then with ethanol/ether (1:l) at -10 "C. The dried residue was dissolved in 150 pl of chilled performic acid and incubated for 2 h at 4 "C. The performic acid was prepared by mixing 30% HZ02 and 98% formic acid (1:9) followed by standing for 1 h at room temperature. The protein solution was diluted with 1 ml of water and lyophilized. The oxidized protein was treated with 30 pg of TPCK-treated trypsin in 0.5 ml of 50 mM NH4HCO3 for 18 h at room temperature. Then, 20 pg of TPCK-treated trypsin was supplemented, and incubation was continued for an additional 4 h. The digest was diluted with 0.5 ml of water and lyophilized. The residue was dissolved with 0.5 ml of water and lyophilized again. The final residue was dissolved in 50 pl of acetic acid/formic acid/water (3:1:16). An aliquot of the digest was spotted on a cellulose thin-layer plate (Merck, 20 X 20 cm), and electrophoresis was carried out for the first dimension at 1000 V for 1 h at 4 "C using a Pharmacia LKB Biotechnology Inc. flat bed apparatus, model FBE 3000, in the presence of the same buffer solution. The peptides were then chromatographed to the second dimension with butanol/pyridine/acetic acid/water (15:103:12) as a solvent, and an autoradiograph was prepared.

Protein Determination-Protein concentrations were determined by densitometric tracing of protein bands on an SDS-polyacrylamide gel with bovine serum albumin as a standard protein as described (29).

RESULTS

Phosphorylation of Bovine Brain smgp21 by Protein Kinase A in a Cell-free System-Purified smg p21 migrated at the position with an M, of about 22,000 on SDS-PAGE as described previously (31) (Fig. l, lane l ) . When smg p21 was first incubated with the catalytic subunit of protein kinase A and then subjected to SDS-PAGE followed by autoradiography, a radioactive band was observed at the position with an M , slightly larger than 22,000 (Fig. 1, lane 2 ) . This radioactive band coincided with the protein staining band of smg p21 preincubated with the catalytic subunit (data not shown). The phosphorylation of smg p21 by the catalytic subunit of protein kinase A was inhibited by its heat-stable protein inhibitor (Fig. 1, lane 3). Moreover, smg p21 was phosphorylated by the holoenzyme of protein kinase A in a CAMP-dependent manner (Fig. 1, lanes 4 and 5 ) . These results indicate that smg p21 is phosphorylated by protein kinase A, and the phosphorylated smg p21 migrated with a slightly lower mo- bility than the nonphosphorylated protein. When smg p21 was incubated with protein kinase C under its standard conditions, a radioactive band was not observed at the position of the phosphorylated smg p21 (Fig. 1, lane 6) . Under these conditions, protein kinase C itself was autophosphorylated as described (46).

Under the conditions in which smg p21 was phosphorylated by protein kinase A, purified c-Ki-ras p21 was phosphorylated by this protein kinase to a small extent, and about 0.1 mol of phosphate was maximally incorporated/mol of this protein (Fig. 1, lane 14). Even when c-Ki-ras p21 was pretreated with alkaline phosphatase to remove possible endogenous phosphate and then incubated with protein kinase A, its phosphorylation did not increase (data not shown). Neither smg p25A nor rho p20 was phosphorylated by protein kinase A (Fig. 1, lanes 8 and 11). None of smg p25A, rho p20, or c-Ki- ras p21 was phosphorylated by protein kinase C (Fig. 1, lanes 9, 12, and 15). None of the small M , G proteins except smg p21 and c-Ki-ras p21 was phosphorylated by protein kinase A or C in the absence or presence of the same concentration of sodium cholate (0.2%) as that used for the phosphorylation of smg p21 and c-Ki-ras p21 (data not shown).

Kinetics for the Phosphorylation of smg p21 by Protein

66-

5 36- 9

20.1-

66-

-; 7

9 5 36-

20.1-

1 2 3

e c

4 5 6

t

7 8 9 10 11 12 13 14 15

FIG. 1. Phosphorylation of bovine brain smgp21 by protein kinase A in a cell-free system. smg p21 (40 ng of protein) was incubated with either the catalytic subunit of protein kinase A (15 ng of protein) for 60 min, its holoenzyme (30 ng of protein) for 10 min, or protein kinase C (20 ng of protein) for 60 min. The heat-stable protein inhibitor of protein kinase A (750 ng of protein) and CAMP (1 p~ at a final concentration) were added as indicated. smg p25A, rho p20, and c-Ki-ras p21 (40 ng of each protein) were incubated with the catalytic subunit of protein kinase A (15 ng of protein) or protein kinase C (20 ng of protein) for 60 min. Lanes I , 7, 10, and 13, protein staining of nonphosphorylated small M, G proteins (500 ng of each protein) with Coomassie Brilliant Blue. Lane 1, smg p21; lane 7, smg p25A; lane 10, rho p20; lane 13, c-Ki-ras p21. Lanes 2-6, 8, 9, 11, 12, 14, and 15, autoradiographs. Lane 2, smg p21 with the catalytic subunit of protein kinase A; lane 3, smg p21 with the catalytic subunit of protein kinase A in the presence of its heat-stable protein inhibitor; lane 4, smg p21 with the holoenzyme of protein kinase A in the presence of CAMP; lane 5, smg p21 with the holoenzyme of protein kinase A in the absence of CAMP; lane 6, smg p21 with protein kinase C; lane 8, smg p25A with the catalytic subunit of protein kinase A; lane 9, smg p25A with protein kinase C; lane 11, rho p20 with the catalytic subunit of protein kinase A; lane 12, rho p20 with protein kinase C; lane 14, c-Ki-ras p21 with the catalytic subunit of protein kinase A; lane 15, c-Ki-ras p21 with protein kinase C. Small and large arrows indicate the autophosphorylation of the regulatory subunit of protein kinases A and C, respectively. The protein markers used were bovine serum albumin (Mr 66,000), glyceraldehyde-3-phosphate de- hydrogenase (M, 36,000), and trypsin inhibitor (M, 20,100).

Kinase A-The phosphorylation of smg p21 by protein kinase A was time dependent (Fig. 2). About 1 mol of phosphate was maximally incorporated into 1 mol of smg p21 even after the readdition of protein kinase A as shown by arrows in the figure. Only serine residue(s) were phosphorylated, and neither threonine nor tyrosine residue was phosphorylated (data not shown). In Fig. 3, the K , and V,,, values for the phosphorylation of smg p21 by protein kinase A were calculated from a least squares fit of the data on double-reciprocal plots. The K, and Vmax values are 0.11 f 0.02 PM and 32 f 2 nmol/ min/mg (mean k S.E.), respectively. The K,,, and V,,, values calculated in the same way for H2B histone, known to be a good substrate for protein kinase A (57, 65), were 130 k 10 pM and 840 & 40 nmol/min/mg (mean * S.E.), respectively' ~~

* The K, value for H2B histone was increased about 4-fold, and

Phosphorylation of srng p21 by Protein Kinase A 15691

7- I

o&- 30 60 6 Incuballon Time imin)

FIG. 2. Time course of the phosphorylation of bovine brain smg p21 by protein kinase A in a cell-free system. smg p21 (40 ng of protein) was phosphorylated by the catalytic subunit of protein kinase A (15 ng of protein) for various periods of time. After the incubation for 40 and 60 min as indicated with arrows, the enzyme (15 ng of protein) and [y3*P]ATP (250 pmol) were added to the reaction mixture, and the incubation was further performed for the indicated periods of time. The results shown are the representatives of three independent experiments.

- - ""LA 0 100 200 300

Concentration of smg p21 (nM)

FIG. 3. Dose-dependent phosphorylation of bovine brain srng p21 by protein kinase A in a cell-free system. Various amounts of smg p21 (10-300 ng of protein) were phosphorylated by the catalytic subunit of protein kinase A (15 ng of protein) for 2 min. All the reaction mixtures contained 0.2% sodium cholate and 58 mM NaC1, which were included in the preparation of smg p21. The inset shows the double-reciprocal plots. The results shown are the representatives of three independent experiments.

(data not shown). The K , value for smg p21 was less than %OOO that for H2B histone, and the V,,, value for smg p21 was about %O that for FI2B histone.

Purified smg p21 was f irst incubated with GTPrS or GDP to prepare the G protein that fully bound each nucleotide. These GTP+- and GDP-bound forms of smg p21 were then phosphorylated by protein kinase A, and their initial velocities for the phosphorylation were compared. The initial velocities were similar between both forms (Table I).

Effect of the Phosphorylation of smgp21 on its P'SJGTPrS- binding and GTPase A'ctiuities-Purified smg p21 was first fully phosphorylated, and then the [35S]GTPyS-binding and GTPase activities of the phosphorylated and nonphosphorylated forms were compared. The phosphorylation of smg p21 affected neither its [35S]GTPyS-binding nor GTPase activity (Fig. 4).

Comparison of the Pe.ptide Maps of Bovine Brain and Hu- man Platelet smgp2ls Phosphorylated by Protein Kinase A in a Cell-free System-We have previously separated at least three small M , G proteins including ras p21 and purified an M , 22,000 G protein to near homogeneity from human platelet membranes (43). Subsequently, we have found that this human platelet M , 22,000 1% protein is identical to bovine brain smg p21 in its physical and kinetic properties (42). Moreover,

the V,,, value for that was decreased by 70% by 0.2% sodium cholate and 58 mM NaCl contained in this reaction mixture.

TABLE I Efficiency of the GTP-yS- and GDP-bound forms of bovine brain smg

p21 for the phosphorylation by protein kinase A smg p21 (40 ng of protein) was first incubated with either 1 pM

GTPyS or GDP for 15 min at 30 "C under the same conditions as described (29) except that NaCl, MgClP, and EDTA were used at the concentrations of 60, 5, and 10 mM, respectively. About 1 mol of GTPyS or GDP bound to 1 mol of smg p21 under these conditions. The GTPyS- and GDP-bound forms of smg p21 were then phosphorylated by the catalytic subunit of protein kinase A (15 ng of protein) for various periods of time. The results shown are the means f S.E. of three independent experiments.

32P incorporated

GTPyS-bound GDP-bound form form

Reaction time

min cpm 5 170 f 10 180 f 15

10 330 f 15 280 k 20 20 540 f 30 580 k 30

10 20 30 60 90 Incubation Time (mm) Incubation Tlme (min)

1 " "ii

FIG. 4. Effect of the phosphorylation of bovine brain smg p21 on its [3SS]GTPyS-binding and GTPase activities. smg p21 (40 ng of protein) was incubated with the catalytic subunit of protein kinase A (30 ng of protein) for 20 min in the presence and absence of nonradioactive ATP. The catalytic subunit of protein kinase A (15 ng of protein) and ATP (250 pmol) were additionally added followed by the further incubation for 10 min. About 1 mol of phosphate was incorporated into 1 mol of smg p21 under these conditions. The [35S] GTPyS-binding and GTPase activities were then assayed. A, [3sS] GTPyS-binding activity; E , GTPase activity; (O), in the presence of ATP; (O), in the absence of ATP. The results shown are the means t S.E. of three independent experiments.

we have found that only srng p21 is phosphorylated by protein kinase A but not by protein kinase C among the three small M, G proteins purified from human platelet membranes as far as tested in a cell-free system (59). Fig. 5 compares the peptide maps of bovine brain and human platelet smg p21s phosphorylated by protein kinase A in a cell-free system. Phosphorylated smg p21s were digested completely with TPCK-treated trypsin, and the digested samples were subjected to two-dimensional peptide mapping followed by autoradiography. Identical peptide maps were obtained for both bovine brain and human platelet smg p21s. An identical peptide map was also obtained when the peptides from phosphorylated bovine brain and human platelet smg p21s were subjected together to two-dimensional peptide mapping (data not shown). These results indicate that bovine brain and human platelet smg p21s are phosphorylated by protein kinase A at the same site(s).

Phosphorylation of smg p21 in Response to PGE, and Bt2cAMP in Intact Human Platelets-When intact human platelets were stimulated by PGE1, four proteins with M , values of 240,000, 50,000, 24,000, and 22,000 were phosphorylated (Fig. 6A, lanes 1 and 2) . These four proteins were also

15692 Phosphorylation of smgp21 by Protein Kinase A

- + Electrophoresis

FIG. 5. Comparison of the peptide maps of bovine brain and human platelet smg p2 1s phosphorylated by protein kinase A in a cell-free system. Bovine brain smg p21 (170 ng of protein) and human platelet smg p21 (170 ng of protein) were phosphorylated by the catalytic subunit of protein kinase A (130 ng of protein). The reaction was terminated by the addition of an SDS-stopping solution, and all the samples were subjected to SDS-PAGE. Phosphorylated bovine brain and human platelet smg p21s were extracted from the gel and digested completely with TPCK-treated trypsin. Aliquots (2000 cpm) of each sample were subjected to two-dimensional peptide mapping followed by autoradiography. Arrows indicate the phosphorylated peptides. Arrowheads indicate the origin. A, bovine brain smg p21; B, human platelet smg p21.

phosphorylated in response to Bt2cAMP (Fig. 6A, lane 3 ) but not in response to thrombin, A23187, or TPA (Fig. 6A, lanes 4-6). Thrombin, A23187, and TPA stimulated the phosphorylation of two different proteins with M I values of 47,000 and 20,000, which have been identified as the substrates for protein kinase C and myosin light chain kinase, respectively (Fig. 6A, lanes 4-6) (46,52). These results are consistent with earlier observations (52, 66). Among these phosphorylated proteins, smg p21 phosphorylated by protein kinase A in a cell-free system comigrated with the M , 22,000 protein phosphorylated in response to PGE, or Bt2cAMP in intact platelets (Fig. 6B).

The M I 22,000 protein phosphorylated in response to PGE, and Bt2cAMP in intact platelets also comigrated with smg p21 phosphorylated in a cell-free system as estimated by O'Farrell's two-dimensional PAGE (Fig. 7). Two radioactive spots with the same M , value but different isoelectric points were observed for phosphorylated smg p21 (Fig. 7A). Fig. 7, B and C, shows the autoradiographs of the proteins phosphorylated in response to PGE, in intact platelets. The M I 22,000 protein also appeared in two spots of which positions were similar to those of phosphorylated smg p21. Moreover, the phosphorylated M I 22,000 protein and smgp21 comigrated (Fig. 70 ) . Similar results were obtained when intact platelets were stimulated by Bt2cAMP instead of PGE, (data not

25

rL 36-

20.1- 1 2 3 4 5 6

B 4 2 4 0

h

& 66- - r' 36-

20.1 - +- 24 + 22

-50 - X

2-

+24 4.22

1 2 3 FIG. 6. One-dimensional SDS-PAGE analysis of the phos-

phorylation of smg p21 in response to PGEl and BtzcAMP in intact human platelets. For the analysis of protein phosphorylation in an intact cell system, the platelets labeled with 32Pi were incubated in the absence of 1 mM CaClz either with 20 p M PGEl for 3 min or 3 mM Bt2cAMP for 10 min, or in the presence of 1 mM CaCI2 either with thrombin (0.04 unit/l X 10' platelets) for 40 s, 1 p M A23187 for 10 s, or 160 nM TPA for 30 s. Aliquots (20 pg of protein) .of the proteins phosphorylated in intact platelets were subjected to one- dimensional SDS-PAGE followed by autoradiography. For the analysis of protein phosphorylation in a cell-free system, purified platelet smg p21 (500 ng of protein) was fully phosphorylated by the catalytic subunit of protein kinase A (380 ng of protein). Aliquots (60 ng of protein) of smg p21 phosphorylated by protein kinase A in a cell-free system were subjected to SDS-PAGE followed by autoradiography. A, autoradiographs of the proteins phosphorylated in intact platelets. Lune 1, control; lune 2, with PGE,; lune 3, with Bt2cAMP; lane 4, with thrombin; lune 5, with A23182 lune 6, with TPA. B, autoradiographs of the proteins phosphorylated in cell-free and intact platelet systems. Lane 1, smg p21 phosphorylated by protein kinase A in a cell-free system; lane 2, smg p21 phosphorylated by protein kinase A in a cell-free system plus the proteins phosphorylated in response to PGE, in intact platelets; lune 3, smg p21 phosphorylated by protein kinase A in a cell-free system plus the proteins phosphorylated in response to Bt2cAMP in intact platelets. Arrows indicate the proteins phosphorylated in response to CAMP in intact platelets.

shown). These results indicate that the M , 22,000 protein phosphorylated in response to PGE, and Bt2cAMP in intact human platelets is smg p21. The reason for the appearance of the two spots in Fig. 7 is not known at present, but it is possible that smg p21 is phosphorylated at two sites, since two-dimensional peptide maps also showed two spots (see Fig. 5). We are currently determining the site(s) of the phosphorylation of smg p21 by protein kinase A.

Comparison of the Peptide Maps of smgp21 Phosphorylated by Protein Kinase A in Cell-free and Intact Platelet Systems- The peptide maps of smg p21 phosphorylated by protein kinase A in cell-free and intact platelet systems were com-

Phosphorylation of smgp21 by Protein Kinase A 15693

FIG. 7. Two-dimensional PAGE analysis of the phosphorylation of smg p21 in response to PGE, in intact human platelets. The phosphorylation of human platelet smg p21 by protein kinase A in cell-free and intact cell systems was performed under the same conditions as described in the legend to Fig. 6. smg p21 (60 ng of protein) and the platelet proteins (10 pg of protein) were subjected to two-dimensional PAGE followed by autoradiography. Arrows indicate smg p21 and the M, 22,000 protein of intact platelets. A, smg p21 phosphorylated by protein kinase A in a cell-free system; B, the proteins phosphorylated in intact platelets as a control; C, the proteins phosphorylated in response to PGEl in intact platelets; D, srng p21 phosphorylated by protein kinase A in a cell-free system plus the proteins phosphorylated in response to PGE, in intact platelets.

! f - 5 .

Acidic 4 t Basic Acidic 4 e Basic

pared. Purified smg p21 was first fully phosphorylated by protein kinase A. In a separate experiment, 32P-labeled platelets were stimulated by PGE,. The samples were then subjected to SDS-PAGE. The proteins corresponding to smg p21 were extracted and digested completely by TPCK-treated trypsin. The digested samples were then subjected to two- dimensional peptide mapping followed by autoradiography. Fig. 8 shows the two-dimensional peptide maps of smg p21 phosphorylated in cell-free and intact cell systems. Similar spots were obtained between the two peptide maps. An identical peptide map was also obtained when the peptides from smg p21 phosphorylated in cell-free and intact cell systems were subjected together to two-dimensional peptide mapping (data not shown). These results indicate that smg p21 is phosphorylated at the same site(s) both in cell-free and intact cell systems.

DISCUSSION

The results presented in this paper indicate that smg p21 purified from bovine brain membranes is phosphorylated by protein kinase A but not by protein kinase C in a cell-free system. Our sample of the catalytic subunit of protein kinase A is more than 95% pure as judged by protein staining on an SDS-polyacrylamide gel. The phosphorylation of smg p21 by the catalytic subunit of protein kinase A is inhibited by its specific heat-stable protein inhibitor. smg p21 is phosphorylated by the holoenzyme of protein kinase A in a CAMP- dependent manner. It is most likely from these results that smg p21 is directly phosphorylated by protein kinase A.

In this paper, we have also shown that human platelet smg p21 is directly phosphorylated by protein kinase A in response to PGE, and BtzcAMP in intact human platelets. It has been shown previously that at least four proteins with apparent MI values of 240,000, 50,000, 24,000, and 22,000 are phosphorylated in response to CAMP-elevating agents in intact platelets (46, 52). Among these four proteins, the M , 240,000 protein has been suggested to be an actin-binding protein, probably filamin (67). The M , 22,000 protein has been partially purified and called thrombolamban (68), but nothing about GTP-

binding activity of this protein has been reported. The other two proteins have not been identified. We have shown previously that the M , 22,000 G protein purified from human platelets is identical to smg p21 purified from bovine brain from their physical and kinetic properties (42) and that this human platelet smg p21 is phosphorylated by protein kinase A when tested in a cell-free system (59). In this paper, we have confirmed the earlier observation that the four proteins described above are phosphorylated in response to PGE, and Bt2cAMP in intact human platelets. We have also shown that the MI 22,000 protein phosphorylated in response to PGEl and Btz cAMP in intact platelets comigrates with smg p21 phosphorylated by protein kinase A in a cell-free system on one- and two-dimensional PAGE. Moreover, we have shown that the tryptic peptide map of the M, 22,000 protein phosphorylated in response to PGEl in intact platelets is identical to that of smg p21 phosphorylated by protein kinase A in a cell-free system. These results indicate that the M, 22,000 protein phosphorylated in response to CAMP-elevating agents in intact platelets described previously (52) is identical to smg p21 and that smg p21 is directly phosphorylated by protein kinase A that is activated by cAMP during the action of PGEl or by Bt2cAMP.

The definitive physiological function of the phosphorylation of smg p21 by protein kinase A remains to be clarified. However, it has been suggested that the M, 22,000 protein is involved in the regulation of Ca2+ uptake across microsomal membranes and that the protein kinase A-mediated phosphorylation of this protein increases the Ca2+ uptake (69,70). Ca2+ and protein kinase C have been shown to be involved in secretion and aggregation of platelets (46). One of the inhib- itory actions of cAMP has been suggested to be due to the phosphorylation of the M , 24,000 and 22,000 proteins (52). On the other hand, there is another line of evidence that GTP enhances Ca2+ release from microsomes in liver and other tissues (see Ref. 71 for review; 72-75). On the basis of these earlier observations, it could be speculated that smg p21 is involved in the release of Ca2+ and that phosphorylation of this protein by protein kinase A inhibits this function. If this

15694 Phosphorylation of smgp21 by Protein Kinase A

- + Electrophoresis

FIG. 8. Comparison of the peptide maps of human platelet srng p21 phosphorylated by protein kinase A in cell-free and intact platelet systems. Purified platelet smg p21 (170 ng of protein) was first fully phosphorylated by protein kinase A (130 ng of protein) in a cell-free system. In a separate experiment, 32P-labeled platelets were stimulated by 20 PM PGE, for 3 min. All the samples were subjected to SDS-PAGE. The proteins corresponding to smg p21 were extracted from the gel and digested completely with TPCK- treated trypsin. Aliquots (2000 cpm) of each sample were then subjected to two-dimensional peptide mapping followed by autoradiography. Symbols are the same as those used in Fig. 5. A, smg p21 phosphorylated by protein kinase A in a cell-free system; B, smg p21 phosphorylated in response to PGE, in intact platelets.

is the case, smg p21 may be involved in the activation of platelets at the processes occurring before or after the protein kinase C/Ca2' systems. Since the phosphorylation of smg p21 affects neither its [3sS]GTPyS-binding nor GTPase activity, it is conceivable that the phosphorylation may affect the interaction of smg p21 with its regulatory or effector proteins.

The modes of activation and action of smg p21 are not known at present, but it is conceivable by analogy with ras p21s that there are GDP-bound inactive and GTP-bound active forms of smg p21, a protein(s) converting the inactive form to the active form, and an effector protein(s) whose function(s) are modulated by the active form of smg p21. The fact that smg p21 has the same putative effector domain as ras p21s implies that smg p21 shares the same effector protein(s) with ras p21s (22, 23, 31). A novel regulatory protein for c-ras p21s, designated as a GTPase-activating protein, has been identified (76). Subsequently, ras p21 GTPase-activating protein has been purified to near homogeneity from bovine brain (77). Recently, the cDNA of ras p21 GTPase-activating protein has been isolated, and its primary structure has been determined (78, 79). ras p21 GTPase-activating protein stim- ulates the GTPase activity of c-ras p21s but not that of the activated ras p21 (76-78). Moreover, it has been shown that one amino acid substitution with another amino acid in the effector domain of ras p21 GTPase-activating protein makes

c-Ha-ras p21 insensitive to this GTPase-activating protein (78, 80, 81). From this result, GTPase-activating protein has been proposed to serve as an effector protein (78, 80, 81). If this is the case, smg p21 may share GTPase-activating protein as a target protein with ras p21s. However, we have not examined this possibility since we have not yet purified ras p21 GTPase-activating protein. Instead, we have recently partially purified from human platelets and bovine brain two GTPase-activating proteins specific for smg p21 which are distinct from ras p21 GTPase-activating protein (82,83). Our preliminary analysis has shown that the phosphorylation of smg p21 does not affect the stimulation of GTPase activity of smg p21 by its specific GTPase-activating protein^.^ The phosphorylation of smg p21 may affect its interaction with regulatory or effector proteins other than GTPase-activating proteins.

In the present paper, we have also shown that other small M, G proteins, which have been purified partially and to near homogeneity from bovine brain membranes, are not phosphorylated by protein kinase A or C. The failure of protein kinase A or C to phosphorylate small M , G proteins other than smg p21 and c-Ki-ras p21 is not simply due to some interfering materials contaminating the sample of each small M, G protein since smg p21 and c-Ki-ras p21 are phosphorylated by protein kinase A in the presence of the samples of other small M, G proteins (data not shown). However, the possibility that other small M, G proteins are phosphorylated by protein kinases A and/or C under the assay conditions other than those used in this paper cannot be completely neglected. In the case of c-Ki-ras p21, we have shown here that about 0.1 mol of phosphate is maximally incorporatedl mol of this protein by protein kinase A but not by protein kinase C when tested in a cell-free system. It has been shown previously that c-Ki-ras p21 is phosphorylated by both protein kinases C and A in both intact cell and cell-free systems (47), that v-Ha-ras p21 is phosphorylated by protein kinase C in a cell-free system (48), that c-Ki- and c-Ha-ras p21s are phosphorylated by both protein kinases C and A in both intact cell and cell-free systems (49), that the yeast RASB protein is phosphorylated by protein kinase A in a cell-free system (50), and that the yeast RASl and RASB proteins are phosphorylated in an intact cell system (51). In these earlier experiments, c-Ki-ras p21 immunoprecipitated from mouse adre- nocortical tumor cell line, Y, (47); v-Ha-ras p21 immunoprecipitated from Escherichia coli (48); c-Ki-ras p21 overproduced in murine early myeloid cell line, 418B; c-Ha- ras p21 purified from E. coli (49); the RAS2 protein purified from E. coli (50); and the RASl and RAS2 proteins overproduced in yeast (51) were used. The reason for the different results between their studies and ours concerning the phosphorylation of ras p21s by protein kinase C is not known. The reason for the low stoichiometry for the phosphorylation of bovine brain c-Ki-ras p21 by protein kinase A is not known either but is not simply due to the endogenous phosphorylation of our sample since treatment of the sample with alkaline phosphatase does not increase this phosphorylation. Since the detailed enzymatic studies on the phosphorylation of ras p21s and the RAS proteins including the stoichiometry of phosphorylation by protein kinase A have not been shown in the earlier papers reported by other investigators, further investigation is necessary for understanding the physiological significance of the phosphorylation of this group of small M , G proteins.

' Y. Hata, T. Sasaki, A. Kikuchi, and Y. Takai, unpublished observation.

Phosphorylation of smg p21 by Protein Kinase A 15695

Acknowledgments-We are grateful to J u n k o Y a m a g u c h i and Fu- miko H iguch i for the i r skillful secretar ia l ass is tance.

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