Phosphorylation of smg p21, a ras p21-like GTP-binding Protein, by Cyclic AMP-dependent Protein Kinase in a Cell-free System and in Response to Prostaglandin E1 in Intact Human Platelets

M Kawata,A Kikuchi,M Hoshijima,K Yamamoto,E Hashimoto,H Yamamura,Y Takai

doi:10.1016/s0021-9258(19)84887-1

Abstract

We have separated multiple small Mr GTP-binding proteins (G proteins) from bovine brain membranes by several column chromatographies and purified to near homogeneity four of them, including a novel Mr 24,000 G protein (smg p25A), a novel Mr 22,000 G protein (smg p21), the rho protein (rho p20), and the c-Ki-ras protein (c-Ki-ras p21). Among these small Mr G proteins, only smg p21 is phosphorylated stoichiometrically by cAMP-dependent protein kinase (protein kinase A), and c-Ki-ras 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 Mr G proteins is phosphorylated by protein kinase A. Neither smg p21 nor other small Mr 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-O-(thio) triphosphate (GTP gamma S)-bound and GDP-bound forms of smg p21 are phosphorylated with the same reaction velocity. The phosphorylation of smg p21 affects neither its GTP gamma S-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 p21 in a cell-free system. When intact human platelets are stimulated by prostaglandin E1 known to elevate the cAMP level, four proteins with apparent Mr 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-O-tetradecanoylphorbol-13-acetate. Among the four proteins, the Mr 22,000 protein is identified as smg p21. The site(s) of phosphorylation of smg p21 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 Mr G proteins, smg p21 is selectively phosphorylated by protein kinase A and that this G protein is also phosphorylated by this protein kinase in response to prostaglandin E1 in intact human platelets.

Highlights

Gprotein, a novel M, 22,000 G protein, therho protein, and the c-Ki-rus protein (c-Ki-rus p21)
Phosphorylation of Bovine Brain smgp2b1y 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 subjected to SDS-PAGE followed by autoradiography, a radioactive band was observed at theposition with an M, slightly larger than 22,000 (Fig.1, lane 2 )
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

Summary

Introduction

Gprotein (smg p25A), a novel M , 22,000 G protein (smgp21), therho protein (rho p20), and the c-Ki-rus protein (c-Ki-rus p21). Phosphorylation of Bovine Brain smgp2b1y 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 subjected to SDS-PAGE followed by autoradiography, a radioactive band was observed at theposition with an M , slightly larger than 22,000 (Fig.1, lane 2 ) .

Results

Conclusion