The antigenicity of platelet proteins was assayed against various monoclonal antibodies (mAbs) that recognize specific epitopes of the ras-encoded p21 protein. mAb M90, which detects the region of p21 protein within amino acids 107-130 and inhibits its GTP-binding activity, strongly reacted with a 22-kDa protein present in the particulate fraction of human platelets. Other mAbs against ras-encoded proteins, including Y13-259, which efficiently detects ras proteins from a variety of organisms, did not recognize the platelet 22-kDa protein. Transfer ofthe platelet 22-kDa protein to nitrocellulose paper showed that the protein binds [a-32P]GTP. Moreover, preincubation of the transferred protein with mAb M90 drastically reduced its GTP-binding activity. Treatment of platelets with ioprost, a prostacyclin analog, caused (i) a time-dependent increase of a 24-kDa protein that is recognized by mAb M90 in particulate and cytosolic fractions and (ii) the gradual decrease of the 22-kDa protein from the particulate fraction. When platelets were labeled with 32P and then treated with iloprost, the 24-kDa protein was found to be phosphorylated. The 32P-labeled 24-kDa protein was specifically immunoprecipitated by mAb M90. These results suggest that appearance of the 24-kDa protein results from phosphorylation of the 22-kDa protein, which shifts its mobility to a higher molecular mass area.In the past two years platelets have been shown to possess distinct GTP-binding proteins with molecular masses between 21 and 31 kDa (1-3). These low molecular weight guanine nucleotide-binding regulatory (G) proteins can be electrophoretically transferred from SDS/polyacrylamide gels to nitrocellulose blots, where they are detected by the binding of [a-32P]GTP (2, 3). One of them, a 21-kDa protein, was recently isolated from platelet membranes; however, this protein is not recognized with monoclonal antibody (mAb) Y13-259 (4), which recognizes all known ras-encoded p21 proteins (1). Also, this antibody did not cross-react with the other low molecular mass G proteins present in platelets (3). The possible physiological significance of these proteins has not yet been elucidated; they could be correlated with regulation of phospholipase C (2) and, recently, we reported that a platelet ras-related protein is phosphorylated by a cAMP-dependent protein kinase (5).Lacal and Aaronson (6) have developed a series of mAbs that recognize epitopes located at different regions in the Harvey (Ha) ras-encoded p21 protein. We have screened platelet proteins against those mAbs and found that a 22-kDa platelet protein is recognized by two of those mAbs. These mAbs, designated M3 and M90, recognize a region of ras p21 protein containing residues 116-119, involved in the interaction with the guanine base (7). By contrast the 22-kDa protein was not recognized by the ras-specific mAb Y13-259. These results suggest that the 22-kDa platelet protein is a member ofthe ras family. Finally, the 22-kDa protein is a substrate for cAMP-dependent protein kinase. Ph...

Rho proteins are a branch of GTPases that belongs to the Ras superfamily which are critical elements of signal transduction pathways leading to a variety of cellular responses. This family of small GTPases has been involved in diverse biological functions such as cytoskeleton organization, cell growth and transformation, cell motility, migration, metastasis, and responses to stress. We report that several human Rho proteins including Rho A, Rho C and Rac 1, are capable of inducing apoptosis in dierent cell systems like murine NIH3T3 ®broblasts and the human erythroleukemia K562 cell line. Since K562 cells are devoid of p53, apoptosis induced by Rho in this system is independent of p53. Rho-dependent apoptosis is mediated by the generation of ceramides, and it is drastically inhibited by ectopic expression of Bcl2, both under in vitro and in vivo conditions. Furthermore, the human oncogenes vav and ost that have been shown to function as guanine exchange factors for Rho proteins, were also able to induce apoptosis under similar conditions. Finally, we also report that the levels of endogenous Rho proteins are increased when U937 myeloid leukemia cells are exposed to apoptosis-inducing conditions such as TNFa treatment. Furthermore, TNFa-induced apoptosis in these cells is inhibited by expression of a dominant negative mutant of Rac 1 but it is not aected by a similar mutant of Rho A. These results suggest that Rho proteins play an important role in the physiological regulation of the apoptotic response to stress-inducing agents.

Expression of the N-ras oncogene under the control of the glucocorticoid-responsive promoter in the pheochromocytoma cell line UR61, a subline of PC-12 cells, has been used to investigate the differentiation process to neuronal cells triggered by ras oncogenes (I. Guerrero, A. Pellicer, and D. E. Burstein, Biochem. Biophys. Res. Commun. 150:1185-1192, 1988). Using ras-inducible cell lines, we observed that expression of the oncogenic N-ras p21 protein interferes with the ability of phorbol esters to induce downregulation of protein kinase C. This effect was associated with the appearance of immunologically detectable protein kinase C as well as the activity of the enzyme as analyzed either by binding of [3H]phorbol-12,13-dibutyrate in intact cells or by in vitro kinase activity. These results indicate a relationship between ras p21 and protein kinase C in neuronal differentiation in this model system. Comparison to the murine fibroblast system suggests that this relationship may be functional.

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Platelet inhibition by agents that increase intracellular levels of cAMP is associated with cAMPdependent phosphorylation of specific intracellular proteins, including a membrane-associated 22-kDa microsomal protein called thrombolamban. In view of recent studies suggesting that platelets also contain 22-kDa GTP-binding proteins that are homologous with ras-encoded p21 proteins, the present work was undertaken to examine the possibility that thrombolamban and the Ras-like proteins were the same. Platelet microsomes were labeled with [y-32P]ATP and the labeled proteins were examined by autoradiography of sodium dodecyl sulfate/polyacrylamide gels. On Western blots of both onedimensional and two-dimensional gels, thrombolamban immunoreacted with M90, a monoclonal antibody that recognizes the GTP-binding domain of Ras p21 proteins, but not with Y13-259, a monoclonal antibody that recognizes another domain and is speciflc for Ras proteins. Overlay experiments with unlabeled platelet microsomes demonstrated numerous low molecular weight proteins that bound [a-32PJGTP, although none could be identified as thrombolamban. Finally, thrombolamban was immunoprecipitated by M90. These studies show that thrombolamban is a low molecular weight protein that is immunologically related to the Ras family of GTPbinding proteins.

Quiescent mouse NIH 3T3 cells responded to microinjection of activated ras p21 with a rapid and sustained rise in intracellular pH (approximately 0.17 pH units). The p21-induced pH change was inhibited by amiloride treatment or growth of cells in medium low in sodium, suggesting a role for the Na+/H+ antiporter. Amiloride was found to suppress p21-induced mitosis, also.

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