A ras-related protein is phosphorylated and translocated by agonists that increase cAMP levels in human platelets.
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 of the platelet 22-kDa protein to nitrocellulose paper showed that the protein binds [alpha-32P]GTP. Moreover, preincubation of the transferred protein with mAb M90 drastically reduced its GTP-binding activity. Treatment of platelets with iloprost, 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.
Lapetina
E G
EG
Division of Cell Biology, Burroughs Wellcome Company, Research Triangle Park, NC 27709.
Lacal
J C
JC
Reep
B R
BR
Molina y Vedia
L
L
D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001665:Binding Sites; D001692:Biological Transport; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000097:blood*; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000276:immunology; D011464:Epoprostenol / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000097:blood; D006801:Humans; D016285:Iloprost; D007163:Immunosorbent Techniques; D008565:Membrane Proteins; D008970:Molecular Weight; D010766:Phosphorylation; D011494:Protein Kinases / Q000097:blood; D011518:Proto-Oncogene Proteins / Q000097:blood*; D016283:Proto-Oncogene Proteins p21(ras)
D000911:Antibodies, Monoclonal; D000939:Epitopes; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D006160:Guanosine Triphosphate; D011464:Epoprostenol; D000242:Cyclic AMP; D011494:Protein Kinases; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D016285:Iloprost