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A ras-related protein is phosphorylated and translocated by agonists that increase cAMP levels in human platelets
E G Lapetina1, J C Lacal, B R Reep
1Division of Cell Biology, Burroughs Wellcome Company, Research Triangle Park, NC 27709.
Abstract:
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.
Insights
Platelet proteins were analyzed for ras-encoded p21 interactions. A 22-kDa protein binds GTP and is recognized by mAb M90, with phosphorylation converting it to a 24-kDa form.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Signaling
Background:
- Platelets contain proteins that interact with ras-encoded proteins.
- Monoclonal antibodies (mAbs) are crucial tools for identifying specific protein epitopes.
Purpose of the Study:
- To investigate the presence and function of ras-encoded p21 proteins in human platelets.
- To characterize a specific platelet protein recognized by mAb M90 and its interaction with GTP.
- To examine the effect of iloprost on this platelet protein.
Main Methods:
- Assaying platelet protein antigenicity with monoclonal antibodies (mAbs).
- Western blotting and GTP-binding assays for protein characterization.
- Analysis of protein changes in response to iloprost treatment.
- Phosphorylation studies using 32P labeling and immunoprecipitation.
Main Results:
- A 22-kDa platelet protein was identified, binding [alpha-32P]GTP and recognized by mAb M90.
- Iloprost treatment induced a time-dependent increase of a 24-kDa protein recognized by mAb M90, with a concomitant decrease in the 22-kDa protein.
- The 24-kDa protein was identified as a phosphorylated form of the 22-kDa protein.
Conclusions:
- Human platelets contain a ras-related GTP-binding protein (22-kDa).
- Iloprost treatment induces phosphorylation of this protein, converting it to a 24-kDa form.
- This phosphorylation event alters the protein's molecular mass and may be involved in platelet signaling pathways.