To investigate the signaling processes induced by recombinant thrombopoietin, we used human platelets to recently show that thrombopoietin induces rapid tyrosine phosphorylation of Jak2, Tyk2, Shc, Stat3, Stat5, and other proteins in human platelets. Because the apparent molecular weight of a major tyrosine-phosphorylated protein in platelets stimulated by thrombopoietin is approximately 120 kD, we examined the possibility that this could be p120(c-cbl), a protein known to be involved in signaling by many growth factors. Specific antisera against p120(c-cbl) recognized the same 120-kD protein in lysates of Jurkat cells, which are known to express p120(c- cbl), and platelets, indicating that platelets have p120(c-cbl). Thrombopoietin induced rapid tyrosine phosphorylation of p120(c-cbl) in platelets. Thrombopoietin also induced tyrosine phosphorylation of p120(c- cbl) in FDCP cells genetically engineered to express the thrombopoietin receptor, c-Mpl. Interestingly, FDCP cells, expressing a truncated c-Mpl devoid of the box-2 domain, proliferate in response to thrombopoietin. However, no increase in tyrosine phosphorylation of p120(c-cbl) was observed upon treatment of these cells with thrombopoietin, indicating that in this system tyrosine phosphorylation of p120(c-cbl) may not be essential for cell proliferation. This suggests that tyrosine phosphorylation of p120(c-cbl) may be required for nonmitogenic responses induced by thrombopoietin in postmitotic cells such as platelets. On the other hand, p120(c-cbl) was not significantly tyrosine-phosphorylated upon treatment of platelets with thrombin. However, it became incorporated into the Triton X-100-insoluble, 10,000g-sedimentable residue in an aggregation-dependent manner, suggesting that it may have a regulatory role in platelet cytoskeletal processes. p120(c-cbl) was constitutively associated with a 28-kD adapter protein, Grb2, that was also incorporated into the Triton X-100-insoluble, sedimentable residue dependent on aggregation. Further, we found that p120(c-cbl) is an endogenous substrate for calpain, a protease that may play a role in postaggregation signaling processes. Our data suggest that p120(c-cbl) may be involved in signal transduction following ligand binding to c-Mpl through its inducible tyrosine phosphorylation, and it may also be involved in signaling during platelet aggregation by its redistribution to the cytoskeleton.
|Original language||English (US)|
|Number of pages||9|
|State||Published - Aug 15 1996|
ASJC Scopus subject areas
- Cell Biology