p120c-cbl is present in human blood platelets and is differentially involved in signaling by thrombopoietin and thrombin.
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 p120c-cbl, a protein known to be involved in signaling by many growth factors. Specific antisera against p120c-cbl recognized the same 120-kD protein in lysates of Jurkat cells, which are known to express p120c-cbl, and platelets, indicating that platelets have p120c-cbl. Thrombopoietin induced rapid tyrosine phosphorylation of p120c-cbl in platelets. Thrombopoietin also induced tyrosine phosphorylation of p120c-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 p120c-cbl was observed upon treatment of these cells with thrombopoietin, indicating that in this system tyrosine phosphorylation of p120c-cbl may not be essential for cell proliferation. This suggests that tyrosine phosphorylation of p120c-cbl may be required for nonmitogenic responses induced by thrombopoietin in postmitotic cells such as platelets. On the other hand, p120c-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. p120c-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 p120c-cbl is an endogenous substrate for calpain, a protease that may play a role in postaggregation signaling processes. Our data suggest that p120c-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.
Oda
A
A
Department of Internal Medicine, Keio University, Tokyo, Japan.
Ozaki
K
K
Druker
B J
BJ
Miyakawa
Y
Y
Miyazaki
H
H
Handa
M
M
Morita
H
H
Ohashi
H
H
Ikeda
Y
Y
D048868:Adaptor Proteins, Signal Transducing; D001792:Blood Platelets / Q000502:physiology*; D002154:Calpain / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism; D051380:GRB2 Adaptor Protein; D006801:Humans; D046911:Macromolecular Substances; D008970:Molecular Weight; D009363:Neoplasm Proteins; D010750:Phosphoproteins / Q000378:metabolism; D019000:Phosphotyrosine / Q000378:metabolism; D010974:Platelet Aggregation; D011506:Proteins / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism / Q000502:physiology*; D050721:Proto-Oncogene Proteins c-cbl; D018121:Receptors, Cytokine; D018179:Receptors, Thrombin / Q000502:physiology*; D053628:Receptors, Thrombopoietin; D015398:Signal Transduction; D013926:Thrombopoietin / Q000502:physiology*; D044767:Ubiquitin-Protein Ligases
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D046911:Macromolecular Substances; D009363:Neoplasm Proteins; D010750:Phosphoproteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D018121:Receptors, Cytokine; D018179:Receptors, Thrombin; D053628:Receptors, Thrombopoietin; C082218:MPL protein, human; D019000:Phosphotyrosine; D013926:Thrombopoietin; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D066246:ErbB Receptors; D002154:Calpain; C067419:CBL protein, human