Discrete protein interactions with the Grb2/c-Cbl complex in SCF- and TPO-mediated myeloid cell proliferation.
Hemopoietic cell proliferation is mediated by non-tyrosine and tyrosine kinases that signal via uncommon and common sets of downstream effector molecules including the Grb2/c-Cbl. In the present study we evaluated tyrosine phosphorylation of c-Cbl and the interaction of the Grb2/c-Cbl complex with signaling proteins upon activation of non-tyrosine (c-Mpl) and tyrosine kinase (c-Kit) receptors leading to myeloid cell proliferation. By using the growth factor dependent M-07e cell line, we found that both c-Mpl and c-Kit ligands, namely: SCF and TPO, induce c-Cbl tyrosine phosphorylation. In these cells the adaptor protein Grb2 constitutively binds a substantial fraction of c-Cbl through the N-terminal SH3 domain. In vitro experiments showed that the stable Grb2/c-Cbl complex interacts, through the Grb2 SH2 domain, with the SCF-activated c-Kit. By contrast stimulation with TPO leads to the formation of a Grb2 complex containing JAK2. In vitro and in vivo experiments support the hypothesis that Grb2 mediates the association of c-Kit with c-Cbl. Moreover we found that, upon SCF stimulation, the Grb2/c-Cbl complex recruits Shc, probably via Grb2. By contrast the Ras exchanger factor (Sos1) was not detected in anti-c-Cbl immunoprecipitates suggesting that Grb2/Sos1 and Grb2/c-Cbl are present in different complexes. Taken together our results demonstrate that c-Cbl plays an important role in coupling both tyrosine and non-tyrosine kinase receptors to downstream effector molecules and that different signaling molecules interact with Grb2/c-Cbl complex when non-tyrosine or tyrosine kinase receptors are activated.
Brizzi
M F
MF
Dipartimento di Medicina Interna, Università di Torino, Italy.
Dentelli
P
P
Lanfrancone
L
L
Rosso
A
A
Pelicci
P G
PG
Pegoraro
L
L
D048868:Adaptor Proteins, Signal Transducing; D002460:Cell Line; D051380:GRB2 Adaptor Protein; D006801:Humans; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D050721:Proto-Oncogene Proteins c-cbl; D019009:Proto-Oncogene Proteins c-kit / Q000378:metabolism*; D019089:Stem Cell Factor / Q000494:pharmacology*; D013926:Thrombopoietin / Q000494:pharmacology*; D014443:Tyrosine / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D011506:Proteins; D011518:Proto-Oncogene Proteins; D019089:Stem Cell Factor; D019000:Phosphotyrosine; D014443:Tyrosine; D013926:Thrombopoietin; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D019009:Proto-Oncogene Proteins c-kit; C067419:CBL protein, human