CrkL is recruited through its SH2 domain to the erythropoietin receptor and plays a role in Lyn-mediated receptor signaling

The erythropoietin (Epo) receptor transduces its signals by activating phys ically associated tyrosine kinases, mainly Jak2 and Lyn, and thereby induci ng tyrosine phosphorylation of various substrates including the Epo recepto r (EpoR) itself. We previously demonstrated that, in Epo-stimulated cells, an adapter protein, CrkL, becomes tyrosine-phosphorylated, physically assoc iates with Shc, SHP-2, and Cb1, and plays a role in activation of the Ras/E rk signaling pathway. Here, we demonstrate that Epo induces binding of CrkL to the tyrosine-phosphorylated EpoR and SHIP1 in 32D/EpoR-Wt cells overexp ressing CrkL. In vitro binding studies showed that the CrkL SH2 domain dire ctly mediates the EpoR binding, which was specifically inhibited by a synth etic phosphopeptide corresponding to the amino acid sequences at Tyr(460) i n the cytoplasmic domain of EpoR. The CrkL SH2 domain was also required for tyrosine phosphorylation of CrkL in Epo-stimulated cells. Overexpression o f Lyn induced constitutive phosphorylation of CrkL and activation of Erk, w hereas that of a Lyn mutant lacking the tyrosine kinase domain attenuated t he Epo-induced phosphorylation of CrkL and activation of Erk. Furthermore, Lyn, but not Jak2, phosphorylated CrkL on tyrosine in in vitro kinase assay s. Together, the present study suggests that, upon Epo stimulation, CrkL is recruited to the EpoR through interaction between the CrkL SH2 domain and phosphorylated Tyr(460) in the EpoR cytoplasmic domain and undergoes tyrosi ne phosphorylation by receptor-associated Lyn to activate the downstream si gnaling pathway leading to the activation of Erk and Elk-1.