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 physically associated tyrosine kinases, mainly Jak2 and Lyn, and thereby inducing tyrosine phosphorylation of various substrates including the Epo receptor (EpoR) itself. We previously demonstrated that, in Epo-stimulated cells, an adapter protein, CrkL, becomes tyrosine-phosphorylated, physically associates with Shc, SHP-2, and Cbl, and plays a role in activation of the Ras/Erk signaling pathway. Here, we demonstrate that Epo induces binding of CrkL to the tyrosine-phosphorylated EpoR and SHIP1 in 32D/EpoR-Wt cells overexpressing CrkL. In vitro binding studies showed that the CrkL SH2 domain directly mediates the EpoR binding, which was specifically inhibited by a synthetic phosphopeptide corresponding to the amino acid sequences at Tyr(460) in the cytoplasmic domain of EpoR. The CrkL SH2 domain was also required for tyrosine phosphorylation of CrkL in Epo-stimulated cells. Overexpression of Lyn induced constitutive phosphorylation of CrkL and activation of Erk, whereas that of a Lyn mutant lacking the tyrosine kinase domain attenuated the Epo-induced phosphorylation of CrkL and activation of Erk. Furthermore, Lyn, but not Jak2, phosphorylated CrkL on tyrosine in in vitro kinase assays. 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 tyrosine phosphorylation by receptor-associated Lyn to activate the downstream signaling pathway leading to the activation of Erk and Elk-1.
Arai
A
A
Department of Hematology, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyoku, Tokyo 113, Japan.
Kanda
E
E
Nosaka
Y
Y
Miyasaka
N
N
Miura
O
O
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002460:Cell Line; D004921:Erythropoietin / Q000494:pharmacology; D017930:Genes, Reporter; D006801:Humans; D053614:Janus Kinase 2; D051379:Mice; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D020239:Receptor Cross-Talk / Q000502:physiology; D017467:Receptors, Erythropoietin / Q000737:chemistry / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection; D014443:Tyrosine; D018909:src Homology Domains; D019061:src-Family Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; C082869:CRKL protein; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D017467:Receptors, Erythropoietin; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D004921:Erythropoietin; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; C507926:Jak2 protein, mouse; D053614:Janus Kinase 2; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases