The SH2B1 adaptor protein associates with a proximal region of the erythropoietin receptor.
Gene targeting experiments have shown that the cytokine erythropoietin (EPO), its cognate erythropoietin receptor (EPO-R), and associated Janus tyrosine kinase, JAK2, are all essential for erythropoiesis. Structural-functional and murine knock-in experiments have suggested that EPO-R Tyr-343 is important in EPO-mediated mitogenesis. Although Stat5 binds to EPO-R phosphotyrosine 343, the initial Stat5-deficient mice did not have profound erythroid abnormalities suggesting that additional Src homology 2 (SH2) domain-containing effectors may bind to EPO-R Tyr-343 and couple to downstream signaling pathways. We have utilized cloning of ligand target (COLT) screening to demonstrate that EPO-R Tyr(P)-343 and Tyr(P)-401 bind to the SH2 domain-containing adaptor protein SH2B1β. Immunoprecipitation and in vitro mixing experiments reveal that EPO-R binds to SH2B1 in an SH2 domain-dependent manner and that the sequence that confers SH2B1 binding to the EPO-R is pYXXL. Previous studies have shown that SH2B1 binds directly to JAK2, but we show that in hematopoietic cells, SH2B1β preferentially associates with the EPO-R. SH2B1 is capable of constitutive association with EPO-R, which is necessary for its optimal SH2-dependent recruitment to EPO-R-Tyr(P)-343/Tyr(P)-401. We also demonstrate that SH2B1 is responsive to EPO stimulation and becomes phosphorylated, most likely on serines/threonines, in an EPO dose- and time-dependent manner. In the absence of SH2B1, we observe enhanced activation of signaling pathways downstream of the EPO-R, indicating that SH2B1 is a negative regulator of EPO signaling.
Ontario Cancer Institute, Campbell Family Cancer Research Institute, Toronto, Ontario M5G 2M9, Canada.
Mojib
M
Javadi
Edda
E
Hofstätter
Natalie
N
Stickle
Bryan K
BK
Beattie
Robert
R
Jaster
Christin
C
Carter-Su
Dwayne L
DL
Barber
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D004900:Erythroblasts / Q000378:metabolism; D004921:Erythropoietin / Q000502:physiology; D006801:Humans; D047468:Immunoprecipitation; D051379:Mice; D008810:Mice, Inbred C57BL; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D061251:Primary Cell Culture; D054730:Protein Interaction Domains and Motifs; D011499:Protein Processing, Post-Translational; D017467:Receptors, Erythropoietin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D015398:Signal Transduction
D048868:Adaptor Proteins, Signal Transducing; D010446:Peptide Fragments; D017467:Receptors, Erythropoietin; C115327:Sh2bpsm1 protein, mouse; D004921:Erythropoietin