The SH2-B Adaptor Protein Negatively Regulates EPO-Dependent Signalling Via Interaction with Erythropoietin Receptor Ptyr-343

Erythropoiesis is a developmentally important process, whereby multipotent hematopoietic stem cells differentiate into mature erythrocytes. Erythropoietin (EPO) is a critical regulator in this process and mediates its signal via the erythropoietin receptor (EPO-R) and the primary associated tyrosine kinase, JAK2. EPO, EPO-R and JAK2 play a crucial role in erythropoiesis, as deficiency in any of these proteins results in an embryonic lethal anemia. Structural-functional studies and murine knock-in models have shown EPO-R pTyr-343 to play a critical role in EPO mediated signalling. STAT-5 is activated by EPO-R pTyr-343, but STAT5^ΔN mice do not have any profound erythroid abnormalities. Such evidence has led our group to hypothesize that other SH2 containing effectors interact with EPO-R pTyr-343. Cloning of Ligand Target screening was utilized to demonstrate that EPO-R pTyr-343 binds to adaptor protein SH2-Bβ. SH2-B contains multiple protein-protein interaction domains including multiple proline-rich regions, a PH domain and an SH2 domain. Although SH2-B does play a role in a number of signaling pathways, it is not required for embryonic development. Since SH2-B is a potent regulator of JAK2 in context of Growth Hormone and Leptin signaling, and it can directly interact with EPO-R pTyr-343, we hypothesize that SH2-B functions as an important adaptor protein downstream of the EPO-R.H2-B constitutively associates to the inactive EPO-R, an interaction that is independent of JAK2 binding to the EPO-R. Upon EPO stimulation, enhanced SH2-dependent binding of SH2-B to pTyr-343 and pTyr-401 of the EPO-R was confirmed utilizing a panel of EPO-R truncation mutants. The EPO mediated interaction between SH2-B and activated EPO-R is both dose and time dependent. EPO stimulation also results in SH2-B serine and threonine phosphorylation. Importantly, the interaction of SH2-B and EPO-R was observed in erythroid cell lines and primary murine splenocytes. The function of SH2-B in EPO signaling was investigated via knocking down SH2-B in Ba/F3-EPO-R cells. Knock down of SH2-B results in hypersensitive EPO-dependent phosphorylation of multiple targets including the EPO-R, JAK2, STAT5 and Erk1/2. It is evident that SH2-B is a global negative regulator of EPO-dependent signaling via its ability to affect EPO-dependent JAK2 activation.