Abstract
The cytokine-inducible SH2 protein-3 (CIS3/SOCS-3/SSI-3) has been shown to inhibit the JAK/STAT pathway and act as a negative regulator of fetal liver erythropoiesis. Here, we studied the molecular mechanisms by which CIS3 regulates the erythropoietin (EPO) receptor (EPOR) signaling in erythroid progenitors and Ba/F3 cells expressing the EPOR (BF-ER). CIS3 binds directly to the EPOR as well as JAK2 and inhibits EPO-dependent proliferation and STAT5 activation. We have identified the region containing Tyr(401) in the cytoplasmic domain of the EPOR as a direct binding site for CIS3. Deletion of the Tyr(401) region of the EPOR reduced the inhibitory effect of CIS3, suggesting that binding of CIS3 to the EPOR augmented the negative effect of CIS3. Both N- and C-terminal regions adjacent to the SH2 domain of CIS3 were necessary for binding to EPOR and JAK2. In the N-terminal region of CIS3, the amino acid Gly(45) was critical for binding to the EPOR but not to JAK2, while Leu(22) was critical for binding to JAK2. The mutation of G45A partially reduced ability of CIS3 to inhibit EPO-dependent proliferation and STAT5 activation, while L22D mutant CIS3 was completely unable to suppress EPOR signaling. Moreover, overexpression of STAT5, which also binds to Tyr(401), reduced the binding of CIS3 to the EPOR, and the inhibitory effect of CIS3 against EPO signaling, while it did not affect JAB/SOCS-1/SSI-1. These data demonstrate that binding of CIS3 to the EPOR augments the inhibitory effect of CIS3. CIS3 binding to both EPOR and JAK2 may explain a specific regulatory role of CIS3 in erythropoiesis.
Highlights
Erythropoietin (EPO)1 is a glycoprotein hormone required for the survival, proliferation, and differentiation of committed erythroid progenitor cells
The critical amino acids necessary for the tight binding to JAK2 and the inhibition of kinase activity were located in the kinase inhibitory region (KIR) domain and were conserved between CIS3 and JAB (Fig. 1A)
CIS3 Binds to the EPO receptor (EPOR) and JAK2—Since CIS3 was indicated as a negative regulator in fetal erythropoiesis [15], we further investigated the precise mechanism of EPO signal inhibition by CIS3 using erythroid progenitor (CFU-E)-enriched splenocytes from phenylhydrazine-treated anemic mice
Summary
Erythropoietin (EPO) is a glycoprotein hormone required for the survival, proliferation, and differentiation of committed erythroid progenitor cells. Molecular Mechanism of EPO Signal Inhibition by CIS3/SOCS3 both the SH2 domain and the C-terminal region (CH domain/ SOCS box), and a data base search identified additional CIS family members (29 –31). Among these members, CIS3 is most similar to JAB. CIS3 and JAB had similar inhibitory effects on cytokine signaling in vivo, the affinity of CIS3 for binding to JAK2 was much weaker than that of JAB, and CIS3 hardly inhibited JAK2 kinase activity in vitro [38, 39] These data suggest that the mechanisms of action between CIS3 and JAB are different
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