Loss of SHIP and CIS recruitment to the granulocyte colony-stimulating factor receptor contribute to hyperproliferative responses in severe congenital neutropenia/acute myelogenous leukemia.

Abstract

Mutations in the G-CSF receptor (G-CSFR) in patients with severe congenital neutropenia (SCN) are postulated to contribute to transformation to acute myelogenous leukemia (AML). These mutations result in defective receptor internalization and sustained cellular activation, suggesting a loss of negative signaling by the G-CSFR. In this paper we investigated the roles of SHIP and cytokine-inducible Src homology 2 protein (CIS) in down-modulating G-CSFR signals and demonstrate that loss of their recruitment as a consequence of receptor mutations leads to aberrant signaling. We show that SHIP binds to phosphopeptides corresponding to Tyr744 and Tyr764 in the G-CSFR and that Tyr764 is required for in vivo phosphorylation of SHIP and the formation of SHIP/Shc complexes. Cells expressing a G-CSFR form lacking Tyr764 exhibited hypersensitivity to G-CSF and enhanced proliferation, but to a lesser degree than observed with the most common mutant G-CSFR form in patients with SCN/AML, prompting us to investigate whether suppressor of cytokine signaling proteins also down-modulate G-CSFR signals. G-CSF was found to induce the expression of CIS and of CIS bound to phosphopeptides corresponding to Tyr729 and Tyr744 of the G-CSFR. The expression of CIS was prolonged in cells with the SCN/AML mutant G-CSFR lacking Tyr729 and Tyr744, which also correlated with increased G-CSFR expression. These findings suggest that SHIP and CIS interact with distal phosphotyrosine residues in the G-CSFR to negatively regulate G-CSFR signaling by limiting proliferation and modulating surface expression of the G-CSFR, respectively. Novel therapeutic approaches targeting inhibitory pathways that limit G-CSFR signaling may have promise in the treatment of patients with SCN/AML.