Suppressor of Cytokine Signaling-3 Is Recruited to the Activated Granulocyte-Colony Stimulating Factor Receptor and Modulates its Signal Transduction

Abstract

G-CSF is a polypeptide growth factor used in treatment following chemotherapy. G-CSF regulates granulopoiesis and acts on its target cells by inducing homodimerization of the G-CSFR, thereby activating intracellular signaling cascades. The G-CSFR encompasses four tyrosine motifs on its cytoplasmic tail that have been shown to recruit a number of regulatory proteins. Suppressor of cytokine signaling 3 (SOCS-3), also referred to as cytokine-inducible Src homolgy 2-containing protein 3, is a member of a recently discovered family of feedback inhibitors that have been shown to inhibit the Janus kinase/STAT pathway. In this study, we demonstrate that human SOCS-3 is rapidly induced by G-CSF in polymorphonuclear neutrophils as well as in the myeloid precursor cell line U937 and that SOCS-3 negatively regulates G-CSFR-mediated STAT activation. Most importantly, we show that SOCS-3 is recruited to the G-CSFR in a phosphorylation-dependent manner and we identify phosphotyrosine (pY)729 as the major recruitment site for SOCS-3. Furthermore, we demonstrate that SOCS-3 directly binds to this pY motif. Surface plasmon resonance analysis reveals a dissociation constant (KD) for this interaction of around 2.8 μM. These findings strongly suggest that the recruitment of SOCS-3 to pY729 is important for the modulation of G-CSFR-mediated signal transduction by SOCS-3.

SOCS3 Is a Physiological Negative Regulator for Granulopoiesis and Granulocyte Colony-stimulating Factor Receptor Signaling

“…The strength of cytokine signals is regulated, in part, by a family of endogenous JAK kinase inhibitor proteins referred to as suppressors of cytokine signaling (SOCS), cytokine-inducible Src homology 2 (SH2) proteins (CIS), or STAT-induced STAT inhibitors (SSI) (3)(4)(5)(6)(7). Among these, SOCS3 is strongly induced by a variety of cytokines and other stimulations, including IL-6, IL-10, granulocyte-colony stimulating factor (G-CSF), EPO, EGF, leptin, and LPS (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). Both SOCS1 and SOCS3 have an N-terminal kinase inhibitory region and inhibit JAK tyrosine kinase activity.…”

“…The G-CSF induces activation of the JAK-STAT (9,10,(15)(16)(17)(18) and the Ras-Raf-ERK pathways (18,19). Overexpression studies have suggested that SOCS3 interacts with the G-CSF receptor and inhibits G-CSFinduced STAT3 activation (9,20).…”

Loss of SHIP and CIS Recruitment to the Granulocyte Colony-Stimulating Factor Receptor Contribute to Hyperproliferative Responses in Severe Congenital Neutropenia/Acute Myelogenous Leukemia

“…SHP-1 and the suppressors of cytokine signaling (SOCS) protein SOCS3 have recently been implicated in negative regulation of G-CSFR signaling (15,16). SOCS proteins can inhibit cytokine signaling by directly binding to either Jak kinases or phosphotyrosine residues in the cytoplasmic domains of cytokine receptors to compete for binding with STAT molecules (17)(18)(19)(20)(21)(22)(23)(24)(25).…”

Regulation of multiple cytokine signalling pathways by SOCS3 is independent of SOCS2

“…Each of these modules contributes to attenuation of the duration and intensity of JAK-STAT signalling. The SH2 domain is responsible for SOCS3 binding to both the phosphotyrosine residues within cytoplasmic domain of the receptor and JAK kinase following cytokine-mediated activation (Dunn et al 2005;Hortner et al 2002a;Hortner et al 2002b;Nicholson et al 2000;Sasaki et al 1999). As a consequence, SOCS3 is thought to inhibit signal transduction by directly inhibiting JAK activity through the kinase inhibitory region (KIR) located in the SOCS3 N-terminus .…”