We have identified Socs1 as a downstream component of the Kit receptor tyrosine kinase signalling pathway. We show that the expression of Socs1 mRNA is rapidly increased in primary bone marrow-derived mast cells following exposure to Steel factor, and Socs1 inducibly binds to the Kit receptor tyrosine kinase via its Src homology 2 (SH2) domain. Previous studies have shown that Socs1 suppresses cytokine-mediated differentiation in M1 cells inhibiting Janus family kinases. In contrast, constitutive expression of Socs1 suppresses the mitogenic potential of Kit while maintaining Steel factordependent cell survival signals. Unlike Janus kinases, Socs1 does not inhibit the catalytic activity of the Kit tyrosine kinase. In order to define the mechanism by which Socs1-mediated suppression of Kit-dependent mitogenesis occurs, we demonstrate that Socs1 binds to the signalling proteins Grb-2 and the Rho-family guanine nucleotide exchange factors Vav. We show that Grb2 binds Socs1 via its SH3 domains to putative diproline determinants located in the N-terminus of Socs1, and Socs1 binds to the N-terminal regulatory region of Vav. These data suggest that Socs1 is an inducible switch which modulates proliferative signals in favour of cell survival signals and functions as an adaptor protein in receptor tyrosine kinase signalling pathways.

Suppressor of cytokine signaling-1 (SOCS1) is an inducible Src homology 2 (SH2)-containing protein that negatively regulates cytokine and growth factor signaling required during thymic development. Recent evidence indicates that SOCS1 interacts with elongins B and C, which are components of a ubiquitin ligase complex, VCB (VHL/elonginC/B), based on the VHL (von Hippel Lindau) tumor suppressor protein. SOCS1 has previously been shown to operate as an inhibitor of Janus kinases. Here we show that SOCS1 has the distinct function of targeting the hematopoietic specific guanine nucleotide exchange factor, VAV, for ubiquitin-mediated protein degradation. VAV and SOCS1 form a protein complex through interactions between the VAV NH 2 -terminal regulatory region and the SH2 domain of SOCS1 in a phosphotyrosine-independent manner. SOCS1 decreases the steady state levels of cotransfected VAV and onco-VAV and reduces the focus forming activity of onco-VAV. SOCS1 stimulates the polyubiquitination of VAV proteins in vivo, which was stabilized by proteasomal inhibitors. These results suggest that SOCS1 programs VAV degradation by acting as a substrate-specific recognition component of a VCB-like ubiquitin ligase complex.The suppressor of cytokine signaling (SOCS) 1 family is composed of eight related SH2-containing proteins and represents a class of adapter molecules that negatively regulate diverse cytokine signaling pathways (1-4). SOCS1 is expressed in the thymus and cells of hematopoietic origin (1, 5). SOCS1 is part of an autoregulatory loop in which SOCS1 induction, following cytokine or growth factor receptor stimulation, attenuates Janus kinase (JAK) activity (1-3, 5). Consistent with this model, targeted disruption of the SOCS1 locus in mice gives rise to a syndrome of perinatal lethality and thymic atrophy resulting from unbridled interferon-␥ signaling (6 -8). SOCS1 inhibits JAK by binding, via its SH2 domain, to the positive regulatory tyrosine in the kinase domain activation helix (2, 9). Although SOCS1 also binds to the receptor tyrosine kinases Kit and Flt3, it does not suppress the kinase activity of these receptors (5). Nevertheless, SOCS1 potently blocks KIT-and FLT3-induced proliferation, suggesting that SOCS1 may modulate signaling through a mechanism distinct from kinase inhibition (5).In addition to its interaction with receptor and non-receptor tyrosine kinases, SOCS1 binds to the hematopoietic-specific guanine nucleotide exchange factor, VAV (5). VAV contains several modular protein domains including an NH 2 -terminal calponin homology (CH), an acidic region, a Dbl-homology (DH) domain, a pleckstrin homology (PH) domain, a cysteine-rich region, and two SH3 domains flanking an SH2 domain (reviewed in Ref. 10 and references therein). VAV converts inactive Rac-GDP into active Rac-GTP, which in turn regulates cytoskeletal reorganization and the activation of the c-jun NH 2 -terminal kinase (JNK) and p38HOG. VAV exchange factor activity is stimulated by tyrosine phosphorylation following antigen and cytokine...

Systemic mastocytosis (SM) is a rare disease caused by an abnormal mast cell accumulation in various tissues. Two classes of constitutive activating c-kit mutations are found in SM. The most frequent class occurs in the catalytic pocket coding region with substitutions at codon 816 and the other in the intracellular juxtamembrane coding region. Therefore, kinase inhibitors that block mutated c-kit activity might be used as therapeutic agents in SM. Here, we show that STI571 inhibits both wild-type and juxtamembrane mutant c-kit kinase activity, but has no effect on the activity of the D816 V mutant. Accordingly, STI571 selectively decreases the survival of normal mast cell and of mast cell lines either with juxtamembrane c-kit mutations, but not that of tumoral mast cell from patient with SM or of mast cell lines with the D816 V mutation. Therefore, STI571 is not a good candidate to treat SM and specific kinase inhibitors should be designed to inhibit constitutive activating mutations at codon 816.

SOCS-1 is an inducible SH2-containing inhibitor of Jak kinases and as such can potently suppress cytokine signaling. SOCS-1 de®cient mice die within the ®rst three weeks of life from a myeloproliferative disorder driven by excessive interferon signaling. We report here that SOCS-1 inhibits proliferation signals induced by a variety of oncogenes active within the hematopoietic system. Ectopic expression of SOCS-1 abolished proliferation mediated by a constitutively active form of the KIT receptor, TEL-JAK2, and v-ABL, and reduced metastasis from BCR-ABL transformed cells. SOCS-1, however, did not interfere with v-SRC or RASV12 mediated cellular transformation. A mutant form of SOCS-1 unable to bind through its SH2 domain to tyrosine phosphorylated proteins could still inhibit KIT, but not TEL-JAK2, indicating multiple mechanisms for SOCS-1-mediated tumor suppression. We show that the steady state levels of TEL-JAK2 and to a greater extent v-ABL are diminished in the presence of SOCS-1. Lastly, we show that SOCS-1 7/7 ®broblasts are more sensitive than wild type ®broblasts to either spontaneous or oncogene-induced transformation. These data suggest that loss-of-function of SOCS-1 may collaborate with a variety of hematopoietic oncogenes to facilitate tumor progression.

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Suppressor of cytokine signaling (SOCS) proteins are a family of Src homology 2-containing adaptor proteins. Cytokine-inducible Src homology domain 2-containing protein, SOCS1, SOCS2, and SOCS3 have been implicated in the down-regulation of cytokine signaling. The function of SOCS4, 5, 6, and 7 are not known. KIT receptor signaling is regulated by protein tyrosine phosphatases and adaptor proteins. We previously reported that SOCS1 inhibited cell proliferation in response to stem cell factor (SCF Cytokines and growth factors regulate the survival, proliferation, differentiation, and migration of hematopoietic cells. Binding of these factors to transmembrane receptors induces receptor activation, which in turn results in the recruitment of signaling complexes in the vicinity of the plasma membrane. The kinetics and magnitude of signal transduction are tightly regulated by multiple mechanisms. Among proteins that modulate signaling, members of the suppressor of cytokine signaling (SOCS) 1 family have been shown to down-regulate the function of cytokines or growth factors (1-3).The eight members of the SOCS family, SOCS1-7 and CIS (cytokine-inducible Src homology domain (SH2)-containing protein), are structurally characterized by a SH2 domain followed by a conserved C-terminal motif, the SOCS box (4). The N-terminal region of SOCS proteins is variable both in length and in the primary amino acid sequence. Although many reports including knock-out studies shed light on the function of CIS (5, 6), SOCS1 (7, 8), SOCS2 (9, 10), and SOCS3 (11-13), very little is known regarding the function of SOCS4, SOCS5, SOCS6, and SOCS7.The mechanisms whereby CIS, SOCS1, and SOCS3 inhibit signaling by classical cytokine receptor (i.e. receptors without catalytic activity that associate with JAK tyrosine kinases) are the best characterized. All three are involved in the downregulation of the JAK/STAT pathway. SOCS1 has a dual function as a direct potent JAK kinase inhibitor (14 -17) and as a component of an E3 ubiquitin-ligase complex recruiting substrates to the protein degradation machinery (18 -20). SOCS3 also inhibits JAK activity but indirectly through recruitment to the cytokine receptors (1, 21). More recently, SOCS3 has been suggested to compete with SHP2 for the same binding sites on glycoprotein 130 (22, 23), erythropoietin receptor (21), and leptin receptor (24). CIS binds to cytokine receptors at STAT5-docking sites, which impairs recruitment of STAT5 to the receptor signaling complex and results in the down-regulation of STAT5 activation (6,25).Mice lacking SOCS6 have been generated, and they developed normally with the exception of a 10% reduction in weight compared with wild-type littermates (26). SOCS6 mRNA was induced by erythropoietin in cell lines (27) and was ubiquitously expressed in murine tissues (26). SOCS6 does not interact with JAKs, but the interaction with elongins B and C suggests that, as all SOCS proteins, it might be part of an E3 ubiquitin-ligase complex (28). Yet, there is no evidence so far suggesti...

IntroductionMast cells are multifunctional hematopoietic cells, important for both innate and specific immunity. 1 Immature mast cells leave the bone marrow to migrate to target tissues, mostly mucosal and connective tissues, where they undergo terminal differentiation and perform their biologic functions. 2 Stem cell factor (SCF) and its receptor Kit are essential for mast cell development in vivo, as shown by the phenotype of mice with null mutations in the Kit (White Spotting or W) or SCF (Steel or Sl) loci. Indeed, these mice lack tissue mast cells and also exhibit defects in hematopoiesis, pigmentation, and reproduction. 3 In bone marrow mast cells (BMMCs), activation of Kit, a type III receptor tyrosine kinase (RTK), stimulates cellular responses such as proliferation, survival, differentiation, chemotaxis, cell adhesion, and degranulation. 4 Upon SCF binding, the Kit receptor dimerizes, autophosphorylates, and subsequently transphosphorylates specific tyrosines (Y). The resulting phosphotyrosine (pY) residues serve as docking sites for Src homology 2 (SH2) domain-containing proteins, which control intracellular signaling pathways such as all 3 mitogen-activated protein kinases (MAPKs; ERK, p38, and c-jun N-terminal kinase [JNK]), phosphatidylinositol 3-kinase (PI-3K), and Janus kinases (JAK)/signal transducers and activators of transcription (STAT) pathways. 4,5 Recruitment of particular targets is mediated by the ability of SH2 domains to recognize specific pY-containing motifs on the activated receptor. Analyses of individual docking sites on Kit have provided valuable information about the activation of pathways emanating from this receptor. 6 Juxtamembrane Y567 and Y569 are critical recruitment sites for different regulatory signaling molecules. These include activators such as Src family kinases (Fyn and Lyn), Shp-2 phosphatase and Shc adaptor protein, or negative modulators such as Shp-1 phosphatase, Csk-homology kinase (Chk), suppressors of cytokine signaling (SOCS) proteins, and APS adaptor protein. 5,7,8 Mutational and "knock-in" studies on Kit Y567/Y569 have revealed their important role in cell development, proliferation, survival, and migration. 6,9-11 However, the biologic significance of these interactions is still poorly understood.Lnk is a member of the adaptor protein family that includes APS and SH2-B. All 3 members share common protein-protein interaction domains and motifs: a dimerization domain at the aminoterminus, a pleckstrin homology (PH) domain, an SH2 domain, and a conserved tyrosine near the carboxy-terminus. 12 Mice deficient for members of this family have demonstrated the positive (SH2-B) and negative (Lnk and APS) role of these adaptors in growth factor, cytokine, and antigen-receptor signaling. [13][14][15][16][17][18] In particular, Lnk nullizygous mice show mainly a profound perturbation in hematopoiesis. These animals exhibit splenomegaly together with fibrosis, expansion of hematopoietic stem cells (HSCs), and myeloid and B lymphoid progenitors. [17][18][19] Recent ana...

Mutations of KIT receptor tyrosine kinase are found in the majority of patients with mastocytosis and in most gastrointestinal stromal tumors. Oncogenic KIT mutations in GISTs are located in the KIT juxtamembrane domain (JMD), while codon 816 in the KIT kinase domain is mutated in systemic mastocytosis. We describe and characterize a mutation in the KIT-JMD named KD27. We show that KD27 mutant is constitutively dimerized and phosphorylated. KD27 ectopic expression renders both the Ba/F3 cell line and primary cultures of bone marrow mast cells independent of cytokines for proliferation and cell survival. The classical signaling pathways activated by wild-type KIT upon ligand stimulation are constitutively activated by KD27 and other JMD mutations. However, a side-to-side comparison revealed differences between the wild-type and JMD mutations. First, in vitro kinase assays reveal a change in peptide substrate specificity. Second, STAT proteins are preferentially phosphorylated by KIT mutants. Third, inhibitors of KIT kinase are more efficient on JMD mutations than on WT KIT. We conclude that KD27 is a new oncogenic KIT mutation showing constitutive activation of downstream signaling pathways, and suggest that specific pathways are activated by oncogenic KIT.