Genetic deletion of murine SPRY domain-containing SOCS box protein 2 (SSB-2) results in very mild thrombocytopenia.

The SSB family is comprised of four highly homologous proteins containing a C-terminal SOCS box motif and a central SPRY domain. No function has yet been ascribed to any member of this family in mammalian species despite a clear role for other SOCS proteins in negative regulation of cytokine signaling. To investigate its physiological role, the murine Ssb-2 gene was deleted by homologous recombination. SSB-2-deficient mice were shown to have a reduced rate of platelet production, resulting in very mild thrombocytopenia (25% decrease in circulating platelets). Tissue histology and other hematological parameters were normal, as was the majority of serum biochemistry, with the exception that blood urea nitrogen (BUN) levels were decreased in mice lacking SSB-2. Quantitative analysis of SSB mRNA levels indicated that SSB-1, -2, and -3 were ubiquitously expressed; however, SSB-4 was only expressed at very low levels. SSB-2 expression was observed in the kidney and in megakaryocytes, a finding consistent with the phenotype of mice lacking this gene. Deletion of SSB-2 thus perturbs the steady-state level of two tightly controlled homeostatic parameters and identifies a critical role for SSB-2 in regulating platelet production and BUN levels.

Cloning and characterization of the genes encoding the ankyrin repeat and SOCS box-containing proteins Asb-1, Asb-2, Asb-3 and Asb-4.

Members of the suppressor of cytokine signalling (SOCS) family of proteins have been shown to inhibit cytokine signalling via direct interactions with JAK kinases or activated cytokine receptors. In addition to their novel amino-terminal regions and SH2 domains that mediate these interactions, the SOCS proteins also contain carboxy-terminal regions of homology called the SOCS box. The SOCS box serves to couple SOCS proteins and their binding partners with the elongin B and C complex, possibly targeting them for degradation. Several other families of proteins also contain SOCS boxes but differ from the SOCS proteins in the type of domain or motif they contain upstream of the SOCS box. We report here the cloning, characterization, mapping and expression analysis of four members of the ankyrin repeat and SOCS box-containing (Asb) protein family.

Gigantism in mice lacking suppressor of cytokine signalling-2.

Suppressor of cytokine signalling-2 (SOCS-2) is a member of the suppressor of cytokine signalling family, a group of related proteins implicated in the negative regulation of cytokine action through inhibition of the Janus kinase (JAK) signal transducers and activators of transcription (STAT) signal-transduction pathway. Here we use mice unable to express SOCS-2 to examine its function in vivo. SOCS-2(-/-) mice grew significantly larger than their wild-type littermates. Increased body weight became evident after weaning and was associated with significantly increased long bone lengths and the proportionate enlargement of most organs. Characteristics of deregulated growth hormone and insulin-like growth factor-I (IGF-I) signalling, including decreased production of major urinary protein, increased local IGF-I production, and collagen accumulation in the dermis, were observed in SOCS-2-deficient mice, indicating that SOCS-2 may have an essential negative regulatory role in the growth hormone/IGF-I pathway.

Suppressors of cytokine signaling (SOCS): negative regulators of signal transduction.

SOCS-1 was originally identified as an inhibitor of interleukin-6 signal transduction and is a member of a family of proteins (SOCS-1 to SOCS-7 and CIS) that contain an SH2 domain and a conserved carboxyl-terminal SOCS box motif. Mutation studies have established that critical contributions from both the amino-terminal and SH2 domains are essential for SOCS-1 and SOCS-3 to inhibit cytokine signaling. Inhibition of cytokine-dependent activation of STAT3 occurred in cells expressing either SOCS-1 or SOCS-3, but unlike SOCS-1, SOCS-3 did not directly interact with or inhibit the activity of JAK kinases. Although the conserved SOCS box motif appeared to be dispensable for SOCS-1 and SOCS-3 action when overexpressed, this domain interacts with elongin proteins and may be important in regulating protein turnover. In gene knockout studies, SOCS-1(-/-) mice were born but failed to thrive and died within 3 weeks of age with fatty degeneration of the liver and hemopoietic infiltration of several organs. The thymus in SOCS-1(-/-) mice was small, the animals were lymphopenic, and deficiencies in B lymphocytes were evident within hemopoietic organs. We propose that the absence of SOCS-1 in these mice prevents lymphocytes and liver cells from appropriately controlling signals from cytokines with cytotoxic side effects.

Twenty proteins containing a C-terminal SOCS box form five structural classes.

The four members of the recently identified suppressor of cytokines signaling family (SOCS-1, SOCS-2, SOCS-3, and CIS, where CIS is cytokine-inducible SH2-containing protein) appear, by various means, to negatively regulate cytokine signal transduction. Structurally, the SOCS proteins are composed of an N-terminal region of variable length and amino acid composition, a central SH2 domain, and a previously unrecognized C-terminal motif that we have called the SOCS box. By using the SOCS box amino acid sequence consensus, we have searched DNA databases and have identified a further 16 proteins that contain this motif. These proteins fall into five classes based on the protein motifs found N-terminal of the SOCS box. In addition to four new SOCS proteins (SOCS-4 to SOCS-7) containing an SH2 domain and a SOCS box, we describe three new families of proteins that contain either WD-40 repeats (WSB-1 and -2), SPRY domains (SSB-1 to -3) or ankyrin repeats (ASB-1 to -3) N-terminal of the SOCS box. In addition, we show that a class of small GTPases also contains a SOCS box. The expression of representative members of each class of proteins differs markedly, as does the regulation of expression by cytokines. The function of the WSB, SSB, and ASB protein families remains to be determined.

A family of cytokine-inducible inhibitors of signalling.

Cytokines are secreted proteins that regulate important cellular responses such as proliferation and differentiation. Key events in cytokine signal transduction are well defined: cytokines induce receptor aggregation, leading to activation of members of the JAK family of cytoplasmic tyrosine kinases. In turn, members of the STAT family of transcription factors are phosphorylated, dimerize and increase the transcription of genes with STAT recognition sites in their promoters. Less is known of how cytokine signal transduction is switched off. We have cloned a complementary DNA encoding a protein SOCS-1, containing an SH2-domain, by its ability to inhibit the macrophage differentiation of M1 cells in response to interleukin-6. Expression of SOCS-1 inhibited both interleukin-6-induced receptor phosphorylation and STAT activation. We have also cloned two relatives of SOCS-1, named SOCS-2 and SOCS-3, which together with the previously described CIS form a new family of proteins. Transcription of all four SOCS genes is increased rapidly in response to interleukin-6, in vitro and in vivo, suggesting they may act in a classic negative feedback loop to regulate cytokine signal transduction.

Purification of a ligand for the EPH-like receptor HEK using a biosensor-based affinity detection approach.

Advances in screening technologies allowing the identification of growth factor receptors solely by virtue of DNA or protein sequence comparison call for novel methods to isolate corresponding ligand growth factors. The EPH-like receptor tyrosine kinase (RTK) HEK (human EPH-like kinase) was identified previously as a membrane antigen on the LK63 human pre-B-cell line and overexpression in leukemic specimens and cell lines suggested a role in oncogenesis. We developed a biosensor-based approach using the immobilized HEK receptor exodomain to detect and monitor purification of the HEK ligand. A protein purification protocol, which included HEK affinity chromatography, achieved a 1.8 X 10(6)-fold purification of an approximately 23-kDa protein from human placental conditioned medium. Analysis of specific sHEK (soluble extracellular domain of HEK) ligand interactions in the first and final purification steps suggested a ligand concentration of 40 pM in the source material and a Kd of 2-3 nM. Since the purified ligand was N-terminally blocked, we generated tryptic peptides and N-terminal amino acid sequence analysis of 7 tryptic fragments of the S-pyridylethylated protein unequivocally matched the sequence for AL-1, a recently reported ligand for the related EPH-like RTK REK7 (Winslow, J.W., Moran, P., Valverde, J., Shih, A., Yuan, J.Q., Wong, S.C., Tsai, S.P., Goddard, A., Henzel, W.J., Hefti, F., Beck, K.D., & Caras, I.W. (1995) Neuron 14, 973-981). Our findings demonstrate the application of biosensor technology in ligand purification and show that AL-1, as has been found for other ligands of the EPH-like RTK family, binds more than one receptor.

Molecular cloning of two novel transmembrane ligands for Eph-related kinases (LERKS) that are related to LERK-2.

A search of the nucleic acid database of expressed sequence tags (ESTs) revealed several partial cDNA sequences that could encode proteins homologous to the ligands for Eph-related kinases (LERKs). Oligonucleotides designed from the ESTs were used to probe a human brain cDNA library and obtain overlapping clones that encoded two different novel LERKS (NLERK-1 and NLERK-2). NLERK-1 and NLERK-2 are most closely related to human LERK-2/Elk-ligand and they form a subclass of LERKs that contain a transmembrane domain and a conserved cytoplasmic domain. Full-length NLERK-1 was expressed as a glycosylated membrane protein in COS cells and was not secreted into the medium. Full-length NLERK-2 was similarly expressed in COS cells but both membrane-bound and a truncated, proteolytically-released form were detected. Engineered forms of both NLERK-1 and NLERK-2 lacking transmembrane and cytoplasmic domains were also expressed in COS cells and each was detected in the extracellular medium.