SOCS (suppressors of cytokine signaling) proteins are negative regulators of cytokine signaling that function primarily at the receptor level. Remarkably, in vitro and in vivo observations revealed both inhibitory and stimulatory effects of SOCS2 on growth hormone signaling, suggesting an additional regulatory level. In this study, we examined the possibility of direct crossmodulation between SOCS proteins and found that SOCS2 could interfere with the inhibitory actions of other SOCS proteins in growth hormone, interferon, and leptin signaling. This SOCS2 effect was SOCS box-dependent, required recruitment of the elongin BC complex, and coincided with degradation of target SOCS proteins. Detailed mammalian protein-protein interaction trap (MAPPIT) analysis indicated that SOCS2 can interact with all members of the SOCS family. SOCS2 may thus function as a molecular bridge between a ubiquitin-protein isopeptide ligase complex and SOCS proteins, targeting them for proteasomal turnover. We furthermore extended these observations to SOCS6 and SOCS7. Our findings point to a unique regulatory role for SOCS2, SOCS6, and SOCS7 within the SOCS family and provide an explanation for the unexpected phenotypes observed in SOCS2 and SOCS6 transgenic mice.Cytokine signaling is typically a transient event, implying rapid and finely tuned attenuation. Receptor binding leads to rapid activation of receptor-associated members of the JAK 3 family. Subsequent phosphorylation of tyrosine residues in the receptor tails enables recruitment of downstream signaling molecules whereby STATs play a prominent role. Activated STATs translocate to the nucleus, where they control cytokineregulated gene transcription. Negative control occurs at many levels and involves receptor down-regulation, protein-tyrosine phosphatases, protein inhibitors of activated STATs, and members of the SOCS (suppressors of cytokine signaling) protein family.The SOCS family consists of eight different members (SOCS1-7 and CIS (cytokine-inducible SH2 domain-containing protein)) characterized by conserved structural features. All SOCS proteins consist of a central SH2 domain flanked by a variable N-terminal region and a conserved C-terminal SOCS box (1, 2). The SH2 domain can inhibit STAT activation by direct competition for the phosphorylated receptor recruitment sites (3-8). SOCS1 and SOCS3 carry an additional kinase inhibitory region (KIR) in their N-terminal domains that acts as a pseudosubstrate for the JAK kinase, thereby blocking signaling (5). The SOCS box was shown to act as an interaction domain for the elongin BC complex (9, 10), which, in turn, is a component of an ubiquitin-protein isopeptide ligase (E3) complex (11). This way, the SOCS box can control protein turnover by marking target proteins for proteasomal degradation (12). However, the significance of the interaction between SOCS proteins and the elongin BC complex is not totally clarified, as some reports propose that elongin association targets SOCS molecules for proteasomal degradation (10,(12)(13)...

Hypothalamic leptin receptor signalling plays a central role in weight regulation by controlling fat storage and energy expenditure. In addition, leptin also has direct effects on peripheral cell types involved in regulation of diverse body functions including immune response, bone formation and reproduction. Previous studies have demonstrated the important role of SOCS3 (suppressor of cytokine signalling 3) in leptin physiology. Here, we show that CIS (cytokine-inducible SH2 protein) and SOCS2 can also interact with the leptin receptor. Using MAPPIT (mammalian protein-protein interaction trap), a cytokine receptor-based two-hybrid method operating in intact cells, we show specific binding of CIS with the conserved Y985 and Y1077 motifs in the cytosolic domain of the leptin receptor. SOCS2 only interacts with the Y1077 motif, but with higher binding affinity and can interfere with CIS and STAT5a prey recruitment at this site. Furthermore, although SOCS2 does not associate with Y985 of the leptin receptor, we find that SOCS2 can block interaction of CIS with this position. This unexpected interference can be explained by the direct binding of SOCS2 on the CIS SOCS box, whereby elongin B/C recruitment is crucial to suppress CIS activity.

αT-catenin is a novel member of the α-catenin family, which shows most abundant expression in cardiomyocytes and in peritubular myoid cells of the testis, pointing to a specific function for αT-catenin in particular muscle tissues. Like other α-catenins, αT-catenin provides an indispensable link between the cadherin-based cell-cell adhesion complex and the cytoskeleton, to mediate cell-cell adhesion. By isolating genomic clones, combined with database sequence analysis, we have been able to determine the structure of the CTNNA3 and Ctnna3 genes, encoding human and mouse αT-catenin, respectively. The positions of the exon-exon boundaries are completely conserved in CTNNA3, Ctnna3, and the αN-catenin encoding CTNNA2 gene. They overlap largely with the boundaries of the CTNNA1 and CTNNAL1 genes encoding αE-catenin and α-catulin, respectively. This emphasizes that these α-catenin genes evolved from the same ancestor gene. Nevertheless, the introns of CTNNA3and Ctnna3 are remarkably large (often more than 100 kb) compared with introns of other CTNNA genes. The CTNNA3 gene was mapped to chromosome band 10q21 by both fluorescence in situ hybridization and polymerasechain-reaction-based hybrid mapping. This region encodes a gene for autosomal dominant familial dilated cardiomyopathy (DCM), a common cause of morbidity and mortality. As αT-catenin is highly expressed in healthy heart tissue, we have considered CTNNA3 as a candidate disease gene in a family showing DCM linkage to the 10q21-q23 locus. Mutation screening of all 18 exons of the CTNNA3 gene in this family has, however, not detected any DCM-linked CTNNA3 mutations.Assessment of the CTNNA3 gene encoding human αT-catenin regarding its involvement in dilated cardiomyopathy

Signaling via the erythropoietin receptor (EpoR) depends on the interaction of several proteins with phosphorylated tyrosine-containing motifs in its cytosolic domain. Detailed mapping of these interactions is required for an accurate insight into Epo signaling. We recently developed a mammalian protein-protein interaction trap (MAPPIT), a cytokine receptorbased 2-hybrid method that operates in intact Hek293-T mammalian cells. As baits, we used intracellular segments of the EpoR containing 1 or 2 tyrosines. Several known signaling molecules, including cytokine-inducible SH2-containing protein (CIS), suppressor of cytokine signaling-2 (SOCS2), phosphatidylinositol 3-kinase (PI3-K), phospholipase C-␥ (PLC-␥), and signal transducer and activator of transcription 5 (STAT5) were used as prey. We also extended the MAPPIT method to enable interaction analysis with wild-type EpoR. In this relay MAPPIT approach, instead of using isolated EpoR fragments as bait, we used the full-length EpoR itself as a "receptor bait." Finally, we introduced MAPPIT in the erythroleukemic TF-1 cell line, which is a more natural setting of the EpoR. With these strategies several known interactions with the EpoR were analyzed and evidence for new interactions was obtained. IntroductionThe glycoprotein hormone erythropoietin (Epo) is essential for red blood cell development because Epo Ϫ/Ϫ and Epo receptor-deficient (EpoR Ϫ/Ϫ ) mice die at an embryonic stage due to severe anemia. 1 Binding of Epo to its receptor prevents apoptosis of erythroid progenitors and promotes proliferation and erythroid maturation. The EpoR is a member of the type I cytokine receptor family 2 characterized by a single ligand-binding cytokine receptor homology domain 3 containing conserved cysteines and a Trp-Ser-X-Trp-Ser motif, 4 and lacking any catalytic activity in the intracellular region. 2 Epo binding to its receptor induces a conformational change of the preformed receptor-dimer resulting in activation and cross-phosphorylation of associated Janus kinase 2 (JAK2) tyrosine kinases. 5 Activated JAK2 molecules then phosphorylate tyrosine residues in the cytosolic tail of the EpoR that serve as docking sites for signaling molecules, thereby leading to the activation of several intracellular pathways. 6,7 These signaling molecules are often phosphorylated by JAK2 or other kinases such as Lyn. 8 Activation of the EpoR can lead to both antiapoptotic and proliferative effects. For example, signal transducers and activators of transcription 5a and 5b (STAT5a/b) bind to multiple phosphorylated tyrosine motifs (pYs) on the receptor, including pY344 and pY402. [9][10][11] On tyrosine phosphorylation by JAK2, STAT5 molecules translocate as dimers to the nucleus where they promote transcription of target genes including the antiapoptotic Bcl xL gene. 12 Several pathways have been described that independently lead to Epo-induced activation of phosphatidylinositol 3Ј-kinase (PI3-K) and phosphorylation of its downstream effectors. The p85 subunit of PI3-K directly associates ...

Despite its growing popularity and use, bottom-up proteomics remains a complex analytical methodology. Its general workflow consists of three main steps: sample preparation, liquid chromatography coupled to tandem mass spectrometry (LC−MS/ MS), and computational data analysis. Quality assessment of the different steps and components of this workflow is instrumental to identify technical flaws and avoid loss of precious measurement time and sample material. However, assessment of the extent of sample losses along with the sample preparation protocol, in particular, after proteolytic digestion, is not yet routinely implemented because of the lack of an accurate and straightforward method to quantify peptides. Here, we report on the use of a microfluidic UV/ visible spectrophotometer to quantify MS-ready peptides directly in the MS-loading solvent, consuming only 2 μL of sample. We compared the performance of the microfluidic spectrophotometer with a standard device and determined the optimal sample amount for LC−MS/MS analysis on a Q Exactive HF mass spectrometer using a dilution series of a commercial K562 cell digest. A careful evaluation of selected LC and MS parameters allowed us to define 3 μg as an optimal peptide amount to be injected into this particular LC−MS/MS system. Finally, using tryptic digests from human HEK293T cells and showing that injecting equal peptide amounts, rather than approximate ones, result in less variable LC−MS/MS and protein quantification data. The obtained quality improvement together with easy implementation of the approach makes it possible to routinely quantify MS-ready peptides as a next step in daily proteomics quality control.

The adipocyte-secreted hormone leptin participates in the regulation of hematopoiesis and enhances proliferation of hematopoietic cells. We used an adaptation of the MAPPIT mammalian two-hybrid method to study leptin signalling in a hematopoietic setting. We confirmed the known interactions of suppressor of cytokine signalling 3 (SOCS3) and STAT5 with the Y985 and Y1077 motifs of the leptin receptor, respectively. We also provide evidence for novel interactions at the Y1077 motif, including phospholipase C gamma and several members of the SOCS protein family, further underscoring the important role of the Y1077 motif in leptin signalling.

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Despite its growing popularity and use, bottom-up proteomics remains a complex analytical methodology. Its general workflow consists of three main steps: sample preparation, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and computational data analysis. Quality assessment of the different steps and components of this workflow is instrumental to identify technical flaws and to avoid loss of precious measurement time and sample material. However, assessment of the extent of sample losses along the sample preparation protocol, in particular after proteolytic digestion, is not yet routinely implemented because of the lack of an accurate and straightforward method to quantify peptides. Here, we report on the use of a microfluidic UV/visible spectrophotometer to quantify MS-ready peptides directly in MS loading solvent, consuming only 2 µl of sample. We determined the optimal peptide amount for LC-MS/MS analysis on a Q Exactive HF mass spectrometer using a dilution series of a commercial K562 cell digest. Careful evaluation of selected LC and MS parameters allowed us to define 3 µg as an optimal peptide amount to be injected on this particular LC-MS/MS system. Finally, using tryptic digests from human HEK293T cells, we showed that injecting equal peptide amounts, rather than approximated ones, results into less variable LC-MS/MS and protein quantification data. The obtained quality improvement together with easy implementation of the approach makes it possible to routinely quantify MS-ready peptides as a next step in daily proteomics quality control.This material is available free of charge via the Internet at