Tyrosine phosphorylated SOCS-3 inhibits STAT activation but binds p120-RasGAP and activates Ras

Suppressors of cytokine signalling (SOCS, also known as CIS and SSI) are encoded by immediate early genes that act in a feedback loop to inhibit cytokine responses and activation of 'signal transducer and activator of transcription' (STAT). Here we show that SOCS-3 is strongly tyrosine-phosphorylated in response to many growth factors, including interleukin-2 (IL-2), erythropoietin (EPO), epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). The principal phosphorylation sites on SOCS-3 are residues 204 and 221 at the carboxy terminus, and upon phosphorylation tyrosine 221 interacts with the Ras inhibitor p120 RasGAP. After IL-2 stimulation, phosphorylated SOCS-3 strongly inhibits STAT5 activation but, by binding to RasGAP, maintains activation of extracellular-signal-regulated kinase (ERK). A tyrosine mutant of SOCS-3 still blocks STAT phosphorylation, but also strongly inhibits IL-2-dependent activation of ERK and cell proliferation. Moreover, it also inhibits EPO- and PDGF-induced proliferation and ERK activation. Therefore, although SOCS proteins inhibit growth-factor responses, tyrosine phosphorylation of SOCS-3 can ensure cell survival and proliferation through the Ras pathway.

ICln, a novel integrin alpha(11b)beta3-associated protein, functionally regulates platelet activation.

A critical role for the conserved -integrin cytoplasmic motif, KVGFFKR, is recognized in the regulation of activation of the platelet integrin IIb3. To understand the molecular mechanisms of this regulation, we sought to determine the nature of the protein interactions with this cytoplasmic motif. We used a tagged synthetic peptide, biotin-KVGFFKR, to probe a high density protein expression array (37,200 recombinant human proteins) for high affinity interactions. A number of potential integrin-binding proteins were identified. One such protein, a chloride channel regulatory protein, ICln, was characterized further because its affinity for the integrin peptide was highest as was its expression in platelets. We verified the presence of ICln in human platelets by PCR, Western blots, immunohistochemistry, and its co-association with IIb3 by surface plasmon resonance. The affinity of this interaction was 82.2 ± 24.4 nM in a cell free assay. ICln co-immunoprecipitates with IIb3 in platelet lysates demonstrating that this interaction is physiologically relevant. Furthermore, immobilized KVGFFKR peptides, but not control KAAAAAR peptides, specifically extract ICln from platelet lysates. Acyclovir (100 µM to 5 mM), a pharmacological inhibitor of the ICln chloride channel, specifically inhibits integrin activation (PAC-1 expression) and platelet aggregation without affecting CD62 P expression confirming a specific role for ICln in integrin activation. In parallel, a cell-permeable peptide corresponding to the potential integrin-recognition domain on ICln (AKFEEE, 10–100 µM) also inhibits platelet function. Thus, we have identified, verified, and characterized a novel functional interaction between the platelet integrin and ICln, in the platelet membrane.

Truncated estrogen receptor alpha 46-kDa isoform in human endothelial cells: relationship to acute activation of nitric oxide synthase.

Background Estrogen acutely activates endothelial nitric oxide synthase (eNOS). However, the identity of the receptors involved in this rapid response remains unclear. Methods and Results We detected an estrogen receptor (ER) transcript in human endothelial cells that encodes a truncated 46-kDa ER (1a-hER-46). A corresponding 46-kDa ER protein was identified in endothelial cell lysates. Transfection of cDNAs encoding the full-length ER (ER-66) and 1a-hER-46 resulted in appropriately sized recombinant proteins identified by anti-ER antibodies. Confocal microscopy revealed that a proportion of both ER-66 and hER-46 was localized outside the nucleus and mediated specific cell-surface binding of estrogen as assessed by FITC-conjugated, BSA-estrogen binding studies. Both ER isoforms colocalized with eNOS and mediated acute activation of eNOS in response to estrogen stimulation. However, estrogen-stimulated transcriptional activation mediated by 1a-hER-46 was much less than with ER-66. Furthermore, 1a-hER-46 inhibited classical hER-66 mediated transcriptional activation in a dominant-negative fashion. Conclusions These findings suggest that expression of an alternatively spliced, truncated ER isoform in human endothelial cells confers a unique ability to mediate acute but not transcriptional responses to estrogen.

Diabetes-induced activation of protein kinase C inhibits store-operated Ca2+ uptake in rat retinal microvascular smooth muscle.

AIMS/HYPOTHESIS: To assess the effects of diabetes-induced activation of protein kinase C (PKC) on voltage-dependent and voltage-independent Ca2+ influx pathways in retinal microvascular smooth muscle cells. METHODS: Cytosolic Ca2+ was estimated in freshly isolated rat retinal arterioles from streptozotocin-induced diabetic and non-diabetic rats using fura-2 microfluorimetry. Voltage-dependent Ca2+ influx was tested by measuring rises in [Ca2+]i with KCl (100 mmol/l) and store-operated Ca2+ influx was assessed by depleting [Ca2+]i stores with Ca2+ free medium containing 5 micromol/l cyclopiazonic acid over 10 min and subsequently measuring the rate of rise in Ca2+ on adding 2 mmol/l or 10 mmol/l Ca2+ solution. RESULTS: Ca2+ entry through voltage-dependent L-type Ca2+ channels was unaffected by diabetes. In contrast, store-operated Ca2+ influx was attenuated. In microvessels from non-diabetic rats 20 mmol/l D-mannitol had no effect on store-operated Ca2+ influx. Diabetic rats injected daily with insulin had store-operated Ca2+ influx rates similar to non-diabetic control rats. The reduced Ca2+ entry in diabetic microvessels was reversed by 2-h exposure to 100 nmol/l staurosporine, a non-specific PKC antagonist and was mimicked in microvessels from non-diabetic rats by 10-min exposure to the PKC activator phorbol myristate acetate (100 nmol/l). The specific PKCbeta antagonist LY379196 (100 nmol/l) also reversed the poor Ca2+ influx although its action was less efficacious than staurosporine. CONCLUSION/INTERPRETATION: These results show that store-operated Ca2+ influx is inhibited in retinal arterioles from rats having sustained increased blood glucose and that PKCbeta seems to play a role in mediating this effect.

Cytohesin Binder and Regulator (Cybr) is Not Essential for T- and Dendritic-Cell Activation and Differentiation

Cybr (also known as Cytip, CASP, and PSCDBP) is an interleukin-12-induced gene expressed exclusively in hematopoietic cells and tissues that associates with Arf guanine nucleotide exchange factors known as cytohesins. Cybr levels are dynamically regulated during T-cell development in the thymus and upon activation of peripheral T cells. In addition, Cybr is induced in activated dendritic cells and has been reported to regulate dendritic cell (DC)-T-cell adhesion. Here we report the generation and characterization of Cybr-deficient mice. Despite the selective expression in hematopoietic cells, there was no intrinsic defect in T- or B-cell development or function in Cybr-deficient mice. The adoptive transfer of Cybr-deficient DCs showed that they migrated efficiently and stimulated proliferation and cytokine production by T cells in vivo. However, competitive stem cell repopulation experiments showed a defect in the abilities of Cybr-deficient T cells to develop in the presence of wild-type precursors. These data suggest that Cybr is not absolutely required for hematopoietic cell development or function, but stem cells lacking Cybr are at a developmental disadvantage compared to wild-type cells. Collectively, these data demonstrate that despite its selective expression in hematopoietic cells, the role of Cybr is limited or largely redundant. Previous in vitro studies using overexpression or short interfering RNA inhibition of the levels of Cybr protein appear to have overestimated its immunological role.

A switch in the cellular localization of macrophage migration inhibitory factor in the rat testis after ethane dimethane sulfonate treatment.

Macrophage migration inhibitory factor (MIF), one of the first cytokines to be discovered, has recently been localized to the Leydig cells in adult rat testes. In the following study, the response of MIF to Leydig cell ablation by the Leydig cell-specific toxin ethane dimethane sulfonate (EDS) was examined in adult male rats. Testicular MIF mRNA and protein in testicular interstitial fluid measured by ELISA and western blot were only marginally reduced by EDS treatment, in spite of the fact that the Leydig cells were completely destroyed within 7 days. Immunohistochemistry using an affinity-purified anti-mouse MIF antibody localized MIF exclusively to the Leydig cells in control testes. At 7 days post-EDS treatment, there were no MIF immunopositive Leydig cells in the interstitium, although distinct MIF immunostaining was observed in the seminiferous tubules, principally in Sertoli cells and residual cytoplasm, and some spermatogonia. A few peritubular and perivascular cells were also labelled at this time, which possibly represented mesenchymal Leydig cell precursors. At 14 and 21 days, Sertoli cell MIF immunoreactivity was observed in only a few tubule cross-sections, while some peritubular and perivascular mesenchymal cells and the re-populating immature Leydig cells were intensely labeled. At 28 days after EDS-treatment, the MIF immunostaining pattern was identical to that of untreated and control testes. The switch in the compartmentalization of MIF protein at 7 days after EDS-treatment was confirmed by western blot analysis of interstitial tissue and seminiferous tubules separated by mechanical dissection. These data establish that Leydig cell-depleted testes continue to produce MIF, and suggest the existence of a mechanism of compensatory cytokine production involving the Sertoli cells. This represents the first demonstration of a hitherto unsuspected pattern of cellular interaction between the Leydig cells and the seminiferous tubules which is consistent with an essential role for MIF in male testicular function.