Mode of regulation of the extracellular signal-regulated kinases in the pancreatic beta-cell line MIN6 and their implication in the regulation of insulin gene transcription.
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Physiological concentrations of glucose that lead to Ca2+ entry and insulin secretion activate extracellular signal-regulated protein kinases (ERK1 and ERK2) in the MIN6 pancreatic beta-cell line. Here we show that this activation is inhibited by the down-regulation of protein kinase C (PKC) and by genistein, an inhibitor of protein tyrosine kinases. In contrast with results obtained in other cell types, neither the epidermal growth factor activity nor the Src family protein tyrosine kinases seem to be involved in the Ca2+-dependent activation of ERKs. inhibition of tyrosine phosphatases by vanadate leads to the activation of ERKs. As observed in the response to glucose, this activation is dependent on Ca2+ entry through L-type voltage-dependent Ca2+ channels. Thus the activation of ERKs in response to glucose depends on PKC and possibly on a tyrosine kinase/tyrosine phosphatase couple. To define the role of ERK activation by glucose we studied the regulation of transcription of the insulin gene. We found that this transcription is regulated in the MIN6 cells in the same range of glucose concentration as in primary islets, and that specific inhibition of mitogen-activated protein kinase kinase, the direct activator of ERK, impaired the response of the insulin gene to glucose. This was observed by analysis of the transfected rat insulin I gene promoter activity and a Northern blot of endogenous insulin mRNA. (+info)
The binding kinetics of the TCR for its interacting ligand and the nature of the resulting signal transduction event determine the fate of a developing thymocyte. The intracellular tyrosine phosphatase SHP-1 is a potential regulator of the TCR signal transduction cascade and may affect thymocyte development. To assess the role of SHP-1 in thymocyte development, we generated T cell-transgenic mice that express a putative dominant negative form of SHP-1, in which a critical cysteine is mutated to serine (SHP-1 C453S). SHP-1 C453S mice that express the 3.L2 TCR transgene are increased in CD4 single positive cells in the thymus and are increased in cells that express the clonotypic TCR. These data suggest that the expression of SHP-1 C453S results in increased positive selection in 3.L2 TCR-transgenic mice and support a role for SHP-1 thymocyte development. (+info)
The NKR-P1B gene product is an inhibitory receptor on SJL/J NK cells.
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The mouse NKR-P1 family includes at least three genes: NKR-P1A, -B, -C. Neither surface expression nor function of the NKR-P1B gene product has previously been shown. Here, we demonstrate that the SJL/J allele of the NKR-P1B gene product is expressed on SJL/J NK cells, and is recognized by PK136 mAb. Interestingly, the same mAb does not recognize the NKR-P1B gene product of C57BL/6. We have also generated a novel mAb, 1C10, that recognizes an activation receptor on SJL/J NK cells. Activation of the NKR-P1B receptor-inhibited 1C10 mAb induced redirected lysis and recruited SHP-1, indicating that NKR-P1B is an inhibitory receptor. Therefore, the mouse NKR-P1 gene family, like the Ly49 family, includes both activation and inhibitory receptors. (+info)
Mouse NKR-P1B, a novel NK1.1 antigen with inhibitory function.
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The mouse NK1.1 Ag originally defined as NK cell receptor (NKR)-P1C (CD161) mediates NK cell activation. Here, we show that another member of the mouse CD161 family, NKR-P1B, represents a novel NK1.1 Ag. In contrast to NKR-P1C, which functions as an activating receptor, NKR-P1B inhibits NK cell activation. Association of NKR-P1B with Src homology 2-containing protein tyrosine phosphatase-1 provides a molecular mechanism for this inhibition. The existence of these two NK1.1 Ags with opposite functions suggests a potential role for NKR-P1 molecules, such as those of the Ly-49 gene family, in regulating NK cell function. (+info)
Retinal axon target selection in Drosophila is regulated by a receptor protein tyrosine phosphatase.
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Different Drosophila photoreceptors (R cells) connect to neurons in different optic lobe layers. R1-R6 axons project to the lamina; R7 and R8 axons project to separate layers of the medulla. We show a receptor tyrosine phosphatase, PTP69D, is required for lamina target specificity. In Ptp69D mutants, R1-R6 project through the lamina, terminating in the medulla. Genetic mosaics, transgene rescue, and immunolocalization indicate PTP69D functions in R1-R6 growth cones. PTP69D overexpression in R7 and R8 does not respecify their connections, suggesting PTP69D acts in combination with other factors to determine target specificity. Structure-function analysis indicates the extracellular fibronectin type III domains and intracellular phosphatase activity are required for targeting. We propose PTP69D promotes R1-R6 targeting in response to extracellular signals by dephosphorylating substrate(s) in R1-R6 growth cones. (+info)
Active nucleocytoplasmic shuttling required for function and regulation of stress-activated kinase Spc1/StyI in fission yeast.
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Transcriptional induction of many stress-response genes is dependent on stress-induced nuclear accumulation of stress-activated protein kinases (SAPKs). In the fission yeast Schizosaccharomyces pombe, nuclear accumulation of the SAPK Spc1 (also known as StyI) requires activating phosphorylation catalyzed by the SAPK kinase Wis1; however, it is unknown whether the localization of Spc1 is regulated by nuclear transport factors. Herein are reported studies that show that Spc1 localization is regulated by active transport mechanisms during osmotic stress. Nuclear import of Spc1 requires Pim1, a homologue of the guanine nucleotide exchange factor RCC1 that is essential for nucleocytoplasmic shuttling of proteins. Nuclear export of Spc1 is regulated by the export factor Crm1. An Spc1-Crm1 complex forms as Spc1 is exported from the nucleus. Wis1 and the tyrosine phosphatases Pyp1 and Pyp2 that inactivate Spc1 are excluded from the nucleus by a Crm1-independent mechanism; hence the nuclear import of Spc1 leads to transient isolation from its regulatory proteins. Thus, active nucleocytoplasmic shuttling is required for both the function and regulation of Spc1 during the osmotic shock response. (+info)
Thrombospondin-1 induces tyrosine phosphorylation of adherens junction proteins and regulates an endothelial paracellular pathway.
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Thrombospondin-1 (TSP) induces endothelial cell (EC) actin reorganization and focal adhesion disassembly and influences multiple EC functions. To determine whether TSP might regulate EC-EC interactions, we studied the effect of exogenous TSP on the movement of albumin across postconfluent EC monolayers. TSP increased transendothelial albumin flux in a dose-dependent manner at concentrations >/=1 microg/ml (2.2 nM). Increases in albumin flux were observed as early as 1 h after exposure to 30 microg/ml (71 nM) TSP. Inhibition of tyrosine kinases with herbimycin A or genistein protected against the TSP-induced barrier dysfunction by >80% and >50%, respectively. TSP-exposed monolayers exhibited actin reorganization and intercellular gap formation, whereas pretreatment with herbimycin A protected against this effect. Increased staining of phosphotyrosine-containing proteins was observed in plaque-like structures and at the intercellular boundaries of TSP-treated cells. In the presence of protein tyrosine phosphatase inhibition, TSP induced dose- and time-dependent increments in levels of phosphotyrosine-containing proteins; these TSP dose and time requirements were compatible with those defined for EC barrier dysfunction. Phosphoproteins that were identified include the adherens junction proteins focal adhesion kinase, paxillin, gamma-catenin, and p120(Cas). These combined data indicate that TSP can modulate endothelial barrier function, in part, through tyrosine phosphorylation of EC proteins. (+info)
Evidence for a calpeptin-sensitive protein-tyrosine phosphatase upstream of the small GTPase Rho. A novel role for the calpain inhibitor calpeptin in the inhibition of protein-tyrosine phosphatases.
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Activation of the thiol protease calpain results in proteolysis of focal adhesion-associated proteins and severing of cytoskeletal-integrin links. We employed a commonly used inhibitor of calpain, calpeptin, to examine a role for this protease in the reorganization of the cytoskeleton under a variety of conditions. Calpeptin induced stress fiber formation in both forskolin-treated REF-52 fibroblasts and serum-starved Swiss 3T3 fibroblasts. Surprisingly, calpeptin was the only calpain inhibitor of several tested with the ability to induce these effects, suggesting that calpeptin may act on targets besides calpain. Here we show that calpeptin inhibits tyrosine phosphatases, enhancing tyrosine phosphorylation particularly of paxillin. Calpeptin preferentially inhibits membrane-associated phosphatase activity. Consistent with this observation, in vitro phosphatase assays using purified glutathione S-transferase fusion proteins demonstrated a preference for the transmembrane protein-tyrosine phosphatase-alpha over the cytosolic protein-tyrosine phosphatase-1B. Furthermore, unlike wide spectrum inhibitors of tyrosine phosphatases such as pervanadate, calpeptin appeared to inhibit a subset of phosphatases. Calpeptin-induced assembly of stress fibers was inhibited by botulinum toxin C3, indicating that calpeptin is acting on a phosphatase upstream of the small GTPase Rho, a protein that controls stress fiber and focal adhesion assembly. Not only does this work reveal that calpeptin is an inhibitor of protein-tyrosine phosphatases, but it suggests that calpeptin will be a valuable tool to identify the phosphatase activity upstream of Rho. (+info)