Dephosphorylation of beta2-syntrophin and Ca2+/mu-calpain-mediated cleavage of ICA512 upon stimulation of insulin secretion

Islet cell autoantigen (ICA) 512 is a receptor-tyrosine phosphatase-like protein associated with the secretory granules of neuroendocrine cells, including pancreatic beta-cells. Binding of its cytoplasmic tail to beta2-syntrophin suggests that ICA512 connects secretory granules to the utrophin complex and the actin cytoskeleton. Here we show that stimulation of insulin secretion from INS-1 cells triggers the biosynthesis of pro-ICA512 and the degradation of its mature form. Inhibition of calpain, which is activated upon stimulation of insulin secretion, prevents the Ca2+-dependent proteolysis of ICA512. In vitro mu-calpain cleaves ICA512 between a putative PEST domain and the beta2-syntrophin binding site, whereas binding of ICA512 to beta2-syntrophin protects the former from cleavage. beta2-syntrophin and its F-actin-binding protein utrophin are enriched in subcellular fractions containing secretory granules. ICA512 preferentially binds phospho-beta2-syntrophin and stimulation of insulin secretion induces the Ca2+-dependent, okadaic acid-sensitive dephosphorylation of beta2-syntrophin. Similarly to calpeptin, okadaic acid inhibits ICA512 proteolysis and insulin secretion. Thus, stimulation of insulin secretion might promote the mobilization of secretory granules by inducing the dissociation of ICA512 from beta2-syntrophin-utrophin complexes and the cleavage of the ICA512 cytoplasmic tail by mu-calpain.     

Expression of the protein tyrosine phosphatase-like protein IA-2 during pancreatic islet development.

A tyrosine phosphatase-like protein, IA-2, is a major autoantigen in Type 1 diabetes but its role in islet function is unclear. Tyrosine phosphorylation mediates regulation of cellular processes such as exocytosis, cell growth, and cell differentiation. To investigate the potential involvement of IA-2 in islet differentiation and insulin secretion, we analyzed by immunohistochemistry expression of IA-2 during islet development in fetal rats and during the maturation of insulin secretory responses after birth. In the fetus, IA-2 immunoreactivity was detected in primitive islets positive for insulin and glucagon at 12 days' gestation. Subsequently, IA-2 was only weakly detectable in the fetal pancreas. In neonatal rat, a progressive increase in IA-2 immunoreactivity was observed in islets from very low levels at 1 day of age to moderate labeling at 10 days. In the adult, relatively high levels of IA-2 were detected in islets, with heterogeneous expression in individual cells within each islet. IA-2 marks a population of endocrine cells that transiently appear early in pancreatic ontogeny. Islet IA-2 expression reappears after birth concomitant with the development of mature insulin secretory responses, consistent with a role for this protein in regulated hormone secretion.     

c-Src tyrosine kinase binds the beta 2-adrenergic receptor via phospho-Tyr-350, phosphorylates G-protein-linked receptor kinase 2, and mediates agonist-induced receptor desensitization

The nonreceptor tyrosine kinase Src has been implicated in the switching of signaling of beta2-adrenergic receptors from adenylylcyclase coupling to the mitogen-activated protein kinase pathway. In the current work, we demonstrate that Src plays an active role in the agonist-induced desensitization of beta2-adrenergic receptors. Both the expression of dominant-negative Src and treatment with the 4-amine-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) inhibitor of Src kinase activity blocks agonist-induced desensitization. Agonist triggers tyrosine phosphorylation of the beta2-adrenergic receptor and recruitment and activation of Src. Because phosphorylation of the Tyr-350 residue of the beta2-adrenergic receptor creates a conditional, canonical SH2-binding site on the receptor, we examined the effect of the Y350F mutation on Src phosphorylation, Src recruitment, and desensitization. Mutant beta2-adrenergic receptors with a Tyr-to-Phe substitution at Tyr-350 do not display agonist-induced desensitization, Src recruitment, or Src activation. Downstream of binding to the receptor, Src phosphorylates and activates G-protein-linked receptor kinase 2 (GRK2), a response obligate for agonist-induced desensitization. Constitutively active Src increases GRK phosphorylation, whereas either expression of dominant-negative Src or treatment with the PP2 inhibitor abolishes tyrosine phosphorylation of GRK and desensitization. Thus, in addition to its role in signal switching to the mitogen-activated protein kinase pathway, Src recruitment to the beta2-adrenergic receptor and activation are obligate for normal agonist-induced desensitization.     

Expression of pancreatic islet MHC class I, insulin, and ICA 512 tyrosine phosphatase in low-dose streptozotocin-induced diabetes in mice.

Activated immune cells contribute to the development of diabetes mellitus in multiple low-dose streptozotocin-treated mice. However, a role in the process for MHC Class I restricted T-cells remains a matter of debate. In this study, we examined by confocal microscopy the pancreatic expression of MHC Class I protein, insulin, and ICA 512 protein tyrosine phosphatase in C57BL/Ks mice given 40 mg/kg bw streptozotocin IP on 5 consecutive days. All animals were hyperglycemic from Day 7 and onwards. A loss of ICA 512 from the central portions of the islets was noted on Day 3. On Day 7, an increase in MHC Class I expression, confined primarily to immune cells in the exocrine pancreas and the periinsular areas, was detected. Later, several MHC class I/glucagon and some MHC class I/insulin double-positive cells were found. The insulitis was maximal on Day 14 and declined thereafter. The induction of MHC Class I expression in endocrine cells, occuring only after the cellular infiltration and when the animals were diabetic, indicates that the immune component of the disease does not depend on MHC Class I-restricted cytotoxic T-cells but rather comprises a non-antigen-specific process. (J Histochem Cytochem 48:761-767, 2000)     

Pasticcino2 is a protein tyrosine phosphatase-like involved in cell proliferation and differentiation in Arabidopsis

The pasticcino2 (pas2) mutant shows impaired embryo and seedling development associated with cell de-differentiation and proliferation. This process is specifically enhanced in presence of cytokinins leading to callus-like structure of the apical part of the seedling. Cell proliferation concerns localized and stochastic nodules of dividing cells. In absence of cytokinins, cell proliferation leads to small calli on stems but, most often, cell proliferation is associated with post-genital organ fusion. The PAS2 gene was identified by positional cloning. PAS2 expression was found in every plant organ and was not regulated by PAS1 and PAS3 genes. PAS2 encodes the Arabidopsis member of the protein tyrosine phosphatase-like (Ptpl) family, a new PTP family originally described in mice and humans and characterized by a mutated PTP active site. This family of proteins has a yeast homolog that is essential for cell viability. The absence of yeast PAS2 homolog can be functionally replaced by the Arabidopsis PAS2 protein, demonstrating that PAS2 function is conserved between higher and lower eukaryotes.     

Glucose stimulates the tyrosine phosphorylation of Crk-associated substrate in pancreatic beta-cells

Several years ago, we demonstrated that glucose induced tyrosine phosphorylation of a 125-kDa protein (p125) in pancreatic beta-cells (Konrad, R. J., Dean, R. M., Young, R. A., Bilings, P. C., and Wolf, B. A. (1996) J. Biol. Chem. 271, 24179-24186). Glucose induced p125 tyrosine phosphorylation in beta-TC3 insulinoma cells, beta-HC9 cells, and in freshly isolated rat islets, whereas increased tyrosine phosphorylation was not observed with other fuel secretagogues. Initial efforts to identify p125 were unsuccessful, so a new approach was taken. The protein was purified from betaTC6,F7 cells via an immunodepletion method. After electrophoresis and colloidal Coomassie Blue staining, the area of the gel corresponding to p125 was excised and subjected to tryptic digestion. Afterward, mass spectrometry was performed and the presence of Crk-associated substrate (Cas) was detected. Commercially available antibodies against Cas were obtained and tested directly in beta-cells, confirming glucose-induced tyrosine phosphorylation of Cas. Further experiments demonstrated that in beta-cells the glucose-induced increase in Cas tyrosine phosphorylation occurs immediately and is not accompanied by increased focal adhesion kinase tyrosine phosphorylation. Finally, it is also demonstrated via Western blotting that Cas is present in normal isolated rat islets. Together, these results show that the identity of the previously described p125 beta-cell protein is Cas and that Cas undergoes rapid glucose-induced tyrosine phosphorylation in beta-cells.     

Functions of the ectodomain and cytoplasmic tyrosine phosphatase domains of receptor protein tyrosine phosphatase Dlar in vivo

The receptor protein tyrosine phosphatase (PTPase) Dlar has an ectodomain consisting of three immunoglobulin (Ig)-like domains and nine fibronectin type III (FnIII) repeats and a cytoplasmic domain consisting of two PTPase domains, membrane-proximal PTP-D1 and C-terminal PTP-D2. A series of mutant Dlar transgenes were introduced into the Drosophila genome via P-element transformation and were then assayed for their capacity to rescue phenotypes caused by homozygous loss-of-function genotypes. The Ig-like domains, but not the FnIII domains, are essential for survival. Conversely, the FnIII domains, but not the Ig-like domains, are required during oogenesis, suggesting that different domains of the Dlar ectodomain are involved in distinct functions during Drosophila development. All detectable PTPase activity maps to PTP-D1 in vitro. The catalytically inactive mutants of Dlar were able to rescue Dlar(-/-) lethality nearly as efficiently as wild-type Dlar transgenes, while this ability was impaired in the PTP-D2 deletion mutants DlarDeltaPTP-D2 and Dlar(bypass). Dlar-C1929S, in which PTP-D2 has been inactivated, increases the frequency of bypass phenotype observed in Dlar(-/-) genotypes, but only if PTP-D1 is catalytically active in the transgene. These results indicate multiple roles for PTP-D2, perhaps by acting as a docking domain for downstream elements and as a regulator of PTP-D1.     

Role of the PDZ domains in Escherichia coli DegP protein

PDZ domains are modular protein interaction domains that are present in metazoans and bacteria. These domains possess unique structural features that allow them to interact with the C-terminal residues of their ligands. The Escherichia coli essential periplasmic protein DegP contains two PDZ domains attached to the C-terminal end of the protease domain. In this study we examined the role of each PDZ domain in the protease and chaperone activities of this protein. Specifically, DegP mutants with either one or both PDZ domains deleted were generated and tested to determine their protease and chaperone activities, as well as their abilities to sequester unfolded substrates. We found that the PDZ domains in DegP have different roles; the PDZ1 domain is essential for protease activity and is responsible for recognizing and sequestering unfolded substrates through C-terminal tags, whereas the PDZ2 domain is mostly involved in maintaining the hexameric cage of DegP. Interestingly, neither of the PDZ domains was required for the chaperone activity of DegP. In addition, we found that the loops connecting the protease domain to PDZ1 and connecting PDZ1 to PDZ2 are also essential for the protease activity of the hexameric DegP protein. New insights into the roles of the PDZ domains in the structure and function of DegP are provided. These results imply that DegP recognizes substrate molecules targeted for degradation and substrate molecules targeted for refolding in different manners and suggest that the substrate recognition mechanisms may play a role in the protease-chaperone switch, dictating whether the substrate is degraded or refolded.     

Expression and function of the extracellular calcium-sensing receptor in pancreatic beta-cells

The extracellular calcium-sensing receptor (CaR) was first identified in tissues involved in systemic Ca2+ homeostasis, where it acts to sense changes in circulating Ca2+. It has since been reported that the CaR is expressed in many tissues that are not associated with Ca2+ homeostasis, including the endocrine cells in pancreatic islets of Langerhans. In the present study we have used an insulin-secreting pancreatic beta-cell line (MIN6) to investigate the expression and function of CaR, using the calcimimetic A568, a CaR agonist that activates the CaR at physiological concentrations of extracellular Ca2+ ([Ca2+]o). Immunocytochemistry, Western blotting and RT-PCR confirmed the expression of CaR in MIN6 cells. CaR activation was associated with rapid and transient increases in [Ca2+]o, which were accompanied by the initiation of a marked but transient insulin secretory response. Stimulation of beta-cell secretory activity had no detectable effect on CaR mRNA levels, but CaR mRNA was markedly reduced by configuring MIN6 cells into islet- like structures. Our data are consistent with an important function for the beta-cell CaR in cell - cell communication within islets to co-ordinate insulin secretory responses.     

Dynamics of glucose-induced membrane recruitment of protein kinase C beta II in living pancreatic islet beta-cells

The mechanisms by which glucose may affect protein kinase C (PKC) activity in the pancreatic islet beta-cell are presently unclear. By developing adenovirally expressed chimeras encoding fusion proteins between green fluorescent protein and conventional (betaII), novel (delta), or atypical (zeta) PKCs, we show that glucose selectively alters the subcellular localization of these enzymes dynamically in primary islet and MIN6 beta-cells. Examined by laser scanning confocal or total internal reflection fluorescence microscopy, elevated glucose concentrations induced oscillatory translocations of PKCbetaII to spatially confined regions of the plasma membrane. Suggesting that increases in free cytosolic Ca(2+) concentrations ([Ca(2+)](c)) were primarily responsible, prevention of [Ca(2+)](c) increases with EGTA or diazoxide completely eliminated membrane recruitment, whereas elevation of cytosolic [Ca(2+)](c) with KCl or tolbutamide was highly effective in redistributing PKCbetaII both to the plasma membrane and to the surface of dense core secretory vesicles. By contrast, the distribution of PKCdelta.EGFP, which binds diacylglycerol but not Ca(2+), was unaffected by glucose. Measurement of [Ca(2+)](c) immediately beneath the plasma membrane with a ratiometric "pericam," fused to synaptic vesicle-associated protein-25, revealed that depolarization induced significantly larger increases in [Ca(2+)](c) in this domain. These data demonstrate that nutrient stimulation of beta-cells causes spatially and temporally complex changes in the subcellular localization of PKCbetaII, possibly resulting from the generation of Ca(2+) microdomains. Localized changes in PKCbetaII activity may thus have a role in the spatial control of insulin exocytosis.     

The PDZ binding motif of the beta 1 adrenergic receptor modulates receptor trafficking and signaling in cardiac myocytes

Beta(1) and beta(2) adrenergic receptors (AR) regulate the intrinsic contraction rate in neonatal mouse cardiac myocytes through distinct signaling pathways. It has been shown that stimulation of beta(1)ARs leads to a protein kinase A-dependent increase in contraction rate. In contrast, stimulation of beta(2)ARs has a biphasic effect on contraction rate, with an initial protein kinase A-independent increase followed by a sustained decrease that is blocked by pertussis toxin. The beta(2)AR undergoes agonist-induced endocytosis in cardiac myocytes while the beta(1)AR remains on the cell surface. It has been shown that a PDZ domain binding motif at the carboxyl terminus of beta(1)AR interacts with the postsynaptic density protein PSD-95 when both are expressed in HEK293 cells. We found that mutation of this PDZ binding motif in the beta(1)AR (beta(1)AR-PDZ) enabled agonist-induced internalization in cardiac myocytes. Moreover, stimulation of beta(1)AR-PDZ had a biphasic effect on the myocyte contraction rate similar to that observed following stimulation of the beta(2)AR. The secondary decrease in the contraction rate was mediated by G(i) and could be blocked by pertussis toxin. Furthermore, a non-selective endocytosis inhibitor, concanavalin A, inhibited the internalization of wild type beta(2)AR and the mutated beta(1)AR-PDZ, and blocked the coupling of both receptors to G(i). Finally, treating myocytes with a membrane-permeable peptide representing beta(1)AR PDZ motif caused the endogenous beta(1)AR to behave like beta(1)AR-PDZ. These studies suggest that association of the beta(1)AR with PSD-95 or a related protein dictates signaling specificity by retaining the receptor at the cell surface and preventing interaction with G(i).     

The zyxin-related protein TRIP6 interacts with PDZ motifs in the adaptor protein RIL and the protein tyrosine phosphatase PTP-BL

The small adaptor protein RIL consists of two segments, the C-terminal LIM and the N-terminal PDZ domain, which mediate multiple protein-protein interactions. The RIL LIM domain can interact with PDZ domains in the protein tyrosine phosphatase PTP-BL and with the PDZ domain of RIL itself. Here, we describe and characterise the interaction of the RIL PDZ domain with the zyxin-related protein TRIP6, a protein containing three C-terminal LIM domains. The second LIM domain in TRIP6 is sufficient for a strong interaction with RIL. A weaker interaction with the third LIM domain in TRIP6, including the proper C-terminus, is also evident. TRIP6 also interacts with the second out of five PDZ motifs in PTP-BL. For this interaction to occur both the third LIM domain and the proper C-terminus are necessary. RNA expression analysis revealed overlapping patterns of expression for TRIP6, RIL and PTP-BL, most notably in tissues of epithelial origin. Furthermore, in transfected epithelial cells TRIP6 can be co-precipitated with RIL and PTP-BL PDZ polypeptides, and a co-localisation of TRIP6 and RIL with Factin structures is evident. Taken together, PTP-BL, RIL and TRIP6 may function as components of multi-protein complexes at actin-based sub-cellular structures.     

Inducible nitric-oxide synthase and nitric oxide donor decrease insulin receptor substrate-2 protein expression by promoting proteasome-dependent degradation in pancreatic beta-cells: involvement of glycogen synthase kinase-3beta

Insulin receptor substrate-2 (IRS-2) plays a critical role in the survival and function of pancreatic β-cells. Gene disruption of IRS-2 results in failure of the β-cell compensatory mechanism and diabetes. Nonetheless, the regulation of IRS-2 protein expression in β-cells remains largely unknown. Inducible nitric-oxide synthase (iNOS), a major mediator of inflammation, has been implicated in β-cell damage in type 1 and type 2 diabetes. The effects of iNOS on IRS-2 expression have not yet been investigated in β-cells. Here, we show that iNOS and NO donor decreased IRS-2 protein expression in INS-1/832 insulinoma cells and mouse islets, whereas IRS-2 mRNA levels were not altered. Interleukin-1β (IL-1β), alone or in combination with interferon-γ (IFN-γ), reduced IRS-2 protein expression in an iNOS-dependent manner without altering IRS-2 mRNA levels. Proteasome inhibitors, MG132 and lactacystin, blocked the NO donor-induced reduction in IRS-2 protein expression. Treatment with NO donor led to activation of glycogen synthase kinase-3β (GSK-3β) and c-Jun N-terminal kinase (JNK/SAPK) in β-cells. Inhibition of GSK-3β by pharmacological inhibitors or siRNA-mediated knockdown significantly prevented NO donor-induced reduction in IRS-2 expression in β-cells. In contrast, a JNK inhibitor, SP600125, did not effectively block reduced IRS-2 expression in NO donor-treated β-cells. These data indicate that iNOS-derived NO reduces IRS-2 expression by promoting protein degradation, at least in part, through a GSK-3β-dependent mechanism. Our findings suggest that iNOS-mediated decreased IRS-2 expression may contribute to the progression and/or exacerbation of β-cell failure in diabetes.     

The molecular mechanism of EGF receptor activation in pancreatic beta-cells by thyrotropin-releasing hormone

Thyrotropin-releasing hormone (TRH) and its receptor subtype TRH receptor-1 (TRHR1) are found in pancreatic beta-cells, and it has been shown that TRH might have potential for autocrine/paracrine regulation through the TRHR1 receptor. In this paper, TRHR1 is studied to find whether it can initiate multiple signal transduction pathways to activate the epidermal growth factor (EGF) receptor in pancreatic beta-cells. By initiating TRHR1 G protein-coupled receptor (GPCR) and dissociated alphabetagamma-complex, TRH (200 nM) activates tyrosine residues at Tyr845 (a known target for Src) and Tyr1068 in the EGF receptor complex of an immortalized mouse beta-cell line, betaTC-6. Through manipulating the activation of Src, PKC, and heparin-binding EGF-like growth factor (HB-EGF), with corresponding individual inhibitors and activators, multiple signal transduction pathways linking TRH to EGF receptors in betaTC-6 cell line have been revealed. The pathways include the activation of Src kinase and the release of HB-EGF as a consequence of matrix metalloproteinase (MMP)-3 activation. Alternatively, TRH inhibited PKC activity by reducing the EGF receptor serine/threonine phosphorylation, thereby enhancing tyrosine phosphorylation. TRH receptor activation of Src may have a central role in mediating the effects of TRH on the EGF receptor. The activation of the EGF receptor by TRH in multiple circumstances may have important implications for pancreatic beta-cell biology.     

The carboxyl terminus of Neph family members binds to the PDZ domain protein zonula occludens-1

The PSD95/Dlg/ZO-1 (PDZ) domain-containing protein zonula occludens-1 (ZO-1) selectively localizes to the cytoplasmic basis of the slit diaphragm, a specialized cell-cell contact in between glomerular podocytes necessary to prevent the loss of protein in the urine. However, the function of ZO-1 at the slit diaphragm has remained elusive. Deletion of Neph1, a slit diaphragm protein of the immunoglobulin superfamily with a cytoplasmic PDZ binding site, causes proteinuria in mice. We demonstrate now that Neph1 binds ZO-1. This interaction was mediated by the first PDZ domain of ZO-1 and involved the conserved PDZ domain binding motif present in the carboxyl terminus of the three known Neph family members. Furthermore, Neph1 co-immunoprecipitates with ZO-1 from lysates of mouse kidneys, demonstrating that this interaction occurs in vivo. Both deletion of the PDZ binding motif of Neph1 as well as threonine-to-glutamate mutation of the threonine within the binding motif abrogated binding of ZO-1, suggesting that phosphorylation may regulate this interaction. ZO-1 binding was associated with a strong increase in tyrosine phosphorylation of the cytoplasmic tail of Neph1 and dramatically accelerated the ability of Neph1 to induce signal transduction. Thus, our data suggest that ZO-1 may organize Neph proteins and recruit signal transduction components to the slit diaphragm of podocytes.     

The protein kinase C-related kinase PRK2 interacts with the protein tyrosine phosphatase PTP-BL via a novel PDZ domain binding motif

Protein tyrosine phosphatase-basophil like (PTP-BL) is a large non-transmembrane protein tyrosine phosphatase implicated in the modulation of the cytoskeleton. Here we describe a novel interaction of PTP-BL with the protein kinase C-related kinase 2 (PRK2), a serine/threonine kinase regulated by the G-protein rho. This interaction is mediated by the PSD-95, Drosophila discs large, zonula occludens (PDZ)3 domain of PTP-BL and the extreme C-terminus of PRK2 as shown by yeast two-hybrid assays and coimmunoprecipitation experiments from transfected HeLa cells. In particular, we demonstrate that a conserved C-terminal cysteine of PRK2 is indispensable for the interaction with PTP-BL. In HeLa cells we demonstrate colocalization of both proteins in lamellipodia like structures. Interaction of PTP-BL with the rho effector kinase PRK2 gives further evidence for a possible function of PTP-BL in the regulation of the actin cytoskeleton.