Vanadyl sulfate-stimulated glycogen synthesis is associated with activation of phosphatidylinositol 3-kinase and is independent of insulin receptor tyrosine phosphorylation

Salts of the trace element vanadium, such as sodium orthovanadate and vanadyl sulfate (VS), exhibit a myriad of insulin-like effects, including stimulation of glycogen synthesis and improvement of glucose homeostasis in type I and type II animal models of diabetes mellitus. However, the cellular mechanism by which these effects are mediated remains poorly characterized. We have shown earlier that different vanadium salts stimulate the MAP kinase pathway and ribosomal-S-6-kinase (p70s6k) in chinese hamster ovary cells overexpressing human insulin receptor (CHO-HIR cells) [Pandey, S. K., Chiasson, J.-L., and Srivastava, A. K. (1995) Mol. Cell. Biochem. 153, 69-78]. In the present studies, we have investigated if similar to insulin, VS also activates phosphatidylinositol 3-kinase (PI3-k) activity, and whether VS-induced activation of the PI3-k, MAP kinase, and p70s6k pathways contributes to glycogen synthesis. Treatment of CHO-HIR cells with VS resulted in increased glycogen synthesis and PI3-k activity which were blocked by pretreatment of the cells with wortmannin and LY294002, two specific inhibitors of PI3-k. On the other hand, PD98059 and rapamycin, specific inhibitors of the MAP kinase pathway and p70s6k, respectively, were unable to inhibit VS-stimulated glycogen synthesis. Moreover, VS-stimulated glycogen synthesis and PI3-k were observed without any change in the tyrosine phosphorylation of insulin receptor (IR) beta-subunit but were associated with increased tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1). In addition, PI3-k activation was detected in IRS-1 immunoprecipitates from VS-stimulated cells, indicating that tyrosine-phosphorylated IRS-1 was able to interact and thereby activate PI3-k in response to VS. Taken together, these results provide evidence that tyrosine phosphorylation of IRS-1 and activation of PI3-k play a key role in mediating the insulinomimetic effect of VS on glycogen synthesis independent of IR-tyrosine phosphorylation.     

Differential effects of heterogeneous nuclear ribonucleoprotein K on Sp1- and Sp3-mediated transcriptional activation of a neuronal nicotinic acetylcholine receptor promoter

The neuronal nicotinic acetylcholine receptor gene family consists of 11 members, alpha2-alpha9 and beta2-beta4. Three of the genes, those encoding the alpha3, alpha5, and beta4 subunits, are clustered tightly within the genome. These three subunits constitute the predominant acetylcholine receptor subtype expressed in the peripheral nervous system. The genomic proximity of the three genes suggests a regulatory mechanism ensuring their coordinate expression. However, it is likely that gene-specific regulatory mechanisms are also functioning because the expression patterns of the three genes, although similar, are not identical. Previously we identified regulatory elements within the beta4 promoter region and demonstrated that these elements interact specifically with nuclear proteins. One of these elements, E1, interacts with the regulatory factor Puralpha as well as three other unidentified DNA-binding proteins with molecular masses of 31, 65, and 114 kDa. Another element, E2, interacts with Sp1 and Sp3. Because E1 and E2 are immediately adjacent to one another, we postulated that the proteins that bind to the elements interact to regulate beta4 gene expression. Here we report the identification of the 65-kDa E1-binding protein as heterogeneous nuclear ribonucleoprotein K and demonstrate that it affects the transactivation of beta4 promoter activity by Sp1 and Sp3 differentially.     

Integrin-mediated activation of focal adhesion kinase is independent of focal adhesion formation or integrin activation. Studies with activated and inhibitory beta3 cytoplasmic domain mutants

Integrin alphaIIbbeta3 functions as the fibrinogen receptor on platelets and mediates platelet aggregation and clot retraction. Among the events that occur during either "inside-out" or "outside-in" signaling through alphaIIbbeta3 is the phosphorylation of focal adhesion kinase (pp125(FAK)) and the association of pp125(FAK) with cytoskeletal components. To examine the role of pp125(FAK) in these integrin-mediated events, pp125(FAK) phosphorylation and association with the cytoskeleton was determined in cells expressing two mutant forms of alphaIIbbeta3: alphaIIbbeta3(D723A/E726A), a constitutively active integrin in which the putative binding site for pp125(FAK) is altered, and alphaIIbbeta3(F727A/K729E/F730A), in which the putative binding site for alpha-actinin is altered. Both mutants were expressed on the cell surface and were able to bind ligand, either spontaneously or upon activation. Whereas cells expressing alphaIIbbeta3(D723A/E726A) were able to form focal adhesions and stress fibers upon adherence to fibrinogen, cells expressing alphaIIbbeta3(F727A/K729E/F730A) adhere to fibrinogen, but had reduced focal adhesions and stress fibers. pp125(FAK) is recruited to focal adhesions in adherent cells expressing alphaIIbbeta3(D723A/E726A) and is phosphorylated in adherent cells or in cells in suspension in the presence of fibrinogen. In adherent cells expressing alphaIIbbeta3(F727A/K729E/F730A), pp125(FAK) was phosphorylated despite reduced formation of focal adhesions and stress fibers. We conclude that activation of pp125(FAK) can be dissociated from two important events in integrin signaling, the assembly of focal adhesions in adherent cells and integrin activation following ligand occupation.     

Transcriptional regulation of lysosomal acid lipase in differentiating monocytes is mediated by transcription factors Sp1 and AP-2

The spectrin membrane skeleton is a ubiquitous cytoskeletal structure with several cellular roles, including the maintenance of cell integrity, determination of cell shape and as a contributor to cell polarity. We have isolated mutations in the gene encoding &bgr ;Heavy-spectrin in Drosophila, and have named this essential locus karst. karst mutant individuals have a pleiotropic phenotype characterized by extensive larval lethality and, in adult escapers, rough eyes, bent wings, tracheal defects and infertility. Within karst mutant eyes, a significant number of ommatidia specifically lack photoreceptor R7 alongside more complex morphological defects. Immunolocalization of betaHeavy-spectrin in wild-type eye-antennal and wing imaginal discs reveals that betaHeavy-spectrin is present in a restricted subdomain of the membrane skeleton that colocalizes with DE-cadherin. We propose a model where normal levels of Sevenless signaling are dependent on tight cell-cell adhesion facilitated by the betaHeavy-spectrin membrane skeleton. Immunolocalization of betaHeavy-spectrin in the adult and larval midgut indicates that it is a terminal web protein, but we see no gross morphological defects in the adult apical brush border in karst mutant flies. Rhodamine phalloidin staining of karst mutant ovaries similarly reveals no conspicuous defect in the actin cytoskeleton or cellular morphology in egg chambers. This is in contrast to mutations in alpha-spectrin, the molecular partner of betaHeavy-spectrin, which affect cellular structure in both the larval gut and adult ovaries. Our results emphasize the fundamental contribution of the spectrin membrane skeleton to normal development and reveals a critical interplay between the integrity of a cell's membrane skeleton, the structure of cell-cell contacts and cell signaling.