Degradation of the cyclic AMP antagonist prostaglandylinositol cyclic phosphate (cyclic PIP) by dephosphorylation

The cAMP antagonist, prostaglandylinositol cyclic phosphate (cyclic PIP), is synthesized from prostaglandin E and activated inositol phosphate. From various tissues only that amount of cyclic PIP can be isolated that constitutes the difference between synthesis and degradation. In order to overcome this drawback, the cyclic PIP degrading enzyme or enzymes had to be characterized prior to searching for inhibitors. Cyclic PIP degrading activities have been found in all rat tissues tested, and are lowest in brain (380 pmol x min(-1) x g(-1) wet weight) and highest in liver (1460 pmol x min(-1) x g(-1) wet weight). They are associated primarily with particulate structures of the cells, but not with the plasma membrane. There appear to be at least two different enzymatic activities involved in the degradation of cyclic PIP, because there are two pH-optima, one between pH 7 and 8 and another between pH 4 and 5. It is assumed that these activities are located in microsomes and lysosomes. Because prostaglandylinositol is the final product obtained in the degradation of cyclic PIP, a phosphodiesterase and a phosphatase should be involved, which could not yet be identified individually. Like alkaline phosphatase, cyclic PIP-degrading enzymes require Mg2+ and they are inhibited by heavy metal ions such as mercuric and copper chloride, by sodium fluoride and interestingly, by prostaglandins.     

Spatial compartmentalization of signal transduction in insulin action

Insulin resistance is thought to be the primary defect in the pathophysiology of type 2 diabetes. Thus, understanding the cellular mechanisms of insulin action may contribute significantly to developing new treatments for this disease. Although the effects of insulin on glucose and lipid metabolism are well documented, gaps remain in our understanding of the precise molecular mechanisms of signal transduction for the hormone. One potential clue to understanding the unique cellular effects of insulin may lie in the compartmentalization of signaling molecules and metabolic enzymes. We review this evidence, and speculate on how PI-3 kinase-independent and -dependent signaling pathways both diverge from the insulin receptor and converge at discrete targets to insure the specificity of insulin action.     

Coordinated action of protein tyrosine phosphatases in insulin signal transduction.

Insulin is the principal regulatory hormone involved in the tight regulation of fuel metabolism. In response to blood glucose levels, it is secreted by the beta cells of the pancreas and exerts its effects by binding to cell surface receptors that are present on virtually all cell types and tissues. In humans, perturbations in insulin function and/or secretion lead to diabetes mellitus, a severe disorder primarily characterized by an inability to maintain blood glucose homeostasis. Furthermore, it is estimated that 90-95% of diabetic patients exhibit resistance to insulin action. Thus an understanding of insulin signal transduction and insulin resistance at the molecular level is crucial to the understanding of the pathogenesis of this disease. The insulin receptor (IR) is a transmembrane tyrosine kinase that becomes activated upon ligand binding. Consequently, the receptor and its downstream substrates become tyrosine phosphorylated. This activates a series of intracellular signaling cascades which coordinately initiate the appropriate biological response. One important mechanism by which insulin signaling is regulated involves the protein tyrosine phosphatases (PTPs), which may either act on the IR itself and/or its substrates. Two well characterized examples include leuckocyte antigen related (LAR) and protein tyrosine phosphatase-1B (PTP-1B). The present review will discuss the current knowledge of these two and other potential PTPs involved in the insulin signaling pathway.     

Phosphatidylinositol transfer proteins: a requirement in signal transduction and vesicle traffic

Phosphatidylinositol transfer protein (PITP) was originally identified and named because of its ability to transport phosphatidylinositol through the aqueous phase from one membrane compartment to another. Recent data, however, indicate unanticipated roles for PITP in the coupling of PIP₂ synthesis to signal transduction reactions and to membrane traffic in mammalian cells. PITP was recently purified on the basis of its ability to restore cellular functions in permeabilized cells depleted of cytosolic proteins. These functions include cell-surface receptor-regulated hydrolysis of PIP₂ by phospholipases Cβ- and γ-isozymes, regulated release of secretory granules, and the budding of constitutive secretory vesicles and immature secretory granules from the trans-Golgi network. In the yeast Saccharomyces cerevisiae, a PITP was identified from a mutant strain with a defect in the secretory pathway (SEC14) and therefore required for cell viability; in Yarrowia lipolytica, PITP is required for differentiation from a yeast to a mycelial growth form. We are just beginning to unravel the intriguing mechanisms by which PITP/SEC14 may accomplish its function in eukaryotic cells in signal transduction and membrane trafficking. BioEssays 20 :423-432, 1998. © 1998 John Wiley & Sons Inc.     

Purification and partial characterization of a putative mediator of insulin action on cyclic AMP-dependent protein kinase

Summary An insulin mediator which inhibits cAMP-dependent protein kinase has been purified approximately 1 000 2 000-fold from skeletal muscle. Following heat treatment, charcoal adsorption and Sephadex G-25 sieving, Sephadex G-15 sieving and HPLC over an anion exchange column were performed. The mediator has characteristics of a relatively low molecular weight peptide or derivatized peptide which acts on cAMP-dependent protein kinase but not on mitochondrial pyruvate dehydrogenase.     

Zinc as a possible mediator of signal transduction in T lymphocytes

Our recent findings indicate that phorbol esters, the specific activators of protein kinase C induce the translocation of heavy metals (mostly: zinc) from the nucleus and mitochondria to the cytosol and microsomes of T lymphocytes. Phorbol ester treatment impairs the action of Ca-ionophores, this effect is mediated by intracellular heavy metal ions (most probably: by zinc). Zinc activates cytosolic protein kinase C, increases its affinity towards phorbol esters and contributes to its binding to plasma membranes. These results suggest that zinc may play a role in the "cross-talk" of second messengers and hence in signal transduction in T lymphocytes.     

蛋白激酶B及其在磷脂酰肌醇3-激酶介导的信号转导中的作用

蛋白激酶Ｂ（ＰＫＢ）是原癌基因ｃ－ａｋｔ的表达产物,它参与由生长因子激活的经磷脂磷肌醇３－激酶（ＰＩ３Ｋ）介导的信号转导过程。与许多蛋白激酶相似,ＰＫＢ分子具有一特殊的ＡＨ／ＰＨ结构域（ＡＨ／ＰＨｄｏｍａｉｎ）,后者能介导信号分子间的相互作用。ＰＫＢ是ＰＩ３Ｋ直接的靶蛋白。ＰＩ３Ｋ产生的脂类第二信使ＰＩ－３,４,Ｐ２和ＰＩ－３,４,５－Ｐ３等均能与ＰＫＢ和磷酸肌醇依赖性蛋白激酶（ＰＤＫ）的ＡＨ／Ｐ     

Role of PI 3-kinase and PIP3 in submandibular gland branching morphogenesis

The mouse submandibular gland (SMG) epithelium undergoes extensive morphogenetic branching during embryonic development as the first step in the establishment of its glandular structure. However, the specific signaling pathways required for SMG branching morphogenesis are not well understood. Using E13 mouse SMG organ cultures, we showed that inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase), wortmannin and LY294002, substantially inhibited branching morphogenesis in SMG. Branching morphogenesis of epithelial rudiments denuded of mesenchyme was inhibited similarly, indicating that PI 3-kinase inhibitors act directly on the epithelium. Immunostaining and Western analysis demonstrated that the p85 isoform of PI 3-kinase is expressed in epithelium at levels higher than in the mesenchyme. A target of PI 3-kinase, Akt/protein kinase B (PKB), showed decreased phosphorylation at Ser(473) by Western analysis in the presence of PI 3-kinase inhibitors. The major lipid product of PI 3-kinase, phosphatidylinositol 3,4,5-trisphosphate (PIP(3)), was added exogenously to SMG via a membrane-transporting carrier in the presence of PI 3-kinase inhibitors and was found to stimulate cleft formation, the first step of branching morphogenesis. Together, these data indicate that PI 3-kinase plays a role in the regulation of epithelial branching morphogenesis in mouse SMG acting through a PIP(3) pathway.     

miRNA15a regulates insulin signal transduction in the retinal vasculature

We previously reported that tumor necrosis factor alpha (TNFα) could inhibit insulin signal transduction in retinal cells. We recently found that miR15a/16 also reduced TNFα in retinal endothelial cells (REC) and in vascular specific miR15a/16 knockout mice. Since in silico programs suggested that miR15a could directly bind the insulin receptor, we wanted to determine whether miR15a altered insulin signal transduction. We used a luciferase-based binding assay to determine whether miR15a directly bound the insulin receptor. We then used Western blotting, ELISA, and qPCR to investigate whether miR15a altered insulin signaling proteins in REC and in both miR15a/16 endothelial cell knockout and overexpressing mice. We also treated some REC with resveratrol to determine if resveratrol could increase miR15a expression, since resveratrol is protective to the diabetic retina. We found that miR15a directly bound the 3’UTR of the insulin receptor. Treatment with resveratrol increased miR15a expression in REC grown in high glucose. While total insulin receptor levels were not altered, insulin signal transduction was reduced in REC grown in high glucose and was restored with treatment with resveratrol. miR15a knockout mice had reduced insulin receptor phosphorylation and Akt2 levels, with increased insulin receptor substrate 1 (IRS-1) phosphorylation on serine 307, a site known to inhibit insulin signaling. In contrast, overexpression of miR15a increased insulin signal transduction. Taken together, these data suggest that miR15a binds the insulin receptor and indirectly regulates insulin receptor actions. It also offers an additional mechanism by which resveratrol is protective to the diabetic retina.     

Phosphatidylinositol 3-kinase is a key regulator of early phase differentiation in keratinocytes

The survival and growth of epithelial cells depend on adhesion to the extracellular matrix. Because epidermal keratinocytes differentiate as they leave the basement membrane, an adhesion signal may regulate the initiation of differentiation. Phosphatidylinositol 3-kinase (PI3K) is a fundamental signaling molecule that regulates the adhesion signal. Transfection of a dominant negative form of PI3K into keratinocytes using an adenovirus vector resulted in significant morphological changes comparable to differentiation and the induction of differentiation markers, keratin (K) 1 and K10. In turn, transfection with the constitutively active form of PI3K almost completely abolished the induction of K1 and K10 by differentiation in suspension cultures using polyhydroxyethylmethacrylate-coated dishes. PI3K activity was lost in suspension culture, except by cells bearing the constitutively active form of PI3K. These data demonstrate that blockade of PI3K results in differentiation and that activation of PI3K prevents differentiation. Furthermore, expression of the dominant negative form of PI3K significantly inhibited keratinocyte adhesion to the extracellular matrix and reduced the surface expression of alpha(6) and beta(1) integrins in suspension culture. Moreover, expression of the active form of PI3K restored the mRNA levels of adhesion molecules that were reduced in suspension culture, including alpha(3), alpha(6), and beta(1) integrins, BP180, and BP230. In conclusion, loss of PI3K activity results in keratinocytes leaving the basement membrane and the initiation of a "default" differentiation mechanism.     

Phosphatidylinositol 3-kinase and its novel product, phosphatidylinositol 3-phosphate, are present in Saccharomyces cerevisiae

The metabolism of polyphosphoinositides has been shown to be an important factor in controlling the proliferation of Saccharomyces cerevisiae. The monophosphate form of phosphatidylinositol has been assumed to be phosphatidylinositol 4-phosphate (PI-4-P). Recent evidence from our laboratory has established that a phosphatidylinositol (PI) kinase, which phosphorylates the D-3 position of the inositol ring (PI 3-kinase), is associated with many activated protein-tyrosine kinases and may play an important role in the signaling of cell proliferation (Auger, K. R., Serunian, L. A., Soltoff, S. P., Libby, P., and Cantley, L. C. (1989) Cell 57, 167-175). To determine the evolutionary conservation of this enzymatic activity, we investigated its presence in yeast. In vitro PI kinase assays of yeast cell homogenates demonstrated that PI 3-kinase activity was present. Preliminary biochemical characterization of the activity suggested that it was quite different from the mammalian enzyme yet catalyzed the same reaction, i.e. phosphorylating the D-3 hydroxyl position of the inositol ring of phosphatidyl-myo-inositol. [3H]Inositol labeling of intact yeast cells with the subsequent extraction, deacylation, and high performance liquid chromatography analysis of the lipids demonstrated that PI-3-P was as abundant as the PI-4-P isomer. The conservation of the enzymatic activity from yeast to man suggests that it has an important functional role in the cell cycle.     

c-Cbl acts as a mediator of Src-induced activation of the PI3K-Akt signal transduction pathway during TRAIL treatment

We have previously observed that TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) induces acquired TRAIL resistance by increasing Akt phosphorylation and Bcl-xL expression. In this study, we report that Src, c-Cbl, and PI3K are involved in the phosphorylation of Akt during TRAIL treatment. Data from immunoprecipitation and immunoblotting assay reveal that Src interacts with c-Cbl and PI3K. Data from immune complex kinase assay demonstrate that Src can directly phosphorylate c-Cbl and PI3K p85 subunit protein. Data from gene knockdown experiments with an RNA interference (RNAi) technique show that c-Cbl is involved in the interaction between Src and PI3K p85 during TRAIL treatment, playing an important role in TRAIL-induced Akt phosphorylation. Taken together, c-Cbl may act as a mediator to regulate the Src-PI3K-Akt signal transduction pathway during TRAIL treatment.     

Growth factor receptor-binding protein 10 (Grb10) as a partner of phosphatidylinositol 3-kinase in metabolic insulin action

The regulation of the metabolic insulin response by mouse growth factor receptor-binding protein 10 (Grb10) has been addressed in this report. We find mouse Grb10 to be a critical component of the insulin receptor (IR) signaling complex that provides a functional link between IR and p85 phosphatidylinositol (PI) 3-kinase and regulates PI 3-kinase activity. This regulatory mechanism parallels the established link between IR and p85 via insulin receptor substrate (IRS) proteins. A direct association was demonstrated between Grb10 and p85 but was not observed between Grb10 and IRS proteins. In addition, no effect of mouse Grb10 was observed on the association between IRS-1 and p85, on IRS-1-associated PI 3-kinase activity, or on insulin-mediated activation of IR or IRS proteins. A critical role of mouse Grb10 was observed in the regulation of PI 3-kinase activity and the resulting metabolic insulin response. Dominant-negative Grb10 domains, in particular the SH2 domain, eliminated the metabolic response to insulin in differentiated 3T3-L1 adipocytes. This was consistently observed for glycogen synthesis, glucose and amino acid transport, and lipogenesis. In parallel, the same metabolic responses were substantially elevated by increased levels of Grb10. A similar role of Grb10 was confirmed in mouse L6 cells. In addition to the SH2 domain, the Pro-rich amino-terminal region of Grb10 was implicated in the regulation of PI 3-kinase catalytic activity. These regulatory roles of Grb10 were extended to specific insulin mediators downstream of PI 3-kinase including PKB/Akt, glycogen synthase kinase, and glycogen synthase. In contrast, a regulatory role of Grb10 in parallel insulin response pathways including p70 S6 kinase, ubiquitin ligase Cbl, or mitogen-activated protein kinase p38 was not observed. The dissection of the interaction of mouse Grb10 with p85 and the resulting regulation of PI 3-kinase activity should help elucidate the complexity of the IR signaling mechanism.     

Involvement of phosphoinositide-3-kinase and phospholipase C transduction systems in the non-genomic action of progesterone in vascular tissue

We investigate the participation of tyrosine kinase, phosphatidylinositol-3-kinase, phospholipase C systems in the intracellular transduction pathways involved in the non-genomic stimulation of vasodilators compounds synthesis induced by progesterone (Pg). Using aortic strips isolated from female fertile Wistar rats, we showed that physiological concentrations of progesterone markedly increase prostacyclin synthesis in a very short time interval (45 s to 10 min) as well as nitric oxide release (5-30 min). The stimulatory action of progesterone on nitric oxide synthase (NOS) activity was maintained even in the presence of an antagonist of progesterone receptor, compound RU486. In contrast, in the presence of tyrosine kinase inhibitor (1 microM genistein) or phosphatidylinositol-3-kinase inhibitor (1 microM LY294002), the enhancement of nitric oxide elicited by 10-100 nM progesterone was completely suppressed. The steroid stimulates phopholipase C activity, inducing significant increase in diacylglycerol generation (5-15-min treatment). The presence of an inhibitor of protein kinase C (PKC) impaired the anti-aggregatory action of the hormone. Due to the fact that phospholipase C activation implies calcium mobilization, we investigate the role of changes in calcium fluxes on progesterone nitric oxide generation. We found that calcium influx from extracellular medium and calcium mobilization from internal pools was required. The present results suggest that, tyrosine kinase and phosphatidylinositol-3-kinase cascades are involved in progesterone nitric oxide synthase stimulation and that diacilglicerol/protein kinase C system may be relevant for physiological regulation of platelet aggregation process.     

Phosphatidylinositol 3-kinase and Akt participate in the FSH-induced meiotic maturation of mouse oocytes

Phosphatidylinositol 3-kinase (PI3K) is known to play critical roles in signal transduction processes related to a variety of cellular activities. In the present study, we investigated the role of PI3K during meiotic maturation in mouse oocytes using a specific inhibitor, LY294002. In follicle-stimulating hormone (FSH)-induced reversal of hypoxanthine-mediated meiotic arrest of cumulus oocyte complexes (COCs), LY294002 suppressed germinal vesicle breakdown (GVBD), first polar body (PB1) emission, and cumulus expansion. To examine the effect of LY294002, denuded oocytes (DOs) were cultured in medium containing follicular fluid meiosis-activating sterol (FF-MAS) since absence of gonadotropin receptors in oocytes has been reported and FSH did not stimulate meiotic maturation of DOs in the presence of hypoxanthine. In FF-MAS-induced maturation of DOs, LY294002 suppressed PB1emission, but not GVBD. In spontaneous gonadotropin-independent oocyte maturation, LY294002 had no effect on COCs and DOs. Akt/protein kinase B, a serine-threonine kinase, is a key downstream effector of the PI3K pathway. Therefore, we also examined the distribution of Akt during FSH-induced meiotic maturation. The distribution of Ser(473) phosphorylated Akt was similar to the localization of microtubules, while Thr(308) phosphorylated Akt was present in the pericentriolar materials (PCM) in metaphase I (MI) and II (MII) oocytes. LY294002 decreased the amount of Thr(308) phosphorylated Akt to very low to undetectable levels in MI and MII oocytes. Ser(473) phosphorylated Akt showed aberrant distribution and very low to undetectable levels of expression in LY294002-treated MI and MII oocytes, respectively. These results suggest that PI3K and Akt participate in mouse meiotic maturation.     

Effect of insulin on the incorporation of3H-inositol into the inositol phospholipids (PI,PIP, PIP2) and Glycosyl-phoshatidylinositols (GPIs) of Tetrahymena pyriformis

The unicellular tetrahymena contains inositol phospholipids (PI, PIP, PIP₂) and GPIs. Treatment with 10^–5M insulin decreases the total³H-inositol incorporation and incorporation into PI. 24 h after 10^–6M insulin treatment there is an elevation of these parameters. Second treatment with 10^–6M insulin doubles and 10^–5M decreases these levels. This means that the effect on phosphoinositide turnover by insulin in Tetrahymena is rather concentration dependent. Inositol incorporation into GPIs is also influenced by insulin.