Insulin receptor substrate and p55 orthologous adaptor proteins function in the Caenorhabditis elegans daf-2/insulin-like signaling pathway

An insulin-like signaling pathway regulates development and lifespan in Caenorhabditis elegans. Genetic screens that identified many components of the C. elegans insulin pathway did not identify homologs of insulin receptor substrates or the phosphoinositide 3-kinase (PI3K) adaptor/regulatory subunit, which are both required for signaling by mammalian insulin/insulin-like growth factor I pathways. The C. elegans genome contains one homolog of each protein. The C. elegans versions of insulin receptor substrate (IST-1) and PI3K p50/p55 (AAP-1) share moderate sequence similarity with their vertebrate and Drosophila counterparts. Genetic experiments show that ist-1 and aap-1 potentiate C. elegans insulin-like signaling, although they are not required for signaling in the pathway under most conditions. Worms lacking AAP-1 activity because of the mutation aap-1(m889) constitutively arrest development at the dauer larval stage when raised at high temperatures. aap-1 mutants also live longer than wild-type animals, a phenotype observed in other C. elegans mutants with defects in DAF-2 signaling. Interestingly, IST-1 appears to be required for signaling through a pathway that may act in parallel to AGE-1/PI3K.     

14-3-3 proteins regulate glycogen synthase 3beta phosphorylation and inhibit cardiomyocyte hypertrophy

14-3-3 proteins are dimeric phophoserine-binding molecules that participate in important cellular processes such as cell proliferation, cell-cycle control and the stress response. In this work, we report that several isoforms of 14-3-3s are expressed in neonatal rat cardiomyocytes. To understand their function, we utilized a general 14-3-3 peptide inhibitor, R18, to disrupt 14-3-3 functions in cardiomyocytes. Cardiomyocytes infected with adenovirus-expressing YFP-R18 (AdR18) exhibited markedly increased protein synthesis and atrial natriuretic peptide production and potentiated the responses to norepinephrine stimulation. This response was blocked by the pretreatment with LY294002, a phosphoinositide 3-kinase (PI3K) inhibitor. Consistent with a role of PI3K in the R18 effect, R18 induced phosphorylation of a protein cloned from the vakt oncogene of retrovirus AKT8 (Akt - also called protein kinase B, PKB) at Ser473 and glycogen synthase 3beta (GSK3beta) at Ser9, but not extracellular signal-regulated kinase 1/2 (ERK1/2). AdR18-induced PKB and GSK3beta phosphorylation was completely blocked by LY294002. In addition, a member of the nuclear factor of activated T cells (NFAT) family, NFAT3, was converted into faster mobility forms and translocated into the nucleus upon the treatment of AdR18. These results suggest that 14-3-3s inhibits cardiomyocytes hypertrophy through regulation of the PI3K/PKB/GSK3beta and NFAT pathway.     

Identification of nonsteroidal anti-inflammatory drug-activated gene (NAG-1) as a novel downstream target of phosphatidylinositol 3-kinase/AKT/GSK-3beta pathway

The signaling pathway of phosphatidylinositol 3-kinase (PI3K)/AKT, which is involved in cell survival, proliferation, and growth, has become a major focus in targeting cancer therapeutics. Nonsteroidal anti-inflammatory drug-activated gene (NAG-1) was previously identified as a gene induced by several anti-tumorigenic compounds including nonsteroidal anti-inflammatory drugs, peroxisome proliferator-activated receptor gamma ligands, and dietary compounds. NAG-1 has been shown to exhibit anti-tumorigenic and/or pro-apoptotic activities in vivo and in vitro. In this report, we showed a PI3K/AKT/glycogen synthase kinase-3beta (GSK-3beta) pathway regulates NAG-1 expression in human colorectal cancer cells as assessed by the inhibition of PI3K, AKT, and GSK-3beta. PI3K inhibition by LY294002 showed an increase in NAG-1 protein and mRNA expression, and 1l-6-hydroxymethyl-chiro-inositol 2(R)-2-O-methyl-3-O-octadecylcarbonate (AKT inhibitor) also induced NAG-1 expression. LY294002 caused increased apoptosis, cell cycle, and cell growth arrest in HCT-116 cells. Inhibition of GSK-3beta, which is negatively regulated by AKT, using AR-A014418 and lithium chloride completely abolished LY294002-induced NAG-1 expression as well as the NAG-1 promoter activity. Furthermore, the down-regulation of GSK-3 gene using small interference RNA resulted in a decline of the NAG-1 expression in the presence of LY294002. These data suggest that expression of NAG-1 is regulated by PI3K/AKT/GSK-3beta pathway in HCT-116 cells and may provide a further understanding of the important role of PI3K/AKT/GSK-3beta pathway in tumorigenesis.     

A conserved PTEN/FOXO pathway regulates neuronal morphology during C. elegans development

The phosphatidylinositol 3-kinase (PI3K) signaling pathway is a conserved signal transduction cascade that is fundamental for the correct development of the nervous system. The major negative regulator of PI3K signaling is the lipid phosphatase DAF-18/PTEN, which can modulate PI3K pathway activity during neurodevelopment. Here, we identify a novel role for DAF-18 in promoting neurite outgrowth during development in Caenorhabditis elegans. We find that DAF-18 modulates the PI3K signaling pathway to activate DAF-16/FOXO and promote developmental neurite outgrowth. This activity of DAF-16 in promoting outgrowth is isoform-specific, being effected by the daf-16b isoform but not the daf-16a or daf-16d/f isoform. We also demonstrate that the capacity of DAF-16/FOXO in regulating neuron morphology is conserved in mammalian neurons. These data provide a novel mechanism by which the conserved PI3K signaling pathway regulates neuronal cell morphology during development through FOXO.     

LET-60 RAS modulates effects of insulin/IGF-1 signaling on development and aging in Caenorhabditis elegans

The DAF-2 insulin/insulin-like growth factor 1 (IGF-1) receptor signals via a phosphatidylinositol 3-kinase (PI3K) pathway to control dauer larva formation and adult longevity in Caenorhabditis elegans. Yet epistasis analysis suggests signal bifurcation downstream of DAF-2. We have used epistasis analysis to test whether the Ras pathway (which plays a role in signaling from mammalian insulin receptors) acts downstream of DAF-2. We find that an activated Ras mutation, let-60(n1046gf), weakly suppresses constitutive dauer diapause in daf-2 and age-1 (PI3K) mutants. Moreover, increased Ras pathway signaling partially suppresses the daf-2 mutant feeding defect, while reduced Ras pathway signaling enhances it. By contrast, activated Ras extends the longevity induced by mutation of daf-2, while reduced Ras pathway signaling partially suppresses it. Thus, Ras pathway signaling appears to act with insulin/IGF-1 signaling during larval development, but against it during aging.     

Insulin regulates alternative splicing of protein kinase C beta II through a phosphatidylinositol 3-kinase-dependent pathway involving the nuclear serine/arginine-rich splicing factor, SRp40, in skeletal muscle cells

Insulin regulates the inclusion of the exon encoding protein kinase C (PKC) betaII mRNA. In this report, we show that insulin regulates this exon inclusion (alternative splicing) via the phosphatidylinositol 3-kinase (PI 3-kinase) signaling pathway through the phosphorylation state of SRp40, a factor required for insulin-regulated splice site selection for PKCbetaII mRNA. By taking advantage of a well known inhibitor of PI 3-kinase, LY294002, we demonstrated that pretreatment of L6 myotubes with LY294002 blocked insulin-induced PKCbetaII exon inclusion as well as phosphorylation of SRp40. In the absence of LY294002, overexpression of SRp40 in L6 cells mimicked insulin-induced exon inclusion. When antisense oligonucleotides targeted to a putative SRp40-binding sequence in the betaII-betaI intron were transfected into L6 cells, insulin effects on splicing and glucose uptake were blocked. Taken together, these results demonstrate a role for SRp40 in insulin-mediated alternative splicing independent of changes in SRp40 concentration but dependent on serine phosphorylation of SRp40 via a PI 3-kinase signaling pathway. This switch in PKC isozyme expression is important for increases in the glucose transport effect of insulin. Significantly, insulin regulation of PKCbetaII exon inclusion occurred in the absence of cell growth and differentiation demonstrating that insulin-induced alternative splicing of PKCbetaII mRNA in L6 cells occurs in response to a metabolic change.     

Phosphatidylinositol 3-kinase is required for integrin-stimulated AKT and Raf-1/mitogen-activated protein kinase pathway activation.

Cell attachment to fibronectin stimulates the integrin-dependent interaction of p85-associated phosphatidylinositol (PI) 3-kinase with integrin-dependent focal adhesion kinase (FAK) as well as activation of the Ras/mitogen-activated protein (MAP) kinase pathway. However, it is not known if this PI 3-kinase-FAK interaction increases the synthesis of the 3-phosphorylated phosphoinositides (3-PPIs) or what role, if any, is played by activated PI 3-kinase in integrin signaling. We demonstrate here the integrin-dependent accumulation of the PI 3-kinase products, PI 3,4-bisphosphate [PI(3,4)P2] and PI(3,4,5)P3, as well as activation of AKT kinase, a serine/threonine kinase that can be stimulated by binding of PI(3,4)P2. The PI 3-kinase inhibitors wortmannin and LY294002 significantly decreased the integrin-induced accumulation of the 3-PPIs and activation of AKT kinase, without having significant effects on the levels of PI(4,5)P2 or tyrosine phosphorylation of paxillin. These inhibitors also reduced cell adhesion/spreading onto fibronectin but had no effect on attachment to polylysine. Interestingly, integrin-mediated Erk-2, Mek-1, and Raf-1 activation, but not Ras-GTP loading, was inhibited at least 80% by wortmannin and LY294002. In support of the pharmacologic results, fibronectin activation of Erk-2 and AKT kinases was completely inhibited by overexpression of a dominant interfering p85 subunit of PI 3-kinase. We conclude that integrin-mediated adhesion to fibronectin results in the accumulation of the PI 3-kinase products PI(3,4)P2 and PI(3,4,5)P3 as well as the PI 3-kinase-dependent activation of the kinases Raf-1, Mek-1, Erk-2, and AKT and that PI 3-kinase may function upstream of Raf-1 but downstream of Ras in integrin activation of Erk-2 MAP and AKT kinases.     

Phosphatidylinositol 3-kinase-mediated regulation of neuronal apoptosis and necrosis by insulin and IGF-I.

We examined effects of two insulin-like growth factors, insulin and insulin-like growth factor-I (IGF-I), against apoptosis, excitotoxicity, and free radical neurotoxicity in cortical cell cultures. Like IGF-I, insulin attenuated serum deprivation-induced neuronal apoptosis in a dose-dependent manner at 10-100 ng/mL. The anti-apoptosis effect of insulin against serum deprivation disappeared by addition of a broad protein kinase inhibitor, staurosporine, but not by calphostin C, a selective protein kinase C inhibitor. Addition of PD98059, a mitogen-activated protein kinase kinase (MAPKK) inhibitor, blocked insulin-induced activation of extracellular signal-regulated protein kinases (ERK1/2) without altering the neuroprotective effect of insulin. Cortical neurons underwent activation of phosphatidylinositol (PI) 3-kinase as early as 1 min after exposure to insulin. Inclusion of wortmannin or LY294002, selective inhibitors of PI 3-K, reversed the insulin effect against apoptosis. In contrast to the anti-apoptosis effect, neither insulin nor IGF-I protected excitotoxic neuronal necrosis following continuous exposure to 15 microM N-methyl-D-aspartate or 40 microM kainate for 24 h. Surprisingly, concurrent inclusion of 50 ng/mL insulin or IGF-I aggravated free radical-induced neuronal necrosis over 24 h following continuous exposure to 10 microM Fe2+ or 100 microM buthionine sulfoximine. Wortmannin or LY294002 also reversed this potentiation effect of insulin. These results suggest that insulin-like growth factors act as anti-apoptosis factor and pro-oxidant depending upon the activation of PI 3-kinase.