No abstract

The estrogen receptor ␣ (ER), a member of the steroid receptor superfamily, contains an N-terminal hormone-independent transcriptional activation function (AF-1) and a C-terminal hormone-dependent transcriptional activation function (AF-2). Here, we used in-gel kinase assays to determine that pp90 rsk1 activated by either epidermal growth factor (EGF) or phorbol myristate acetate specifically phosphorylates Ser-167 within AF-1. In vitro kinase assays demonstrated that pp90 rsk1 phosphorylates the N terminus of the wild-type ER but not of a mutant ER in which Ser-167 was replaced by Ala. In vivo, EGF stimulated phosphorylation of Ser-167 as well as Ser-118. Ectopic expression of active pp90 rsk1 increased the level of phosphorylation of Ser-167 compared to that of either a mutant pp90 rsk1 , which is catalytically inactive in the N-terminal kinase domain, or to that of vector control. The ER formed a stable complex with the mutant pp90 rsk1 in vivo. Transfection of the mutant pp90 rsk1 depressed ER-dependent transcription of both a wild-type ER and a mutant ER that had a defective AF-2 domain (ER TAF-1). Furthermore, replacing either Ser-118 or Ser-167 with Ala in ER TAF-1 showed similar decreases in transcription levels. A double mutant in which both Ser-118 and Ser-167 were replaced with Ala demonstrated a further decrease in transcription compared to either of the single mutations. Taken together, our results strongly suggest that pp90 rsk1 phosphorylates Ser-167 of the human ER in vivo and that Ser-167 aids in regulating the transcriptional activity of AF-1 in the ER.

Phosphatidylinositol 3-kinase (PI-3 kinase) is implicated in the regulation of diverse cellular processes, including insulin-stimulated glucose transport. PI-3 kinase is composed of a 110-kDa catalytic subunit and an 85-kDa regulatory subunit. Here, we describe p55 PIK , a new regulatory subunit that was isolated by screening expression libraries with tyrosine-phosphorylated insulin receptor substrate 1 (IRS-1). p55PIK is composed of a unique 30-residue NH 2 terminus followed by a proline-rich motif and two Src homology 2 (SH2) domains with significant sequence identity to those in p85. Phosphatidylinositol 3-kinase (PI-3 kinase) is a common signaling element which plays a role in the regulation of a broad array of biological responses by activated receptors for hormones, growth factors, cytokines, and antigens (6,13,15,42,46,48,61). It is composed of a 110-kDa catalytic subunit (p110) associated with an 85-kDa regulatory subunit (p85) that contains one Src homology 3 (SH3) domain, homology to the breakpoint cluster region (bcr) gene, two proline-rich motifs, and two SH2 domains (11). Interestingly, p110␣ displays dual catalytic specificity, as it phosphorylates the D-3 position of phosphatidylinositol and its phosphorylated derivatives and serine residues in p85 and insulin receptor substrate 1 (IRS-1) (12, 32). Mammalian p110 is homologous to VPS34, a Saccharomyces cerevisiae PI-3 kinase which is involved in vacuolar protein sorting (49); however, the molecular role of PI-3 kinase in mammalian cells is unclear (22).The p85 regulatory subunit has a broad potential to couple the PI-3 kinase to multiple signaling elements by employing its SH3 domain, proline-rich motifs, bcr homology region, or SH2 domains (24). Most activated receptors with tyrosine kinase activity engage the SH2 domains in p85 through phosphorylated YXXM motifs in the receptors themselves or a closely associated subunit (52). The platelet-derived growth factor receptor, one of the best-characterized systems, associates directly with the SH2 domains in p85 at a phosphorylated YMDM motif in the kinase insert region (61). Inhibition of PI-3 kinase catalytic activity with wortmannin or disruption of p85 function by site-directed mutagenesis blocks several growth factor-stimulated processes, including mitogenesis and antiapoptosis (61, 67), differentiation (27), receptor trafficking (23), chemotaxis (31, 45), membrane ruffling (29, 64), and insulin-stimulated glucose transport (7,16,44) and Xenopus oocyte maturation (8, 9). In addition, PI-3 kinase appears to be required for the stimulation of p70S6k by platelet-derived growth factor and insulin and probably other growth factors (7,10,40). On the basis of these results, PI-3 kinase plays a central role in cellular signaling.Insulin regulates PI-3 kinase by tyrosine phosphorylation of IRS-1 and IRS-2, multipotential docking proteins which contain multiple potential tyrosine phosphorylation sites, including several YXXM motifs (41, 58). In addition to insulin and insulin-like growth factor 1 (IGF-1), ...

Mitogen-activated protein kinase and one of its targets, pp90rsk (ribosomal S6 kinase [RSK]), represent two serine/threonine kinases in the Ras-activated signalling cascade that are capable of directly regulating gene expression. pp90rsk has been shown to have two highly conserved and distinct catalytic domains. However, whether both domains are active and which domain is responsible for its various identified phosphotransferase activities have not been determined. Here we demonstrate that the N-terminal domain is responsible for its phosphotransferase activity towards a variety of substrates which contain an RXXS motif at the site of in vitro phosphorylation, including serum response factor, c-Fos, Nur77, and the 40S ribosomal protein S6. We also provide evidence that the C-terminal domain is catalytically active and can be further activated by mitogen-activated protein kinase phosphorylation.

The insulin receptor substrate 2 (Irs2) branch of the insulin/insulin-like growth factor-signaling cascade prevents diabetes in mice because it promotes ␤ cell replication, function, and survival, especially during metabolic stress. Because exendin-4 (Ex4), a long acting glucagon-like peptide 1 receptor agonist, has similar effects upon ␤ cells in rodents and humans, we investigated whether Irs2 signaling was required for Ex4 action in isolated ␤ cells and in Irs2 ؊/؊ mice. Ex4 increased cAMP levels in human islets and Min6 cells, which promoted Irs2 expression and stimulated Akt phosphorylation. In wild type mice Ex4 administered continuously for 28 days increased ␤ cell mass 2-fold. By contrast, Ex4 failed to arrest the progressive ␤ cell loss in Irs2 ؊/؊ mice, which culminated in fatal diabetes; however, Ex4 delayed the progression of diabetes by 3 weeks by promoting insulin secretion from the remaining islets. We conclude that some short term therapeutic effects of glucagon-like peptide 1 receptor agonists can be independent of Irs2, but its long term effects upon ␤ cell growth and survival are mediated by the Irs2 branch of the insulin/insulin-like growth factor signaling cascade.Diabetes mellitus is a complex disorder that arises from various causes, including dysregulated glucose sensing and impaired insulin secretion (maturity-onset diabetes of youth, MODY), autoimmune-mediated ␤ cell destruction (type 1), or insufficient compensation for peripheral insulin resistance (type 2) (1). Type 2 diabetes is the most prevalent form. It usually occurs at middle age and afflicts more than 30 million people over the age of 65 but is appearing with greater frequency in children and adolescents (2). Dysregulated insulin signaling exacerbated by chronic hyperglycemia promotes a cohort of systemic disorders, including dyslipidemia, hypertension, cardiovascular disease, and female infertility (3, 4). The search for strategies to promote ␤ cell function and regeneration has lead to the discovery that glucagon-like peptide-1 (GLP1) 2 receptor agonists increase insulin biosynthesis and secretion from ␤ cells, inhibit glucagon secretion from ␣-cells, and promote peripheral insulin sensitivity and satiety in type 2 diabetics (5-9). During a meal, GLP1 is secreted into the circulation from L cells located in the intestine (10); however, GLP1 is quickly inactivated by circulating dipeptidyl-peptidase IV, which diminishing its usefulness as an injectable therapeutic. Compounds that inhibit dipeptidyl-peptidase IV or GLP1 homologs like exendin-4 (Ex4) that are not degraded by dipeptidyl-peptidase IV display improved therapeutic efficacy (11-16). Administration of Ex4 to rodents or humans with type 2 diabetes increases first-phase insulin secretion and increases ␤ cell mass, which can compensate for peripheral insulin resistance (8,9,17,18). Recently, a synthetic Ex4 called Exenatide (Byetta, Amylin/Lily) has gained Food and Drug Administration approval as an injectable treatment for type 2 diabetes (15). Because Exenatide is the...

To understand how extracellular signals may produce long-term effects in neural cells, we have analyzed the mechanism by which neurotransmitters and growth factors induce phosphorylation of the transcription factor cAMP response element binding protein (CREB) in cortical oligodendrocyte progenitor (OP) cells. Activation of glutamate receptor channels by kainate, as well as stimulation of G-protein-coupled cholinergic receptors by carbachol and tyrosine kinase receptors by basic fibroblast growth factor (bFGF), rapidly leads to mitogen-activated protein kinase (MAPK) phosphorylation and ribosomal S6 kinase (RSK) activation. Kainate and carbachol activation of the MAPK pathway requires extracellular calcium influx and is accompanied by protein kinase C (PKC) induction, with no significant increase in GTP binding to Ras. Conversely, growth factor-stimulated MAPK phosphorylation is independent of extracellular calcium and is accompanied by Ras activation. Both basal and stimulated MAPK activity in OP cells are influenced by cytoplasmic calcium levels, as shown by their sensitivity to the calcium chelator bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid. The kinetics of CREB phosphorylation in response to the various agonists corresponds to that of MAPK activation. Moreover, CREB phosphorylation and MAPK activation are similarly affected by calcium ions. The MEK inhibitor PD 098059, which selectively prevents activation of the MAPK pathway, strongly reduces induction of CREB phosphorylation by kainate, carbachol, bFGF, and the phorbol ester TPA. We propose that in OPs the MAPK/RSK pathway mediates CREB phosphorylation in response to calcium influx, PKC activation, and growth factor stimulation.

The intracellular distribution and metabolism of microinjected fluorescently-labeled oligonucleotides (ODNs) have been evaluated using confocal fluorescence microscopy. Fluorescent phosphodiester ODNs, microinjected into the cytoplasm of mammalian cells, rapidly accumulate within the nucleus; the fluorescence disappears with a half-life of 15-20 minutes. Microinjected fluorescent phosphorothioate ODNs remain in the nucleus for more than 24 hours. The persistence of fluorescence depends on the length of the ODN. Modification of the 3' end of phosphodiester ODNs does not significantly slow the rapid disappearance of fluorescence, although certain 3' modifications localize ODNs into the cytoplasm. Using specially designed ODNs, endonuclease activity is demonstrated to exist in the cytoplasm and nucleus. Modification of the 2' position of the ribose rings of a fluorescent phosphodiester oligodeoxynucleotide with O-methyl or O-allyl does not alter its intracellular distribution; however, the 2'-O-allyl modification stabilizes the persistence of fluorescence more than 60-fold compared to the 2'-deoxy control. Thus, the experiments indicate that somatic cells contain nucleolytic activities which degrade microinjected ODNs; however, chemical modification can dramatically circumvent this process.

Insulin and insulin-like growth factor-1 (IGF-1) regulate metabolism and body growth through homologous receptor tyrosine kinases that phosphorylate the insulin receptor substrate (IRS) proteins. IRS-2 is an important IRS protein, as it mediates peripheral insulin action and ␤-cell survival. In this study, we show that insulin, IGF-1, or osmotic stress promoted ubiquitin/proteasome-mediated degradation of IRS-2 in 3T3-L1 cells, Fao hepatoma, cells and mouse embryo fibroblasts; however, insulin/IGF-1 did not promote degradation of IRS-1 in 3T3-L1 preadipocytes or mouse embryo fibroblasts. MG132 or lactacystin, specific inhibitors of 26S proteasome, blocked insulin/IGF-1-induced degradation of IRS-2 and enhanced the detection of ubiquitinated IRS-2. Insulin/IGF1-induced ubiquitination and degradation of IRS-2 was blocked by inhibitors of phosphatidylinositol 3-kinase (wortmannin or LY294002) or mTOR (rapamycin). Chronic insulin or IGF-1 treatment of IRS-1-deficient mouse embryo fibroblasts inhibited IRS-2-mediated activation of Akt and ERK1/2, which was reversed by lactacystin pretreatment. By contrast, IRS-1 activation of Akt and ERK1/2 was not inhibited by chronic insulin/IGF-1 stimulation in IRS-2-deficient mouse embryo fibroblasts. Thus, we identified a novel negative feedback mechanism by which the ubiquitin/ proteasome-mediated degradation of IRS-2 limits the magnitude and duration of the response to insulin or IGF-1.Insulin and insulin-like growth factor 1 (IGF-1) 1 regulate a variety of biological functions through homologous tyrosine kinases that phosphorylate the insulin receptor substrate (IRS) proteins. IRS proteins mediate signal specificity and diversity in various cellular backgrounds (1). Upon ligand binding, the activated insulin/IGF-1 receptors engage IRS proteins and promote the phosphorylation of multiple tyrosine residues, which activate various downstream signaling pathways, including the phosphatidylinositol (PI) 3-kinase and MAP kinase cascades (1, 2). These signaling pathways coordinate a network of protein kinases that regulate the activity of cytoplasmic enzymes and nuclear transcription factors (1-4). Four IRS proteins are known, including IRS-1 and IRS-2, which play a central role in many tissue and organ systems. Deletion of IRS-1 in mice impairs somatic growth and causes peripheral insulin resistance, but diabetes rarely develops owing to lifelong compensatory hyperinsulinemia (5, 6). By contrast, IRS-2 is essential for normal nutrient homeostasis because it mediates both peripheral insulin action and the effect of IGF-1 on ␤-cell growth; mice lacking IRS-2 fail to maintain sufficient compensatory insulin secretion and develop diabetes as young adults (7). Moreover, female mice lacking IRS-2 are hyperplasic and infertile, owing to a failure of the hypothalamic-pituitaryovarian axis (8).Many mechanisms are proposed to explain the inhibition of IRS protein signaling, including phosphotyrosine dephosphorylation, serine/threonine phosphorylation, and degradation (9 -14). Recent studies s...