P85 Subunit Of Phosphatidylinositol 3-kinase Research Articles

Mammary Epithelial-specific Deletion of the Focal Adhesion Kinase Gene Leads to Severe Lobulo-Alveolar Hypoplasia and Secretory Immaturity of the Murine Mammary Gland

Integrin-mediated cell adhesion and signaling is required for mammary gland development and functions. As a major mediator of integrin signaling, focal adhesion kinase (FAK) has been implicated to play a role in the survival, proliferation, and differentiation of mammary epithelial cells in previously studies in vitro. To assess the role of FAK in vivo, we created mice in which FAK is selectively deleted in mammary epithelial cells. The mammary gland FAK conditional knock-out (MFCKO) mice are viable, fertile, and macroscopically indistinguishable from the control littermates. In virgin MFCKO mice, mammary ductal elongation is retarded at 5 weeks of age but reaches the full extent by 8 weeks of age compared with the control mice. However, the MFCKO females are unable to nurse their pups due to severe lobulo-alveolar hypoplasia and secretory immaturity during pregnancy and lactation. Analysis of the mammary epithelial cells in MFCKO mice showed reduced Erk phosphorylation, expression of cyclin D1, and a corresponding decrease in proliferative capability compared with the littermate controls. In addition, phosphorylation of STAT5 and expression of whey acidic protein are significantly reduced in the mammary glands of MFCKO mice, suggesting defective secretory maturation in these mice. Therefore, the combination of the severe lobulo-alveolar hypoplasia and defective secretory differentiation is responsible for the inability of the MFCKO females to nurse their pups. Together, these results provide strong support for a role of FAK in the mammary gland development and function in vivo.

SH3 Binding Motif 1 in Influenza A Virus NS1 Protein Is Essential for PI3K/Akt Signaling Pathway Activation

Recent studies have demonstrated that influenza A virus infection activates the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway by binding of influenza NS1 protein to the p85 regulatory subunit of PI3K. Our previous study proposed that two polyproline motifs in NS1 (amino acids 164 to 167 [PXXP], SH3 binding motif 1, and amino acids 213 to 216 [PPXXP], SH3 binding motif 2) may mediate binding to the p85 subunit of PI3K. Here we performed individual mutational analyses on these two motifs and demonstrated that SH3 binding motif 1 contributes to the interactions of NS1 with p85beta, whereas SH3 binding motif 2 is not required for this process. Mutant viruses carrying NS1 with mutations in SH3 binding motif 1 failed to interact with p85beta and induce the subsequent activation of PI3K/Akt pathway. Mutant virus bearing mutations in SH3 binding motif 2 exhibited similar phenotype as the wild-type (WT) virus. Furthermore, viruses with mutations in SH3 binding motif 1 induced more severe apoptosis than did the WT virus. Our data suggest that SH3 binding motif 1 in NS1 protein is required for NS1-p85beta interaction and PI3K/Akt activation. Activation of PI3K/Akt pathway is beneficial for virus replication by inhibiting virus induced apoptosis through phosphorylation of caspase-9.

Insulin signaling in the liver and uterus of ovariectomized rats treated with estradiol

We used rat hepatic and uterine tissues to examine the impact of estradiol (E2) on insulin (INS) signaling. Ovariectomized (OVX) female Wistar rats were treated with E2 (20 μg/kg b.wt., i.p.) and used for the experiment 6 h after E2 administration. To highlight E2 effects on tyrosine phosphorylation of INS receptor (IR) and INS receptor substrates (IRSs) and IRSs association with p85 subunit of phosphatidylinositol 3-kinase (PI3-K) in the context of INS signaling, E2-treated OVX rats were also injected with INS (20 IU, i.p.), 30 min before the experiment. Treatment with E2 did not change the levels of plasma INS and glucose (Glu). However, it significantly decreased the free fatty acid (FFA) level and increased uterine weight. Furthermore, the results show that E2 had no effect on the content of hepatic IR protein, but significantly increased IR protein content in the uterus and decreased IR tyrosine phosphorylation in both the liver and uterus. Compared to the control, hepatic IRS-1 and IRS-2 were significantly decreased and increased, respectively, after E2 treatment. Protein content of both molecules, IRS-1 and IRS-2, was increased in uterine tissue after E2 administration. Protein content of the p85 subunit of PI3-K and that of protein kinase B (Akt) were increased in the uterus, with no changes in the liver. The results suggest that E2 treatment induces tissue-specific changes in INS signaling. The consequences of E2 treatment on INS signaling molecules are more apparent in the uterus, but their physiological relevance for INS action is probably greater in the liver.

511 POSTER Tissue microarray immunohistochemical profiling of metastatic colorectal cancer

Insulin resistance of skeletal muscle glucose transport due to prolonged loss of ovarian function in ovariectomized (OVX) rats is accompanied by other features of the metabolic syndrome and may be confounded by increased calorie consumption. In this study, we investigated the role of calorie consumption in the development of insulin resistance in OVX rats. In addition, we examined the cellular mechanisms underlying skeletal muscle insulin resistance in OVX rats. Female Sprague-Dawley rats were ovariectomized (OVX) or sham-operated (SHAM). OVX rats either had free access to food, pair feeding (PF) with SHAM or received a 35% reduction in food intake (calorie restriction; CR) for 12 weeks. Compared with SHAM, ovariectomy induced skeletal muscle insulin resistance, which was associated with decreases (32–70%) in tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 (IRS-1), IRS-1 associated p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase), and Akt Ser473 phosphorylation whereas insulin-stimulated phosphorylation of IRS-1 Ser307, SAPK/JNK Thr183/Tyr185, and p38 mitogen-activated protein kinase (MAPK) Thr180/Tyr182 was increased (24–62%). PF improved the serum lipid profile but did not restore insulin-stimulated glucose transport, indicating that insulin resistance in OVX rats is a consequence of ovarian hormone deprivation. In contrast, impaired insulin sensitivity and defective insulin signaling were not observed in the skeletal muscle of OVX + CR rats. Therefore, we provide evidence for the first time that CR effectively prevents the development of insulin resistance and impaired insulin signaling in the skeletal muscle of OVX rats.

Association of PI3K-Akt signaling pathway with digitalis-induced hypertrophy of cardiac myocytes

Our previous studies on cardiac myocytes showed that positive inotropic concentrations of the digitalis drug ouabain activated signaling pathways linked to Na(+)-K(+)-ATPase through Src and epidermal growth factor receptor (EGFR) and led to myocyte hypertrophy. In view of the known involvement of phosphatidylinositol 3-kinase (PI3K)-Akt pathways in cardiac hypertrophy, the aim of the present study was to determine whether these pathways are also linked to cardiac Na(+)-K(+)-ATPase and, if so, to assess their role in ouabain-induced myocyte growth. In a dose- and time-dependent manner, ouabain activated Akt and phosphorylation of its substrates mammalian target of rapamycin and glycogen synthase kinase in neonatal rat cardiac myocytes. Akt activation by ouabain was sensitive to PI3K inhibitors and was also noted in adult myocytes and isolated hearts. Ouabain caused a transient increase of phosphatidylinositol 3,4,5-trisphosphate content of neonatal myocytes, activated class IA, but not class IB, PI3K, and increased coimmunoprecipitation of the alpha-subunit of Na(+)-K(+)-ATPase with the p85 subunit of class IA PI3K. Ouabain-induced activation of ERK1/2 was prevented by Src, EGFR, and MEK inhibitors, but not by PI3K inhibitors. Activation of Akt by ouabain, however, was sensitive to inhibitors of PI3K and Src, but not to inhibitors of EGFR and MEK. Similarly, ouabain-induced myocyte hypertrophy was prevented by PI3K and Src inhibitors, but not by an EGFR inhibitor. These findings 1) establish the linkage of the class IA PI3K-Akt pathway to Na(+)-K(+)-ATPase and the essential role of this linkage to ouabain-induced myocyte hypertrophy and 2) suggest cross talk between these PI3K-Akt pathways and the signaling cascades previously identified to be associated with cardiac Na(+)-K(+)-ATPase.

Regulation of Hepatitis B Virus Replication by the Phosphatidylinositol 3-Kinase-Akt Signal Transduction Pathway

The phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) signaling pathway is one of the major oncogenic pathways and is activated in many types of human cancers, including hepatocellular carcinoma. It can also be activated by the hepatitis C virus (HCV) nonstructural 5A (NS5A) protein. In the present study, we set out to determine the regulatory effects of this pathway on the replication of hepatitis B virus (HBV). Our results demonstrate that the expression of a constitutively active Akt1 profoundly inhibited HBV RNA transcription and consequently reduced HBV DNA replication in HepG2 cells. This suppression of HBV gene transcription was apparently mediated by the activation of mTOR, as it was abolished by the mTOR inhibitor rapamycin. Moreover, treatment of HBV-expressing HepG2.2.15 cells with inhibitors of PI3K, Akt, and mTOR increased the transcription of 3.5-kb and 2.4-kb viral RNA as well as the replication of HBV DNA. This observation implies that the basal level activation of this pathway in HepG2 cells regulated HBV replication. Consistent with previous reports showing that the HCV NS5A protein could bind to the p85 subunit of PI3K and activate the PI3K-Akt signal transduction pathway, our results showed that expression of this protein could inhibit HBV RNA transcription and reduce HBV DNA replication in HepG2 cells. Taken together, our results suggest that the activation of the PI3K-Akt pathway during liver oncogenesis may be at least partially responsible for the elimination of HBV replication from tumor cells and may also provide an explanation for the observed suppression of HBV replication by HCV coinfection.

Dilinoleoyl-phosphatidic acid mediates reduced IRS-1 tyrosine phosphorylation in rat skeletal muscle cells and mouse muscle

Insulin resistance in skeletal muscle is strongly associated with lipid oversupply, but the intracellular metabolites and underlying mechanisms are unclear. We therefore sought to identify the lipid intermediates through which the common unsaturated fatty acid linoleate causes defects in IRS-1 signalling in L6 myotubes and mouse skeletal muscle. Cells were pre-treated with 1 mmol/l linoleate for 24 h. Subsequent insulin-stimulated IRS-1 tyrosine phosphorylation and its association with the p85 subunit of phosphatidylinositol 3-kinase were determined by immunoblotting. Intracellular lipid species and protein kinase C activation were modulated by overexpression of diacylglycerol kinase epsilon, which preferentially converts unsaturated diacylglycerol into phosphatidic acid, or by inhibition of lysophosphatidic acid acyl transferase with lisofylline, which reduces phosphatidic acid synthesis. Phosphatidic acid species in linoleate-treated cells or muscle from insulin-resistant mice fed a safflower oil-based high-fat diet that was rich in linoleate were analysed by mass spectrometry. Linoleate pretreatment reduced IRS-1 tyrosine phosphorylation and p85 association. Overexpression of diacylglycerol kinase epsilon reversed the activation of protein kinase C isoforms by linoleate, but paradoxically further diminished IRS-1 tyrosine phosphorylation. Conversely, lisofylline treatment restored IRS-1 phosphorylation. Mass spectrometry indicated that the dilinoleoyl-phosphatidic acid content increased from undetectable levels to almost 20% of total phosphatidic acid in L6 cells and to 8% of total in the muscle of mice fed a high-fat diet. Micelles containing dilinoleoyl-phosphatidic acid specifically inhibited IRS-1 tyrosine phosphorylation and glycogen synthesis in L6 cells. These data indicate that linoleate-derived phosphatidic acid is a novel lipid species that contributes independently of protein kinase C to IRS-1 signalling defects in muscle cells in response to lipid oversupply.

The adipose cell lineage is not intrinsically insulin resistant in polycystic ovary syndrome

Selective resistance to the effects of insulin on glucose metabolism in skeletal muscle and adipose tissue is a key feature of polycystic ovary syndrome (PCOS). The pathogenesis of insulin resistance in skeletal muscle in PCOS involves interaction of in vivo environmental factors with intrinsic defects in insulin signaling. We aimed to determine whether (1) intrinsic defects in insulin action/signaling and cytokine secretion were present in adipose cells in PCOS and (2) insulin resistance can be induced in control adipose cells by culture in medium conditioned by insulin-resistant PCOS fibroblasts. Subcutaneous abdominal preadipocytes from obese women with PCOS (n = 7) and age- and body mass index–matched controls (n = 5) were cultured for several generations in vitro. Basal and insulin-stimulated glycogen synthesis and basal glucose transport did not differ in the preadipocytes from women with PCOS and controls. Abundance of insulin receptor (IR) β subunit, insulin receptor substrate (IRS) 1 and 2, p85 subunit of phosphatidylinositol 3-kinase, and extracellular signal-regulated kinase (ERK)1/2 activation did not differ. Secretion of tumor necrosis factor α and interleukin 6 did not differ. Insulin action on glycogen synthesis in control preadipocytes was not altered by coculture with or growth in media conditioned by PCOS skin fibroblasts with constitutive serine phosphorylation of IR β subunit (IR ser+), indicating that IR ser+ cells do not secrete an insulin resistance–inducing factor. We conclude that in contrast to skeletal muscle and skin fibroblasts, there is no evidence for intrinsic defects in insulin signaling in the PCOS adipose cell lineage, indicating that insulin resistance in these cells is likely due to factors in the in vivo environment.

CD2AP/CIN85 Balance Determines Receptor Tyrosine Kinase Signaling Response in Podocytes

Defects in podocyte signaling are the basis of many inherited glomerular diseases leading to glomerulosclerosis. CD2-associated protein (CD2AP) is highly expressed in podocytes and is considered to play an important role in the maintenance of the glomerular slit diaphragm. Mice deficient for CD2AP (CD2AP(-/-)) appear normal at birth but develop a rapid onset nephrotic syndrome at 3 weeks of age. We demonstrate that impaired intracellular signaling with subsequent podocyte damage is the reason for this delayed podocyte injury in CD2AP(-/-) mice. We document that CD2AP deficiency in podocytes leads to diminished signal initiation and termination of signaling pathways mediated by receptor tyrosine kinases (RTKs). In addition, we demonstrate that CIN85, a paralog of CD2AP, is involved in termination of RTK signaling in podocytes. CIN85 protein expression is increased in CD2AP(-/-) podocytes in vitro. Stimulation of CD2AP(-/-) podocytes with various growth factors, including insulin-like growth factor 1, vascular endothelial growth factor, and fibroblast growth factor, resulted in a significantly decreased phosphatidylinositol 3-kinase/AKT and ERK signaling response. Moreover, increased CIN85 protein is detectable in podocytes in diseased CD2AP(-/-) mice, leading to decreased base-line activation of ERK and decreased phosphorylation after growth factor stimulation in vivo. Because repression of CIN85 protein leads to a restored RTK signaling response, our results support an important role of CD2AP/CIN85 protein balance in the normal signaling response of podocytes.

Adhesion-Related Kinase Induction of Migration Requires Phosphatidylinositol-3-Kinase and Ras Stimulation of Rac Activity in Immortalized Gonadotropin-Releasing Hormone Neuronal Cells

GnRH neurons migrate into the hypothalamus during development. Although migratory defects may result in disordered activation of the reproductive axis and lead to delayed or absent sexual maturation, specific factors regulating GnRH neuronal migration remain largely unknown. The receptor tyrosine kinase, adhesion-related kinase (Ark) (also known as Axl, UFO, and Tyro7), has been implicated in the migration of GnRH neuronal cells. Binding of its ligand, growth arrest-specific gene 6 (Gas6), promotes cytoskeletal remodeling and migration of NLT GnRH neuronal cells via Rac and p38 MAPK. Here, we examined the Axl effectors proximal to Rac in the signaling pathway. Gas6/Axl-induced lamellipodia formation and migration were blocked after phosphatidylinositol-3-kinase (PI3K) inhibition in GnRH neuronal cells. The p85 subunit of PI3K coimmunoprecipitated with Axl and was phosphorylated in a Gas6-sensitive manner. In addition, PI3K inhibition in GnRH neuronal cells diminished Gas6-induced Rac activation. Exogenous expression of a dominant-negative form of Ras also decreased GnRH neuronal lamellipodia formation, migration, and Rac activation. PI3K inhibition blocked Ras in addition to Rac activation and migration. In contrast, pharmacological blockade of the phospholipase C gamma effectors, protein kinase C or calcium/calmodulin protein kinase II, had no effect on Gas6/Axl signaling to promote Rac activation or stimulate cytoskeletal reorganization and migration. Together, these data show that the PI3K-Ras pathway is a major mediator of Axl actions upstream of Rac to induce GnRH neuronal cell migration.

C-reactive protein induces phosphorylation of insulin receptor substrate-1 on Ser307 and Ser612 in L6 myocytes, thereby impairing the insulin signalling pathway that promotes glucose transport

C-reactive protein (CRP) is associated with insulin resistance and predicts development of type 2 diabetes. However, it is unknown whether CRP directly affects insulin signalling action. To this aim, we determined the effects of human recombinant CRP (hrCRP) on insulin signalling involved in glucose transport in L6 myotubes. L6 myotubes were exposed to endotoxin-free hrCRP and insulin-stimulated activation of signal molecules, glucose uptake and glycogen synthesis were assessed. We found that hrCRP stimulates both c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK)1/2 activity. These effects were paralleled by a concomitant increase in IRS-1 phosphorylation at Ser(307) and Ser(612), respectively. The stimulatory effects of hrCRP on IRS-1 phosphorylation at Ser(307) and Ser(612) were partially reversed by treatment with specific JNK and ERK1/2 inhibitors, respectively. Exposure of L6 myotubes to hrCRP reduced insulin-stimulated phosphorylation of IRS-1 at Tyr(632), a site essential for engaging p85 subunit of phosphatidylinositol-3 kinase (PI-3K), protein kinase B (Akt) activation and glycogen synthase kinase-3 (GSK-3) phosphorylation. These events were accompanied by a decrease in insulin-stimulated glucose transporter (GLUT) 4 translocation to the plasma membrane, glucose uptake and glucose incorporation into glycogen. The inhibitory effects of hrCRP on insulin signalling and insulin-stimulated GLUT4 translocation were reversed by treatment with JNK inhibitor I and the mitogen-activated protein kinase inhibitor, PD98059. Our data suggest that hrCRP may cause insulin resistance by increasing IRS-1 phosphorylation at Ser(307) and Ser(612) via JNK and ERK1/2, respectively, leading to impaired insulin-stimulated glucose uptake, GLUT4 translocation, and glycogen synthesis mediated by the IRS-1/PI-3K/Akt/GSK-3 pathway.

Influenza A virus NS1 protein activates the phosphatidylinositol 3-kinase (PI3K)/Akt pathway by direct interaction with the p85 subunit of PI3K

Influenza A virus infection activates the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, but the mechanism is not clear. Here, it is reported that influenza A virus NS1 protein is responsible for PI3K/Akt pathway activation. It was demonstrated that the NS1 protein interacts with the p85 regulatory subunit of PI3K via direct binding to the SH3 and C-terminal SH2 domains of p85. Consensus binding motifs for SH3 and SH2 domains were found in influenza A virus NS1, namely an SH2-binding motif (YXXXM) at aa 89, SH3-binding motif 1 (PXXP) around aa 164 and SH3-binding motif 2 around aa 212. Mutant virus encoding NS1 protein with mutations in the SH-binding motifs failed to interact with SH domains of p85 and did not activate the PI3K/Akt pathway. The mutant virus is attenuated in Madin-Darby canine kidney cells. Our study has established a novel function of NS1: by interacting with p85 via the SH-binding motifs, NS1 can activate the PI3K/Akt pathway.

An inherent role of microtubule network in the action of nuclear receptor

Estrogen receptor alpha (ERalpha) functions as both a transcription factor and a mediator of rapid estrogen signaling. Recent studies have shown a role for ERalpha-interacting membranous and cytosolic proteins in ERalpha action, but our understanding of the role of the microtubule network in the modulation of ERalpha signaling remains unclear. Here we found that endogenous ERalpha associates with microtubules through the microtubule-binding protein hematopoietic PBX-interaction protein (HPIP). Biochemical and RNA-interference studies demonstrated that HPIP influences ERalpha-dependent rapid estrogen signaling by acting as a scaffold protein and recruits Src kinase and the p85 subunit of phosphatidylinositol 3-kinase to a complex with ERalpha, which in turn stimulates AKT and MAPK. We also found that ERalpha interacts with beta-tubulin through HPIP. Destabilization of microtubules activated ERalpha signaling, whereas stabilization of microtubules repressed ERalpha transcriptional activity in a HPIP-dependent manner. These findings revealed a role for HPIP-microtubule complex in regulating 17beta-estradiol-ERalpha responses in mammalian cells and discovered an inherent role of microtubules in the action of nuclear receptor.

Transforming Growth Factor β Activation of c-Abl Is Independent of Receptor Internalization and Regulated by Phosphatidylinositol 3-Kinase and PAK2 in Mesenchymal Cultures

Transforming growth factor beta (TGF-beta) modulates a number of cellular phenotypes as divergent as growth stimulation and growth inhibition. Although the Smad pathway is critical for many of these responses, recent evidence indicates that Smad-independent pathways may also have a critical role. One such protein previously shown to regulate TGF-beta action independent of the Smad proteins is the c-Abl nonreceptor tyrosine kinase. In the current study we determined that TGF-beta receptor signaling activates c-Abl kinase activity in a subset of fibroblast but not epithelial cultures. This cell type-specific response occurs in a membrane-proximal locale independent of receptor internalization and upstream of dynamin action. Although c-Abl activation by TGF-beta is independent of Smad2 or Smad3, it is prevented by inhibitors of phosphatidylinositol 3-kinase or PAK2. Thus, c-Abl represents a target downstream of phosphatidylinositol 3-kinase-activated PAK2, which differentiates TGF-beta signaling in fibroblasts and epithelial cell lines and integrates serine/threonine receptor kinases with tyrosine kinase pathways.

Hsp90 inhibition transiently activates Src kinase and promotes Src-dependent Akt and Erk activation

Hsp90 plays an essential role in maintaining stability and activity of its clients, including oncogenic signaling proteins that regulate key signal transduction nodes. Hsp90 inhibitors interfere with diverse signaling pathways by destabilizing and attenuating activity of such proteins, and thus they exhibit antitumor activity. However, Hsp90 inhibition has recently been reported to activate Akt and Erk and potentiate Akt activation induced by insulin-like growth factor 1 and insulin, raising the concern that clinical use of Hsp90 inhibitors might promote tumor progression under certain circumstances. Here, we show that the prototypical Hsp90 inhibitor geldanamycin induces Akt and Erk activation that is independent of PTEN status and is mediated by transient activation of Src kinase. Activated Src phosphorylates Cbl, which recruits the p85 subunit of phosphatidylinositol 3-kinase, resulting in phosphatidylinositol 3-kinase activation and eventually the activation of Akt and Erk. We show that geldanamycin rapidly disrupts Src association with Hsp90, suggesting that Src activation results directly from dissociation of the chaperone. These data suggest that, under certain circumstances, dual inhibition of Hsp90 and Src may be warranted.

Negative Regulation of FcϵRI-mediated Signaling and Mast Cell Function by the Adaptor Protein LAX

LAX is a transmembrane adaptor protein that is expressed in both T and B cells. Upon stimulation via the antigen receptors, it is tyrosine-phosphorylated and binds Grb2 and the p85 subunit of phosphatidylinositol 3-kinase. Disruption of the Lax gene causes hyperresponsiveness in T and B lymphocytes. Here, we showed that LAX was also expressed in mast cells. Upon engagement of the Fc epsilonRI, LAX was also phosphorylated and interacted with Grb2 and p85. LAX-deficient mast cells were hyperresponsive to stimulation via the Fc epsilonRI, as evidenced by enhanced degranulation, p38 MAPK, Akt, and phosphatidylinositol 3-kinase activation. This hyperresponsiveness was likely a consequence of reduced LAB expression after sensitization of mast cells with anti-dinitrophenyl IgE. In addition, Fc epsilonRI-mediated cytokine production and cell survival were also enhanced. These data suggested that LAX negatively regulates mast cell function.

Neuregulin-dependent protein synthesis in C2C12 myotubes and rat diaphragm muscle

The nerve-derived trophic factor neuregulin (NRG) is a prime candidate molecule for modulating muscle fiber growth. NRG regulates signal transduction in skeletal muscle through activation of ErbB receptors present at the neuromuscular junction. In this study, we hypothesize that NRG increases protein synthesis in maturing muscle via a phosphatidylinositol 3-kinase (PI3K)-dependent mechanism. NRG signal transduction and its ability to stimulate protein synthesis (measured by incorporation of [(3)H]phenylalanine into the protein pool) were investigated in differentiated C(2)C(12) myotubes and rat diaphragm muscle (DIAm). In C(2)C(12) myotubes, NRG dose dependently increased phosphorylation of ErbB3 and recruitment of the p85 subunit of PI3K. NRG also increased phosphorylation of Akt, a downstream effector of PI3K. NRG treatment increased total protein synthesis by 35% compared with untreated control myotubes. This NRG-induced increase in Akt phosphorylation and protein synthesis was completely blocked by wortmannin, an inhibitor of PI3K but was unaffected by PD-98059, an inhibitor of MEK. In DIAm obtained from 3-day-old rat pups, Akt phosphorylation increased approximately 30-fold with NRG treatment (vs. untreated DIAm). NRG treatment also significantly increased protein synthesis in the DIAm by 29% after 3 h of incubation with [(3)H]phenylalanine (vs. untreated DIAm). Pretreatment with wortmannin abolished the NRG-induced increase in protein synthesis, suggesting a critical role for PI3K in this response. The results of the present study support the hypothesis that nerve-derived NRG contributes to the regulation of skeletal muscle mass by increasing protein synthesis via activation of PI3K.

Downregulation of the PI3K/Akt survival pathway in cells with deregulated expression of c-Myc

Oncogenic transformation leads to an increased sensitivity to apoptosis, a characteristic that is selectively lost during tumor progression. The sensitization process affects the mitochondrial pathway of apoptosis through signaling events that are poorly defined. We previously showed that a deregulated expression of c-Myc in cells treated with toxic agents caused an enhanced activation of p38 that acts in a death-promoting pathway. Here, we show that deregulated expression of c-Myc causes a severe reduction in the basal activity of Akt, which was further accelerated by serum deprivation. Furthermore, c-Myc expression repressed the activation of Akt induced by the toxic agents doxorubicin, cisplatin and H(2)O(2), and also by the physiological agonists PDGF and insulin. We determined that the activation of Akt was inhibited as a result of the action of c-Myc upstream of phosphatidylinositol 3-kinase (PI3K) activation. c-Myc overexpression impaired the induced association of the p85 subunit of PI3K with phosphotyrosine containing proteins, causing a reduction in the activation of PI3K and recruitment of Akt to the membrane. Inhibiting Akt in addition to enhancing p38 further exacerbate the imbalance between the death and survival signals and results in an enhanced sensitivity to apoptosis.

Biochemical and biological characterization of a neuroendocrine‐associated phosphatase

The biochemical and biological properties of a novel neuroendocrine-associated phosphatase (NEAP) were characterized. NEAP had a sequence characteristic of a dual-specificity phosphatase (DSP), and was preferentially expressed in neuroendocrine cells/tissues as well as in skeletal muscle and heart. Expression of NEAP was up-regulated in nerve growth factor (NGF)-treated, differentiated PC12 cells. NEAP was cytosolic and did not apparently have effects against extracellular signal-regulated kinase, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase activated by various stimuli. Although NEAP and MAPK phosphatase (MPK)-1 showed similar phosphatase activity towards p-nitro phenylphosphate (pNPP), in contrast to MKP-1, NEAP did not dephosphorylate JNK and p38-MAPK in vitro. Overexpression of NEAP, but not the C152S mutant, in PC12 cells suppressed NGF-induced phosphorylation of the p85 subunit of phosphatidylinositol 3-kinase (PI3K) and Akt activation. Overexpression of NEAP also suppressed neurite outgrowth induced by NGF and sensitized PC12 cells to cisplatin-induced apoptosis. Suppression of NEAP by RNA interference enhanced NGF-induced neurite outgrowth and Akt activation. Our results indicated that, unlike other DSPs, down-regulation of conventional MAPKs was not the major function of NEAP. Furthermore, NEAP might be involved in neuronal differentiation via regulation of the PI3K/Akt signaling.

Keeping LAT Out

T cell anergy prevents self-reactive T cells that escape elimination in the thymus from responding. T cell anergy is associated with decreased interleukin 2 (IL-2) production and decreased proliferation in response to antigen-specific stimulation. In a cultured cell system and an in vivo model, Hundt et al. show that, although phosphorylation of the tyrosine kinase ZAP-70 is not impaired, phosphorylation of the ZAP-70 substrate linker of activated T cells (LAT) is decreased. LAT serves as a scaffold recruiting various downstream effectors to the immunological synapse. Thus, lack of LAT phosphorylation prevents activation of phospholipase C-γ1 (PLC-γ1) and phosphatidylinositol 3-kinase (PI3K). Indeed, Hundt et al. observed decreased coimmunoprecipitation of LAT with the p85 subunit of PI3K and decreased phosphorylation of PLC-γ1 in the anergic T cells. There was no decrease in LAT abundance in the anergic cells; however, fluorescence microscopy experiments showed that LAT was selectively excluded from the immunological synapse, and cell fractionation studies showed that LAT was specifically excluded from detergent-resistant membrane fractions. In both models of anergy, palmitoylation of LAT, which is required for localization to detergent-resistant membranes, was reduced. The palmitoylation defect was specific for LAT; palmitoylation of Fyn was unaffected. Thus, exclusion of LAT from the immunological synapse by depalmitoylation of LAT promotes the formation of an immunological synapse with altered signaling properties in anergic T cells compared with the signaling properties of activated T cells (see Lineberry and Fathman for discussion). M. Hundt, H. Tabata, M.-S. Jeon, K. Hayashi, Y. Tanaka, R. Krishna, L. De Giorgio, Y.-C. Liu, M. Fukata, A. Altman, Impaired activation and localization of LAT in anergic T cells as a consequence of a selective palmitoylation defect. Immunity 24 , 513-522 (2006). [PubMed] N. Lineberry, C. G. Fathman, T cell anergy: Where it's LAT. Immunity 24 , 501-503 (2006). [PubMed]