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At least 703 records · Page 39Linked to original sources
PubMed2005

Individual phosphoinositide 3-kinase C2alpha domain activities independently regulate clathrin function.

Phosphoinositide 3-kinase C2alpha (PI3K-C2alpha) is a member of the class II PI-3 kinases, defined by the presence of a second C2 domain at their C termini. The cellular functions of the class II enzymes are incompletely understood, though they have been implicated in receptor activation pathways initiated by epidermal growth factor, insulin, and chemokines. PI3K-C2alpha was recently found to be localized to clathrin-coated membranes in the trans-Golgi network and at endocytic sites on the plasma membrane. Further, a specific binding site was identified for clathrin on the N terminus of PI3K-C2alpha, whose occupancy resulted in lipid kinase activation. Expression of PI3K-C2alpha in cells dramatically affected clathrin distribution and function in cells, leading to accumulation of intracellular clathrin-coated structures, which are visualized here at the ultrastructural level, and inhibition of clathrin-mediated transport from both the plasma membrane and the trans-Golgi network. In this study we have demonstrated that the isolated clathrin binding domain of PI3K-C2alpha can drive clathrin lattice assembly and that both it and the lipid kinase activity of the protein can independently modulate clathrin distribution and function when expressed in cells. Together, these results suggest that PI3K-C2alpha employs both protein-protein interaction and localized production of 3-phosphoinositides to affect clathrin dynamics at sites of membrane budding and targeting.

Animals↗
PubMed2006

A phosphoinositide 3-kinase-AKT-nitric oxide-cGMP signaling pathway in stimulating platelet secretion and aggregation.

Phosphoinositide 3-kinase (PI3K) and Akt play important roles in platelet activation. However, the downstream mechanisms mediating their functions are unclear. We have recently shown that nitric-oxide (NO) synthase 3 and cGMP-dependent protein kinase stimulate platelet secretion and aggregation. Here we show that PI3K-mediated Akt activation plays an important role in agonist-stimulated platelet NO synthesis and cGMP elevation. Agonist-induced elevation of NO and cGMP was inhibited by Akt inhibitors and reduced in Akt-1 knock-out platelets. Akt-1 knock-out or Akt inhibitor-treated platelets showed reduced platelet secretion and aggregation in response to low concentrations of agonists, which can be reversed by low concentrations of 8-bromo-cGMP or sodium nitroprusside (an NO donor). Similarly, PI3K inhibitors diminished elevation of cGMP and inhibited platelet secretion and the second wave platelet aggregation, which was also partially reversed by 8-bromo-cGMP. These results indicate that the NO-cGMP pathway is an important downstream mechanism mediating PI3K and Akt signals leading to platelet secretion and aggregation. Conversely, the PI3K-Akt pathway is the major upstream mechanism responsible for activating the NO-cGMP pathway in platelets. Thus, this study delineates a novel platelet activation pathway involving sequential activation of PI3K, Akt, nitric-oxide synthase 3, sGC, and cGMP-dependent protein kinase.

Blood Platelets↗
PubMed2006

A Phosphatidylinositol 3-kinase-regulated Akt-independent signaling promotes cigarette smoke-induced FRA-1 expression.

The FRA-1 proto-oncogene is overexpressed in a variety of human tumors and is known to up-regulate the expression of genes involved in tumor progression and invasion. The phosphatidylinositol 3-kinase (PI3K)-Akt pathway is also known to regulate these cellular processes. More importantly, respiratory toxicants and carcinogens activate both the PI3K-Akt pathway and FRA-1 expression in human bronchial epithelial (HBE) cells. In this study we investigated a potential link between the PI3K-Akt pathway and the cigarette smoke (CS)-stimulated epidermal growth factor receptor-mediated FRA-1 induction in non-oncogenic HBE cells. Treatment of cells with LY294002, an inhibitor of the PI3K-Akt pathway, completely blocked CS-induced FRA-1 expression. Surprisingly pharmacological inhibition of Akt had no significant effect on CS-induced FRA-1 expression. Likewise the inhibition of protein kinase C zeta, which is a known downstream effector of PI3K, did not alter FRA-1 expression. We found that the PI3K through p21-activated kinase 1 regulates FRA-1 proto-oncogene induction by CS and the subsequent activation of the Elk1 and cAMP-response element-binding protein transcription factors that are bound to the promoter in HBE cells.

Carcinogens↗
PubMed2003

Critical role for scaffolding adapter Gab2 in Fc gamma R-mediated phagocytosis.

Grb2-associated binder 2 (Gab2), a member of the Dos/Gab subfamily scaffolding molecules, plays important roles in regulating the growth, differentiation, and function of many hematopoietic cell types. In this paper, we reveal a novel function of Gab2 in Fcgamma receptor (FcgammaR)-initiated phagocytosis in macrophages. Upon FcgammaR activation, Gab2 becomes tyrosyl phosphorylated and associated with p85, the regulatory subunit of phosphoinositide 3-kinase (PI3K), and the protein-tyrosine phosphatidylinositol Shp-2. FcgammaR-mediated phagocytosis is severely impaired in bone marrow-derived macrophages from Gab2-/- mice. The defect in phagocytosis correlates with decreased FcgammaR-evoked activation of Akt, a downstream target of PI3K. Using confocal fluorescence microscopy, we find that Gab2 is recruited to the nascent phagosome, where de novo PI3K lipid production occurs. Gab2 recruitment requires the pleckstrin homology domain of Gab2 and is sensitive to treatment with the PI3K inhibitor wortmannin. The Grb2 binding site on Gab2 also plays an auxiliary role in recruitment to the phagosome. Because PI3K activity is required for FcgammaR-mediated phagocytosis, our results indicate that Gab2 acts as a key component of FcgammaR-mediated phagocytosis, most likely by amplifying PI3K signaling in the nascent phagosome.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Constitutively activated Akt-1 is vital for the survival of human monocyte-differentiated macrophages. Role of Mcl-1, independent of nuclear factor (NF)-kappaB, Bad, or caspase activation.

Recent data from mice deficient for phosphatase and tensin homologue deleted from chromosome 10 or src homology 2 domain-containing 5' inositol phosphatase, phosphatases that negatively regulate the phosphatidylinositol 3-kinase (PI3K) pathway, revealed an increased number of macrophages in these animals, suggesting an essential role for the PI3K pathway for macro-phage survival. Here, we focused on the role of the PI3K-regulated serine/threonine kinase Akt-1 in modulating macrophage survival. Akt-1 was constitutively activated in human macrophages and addition of the PI3K inhibitor, LY294002, suppressed the activation of Akt-1 and induced cell death. Furthermore, suppression of Akt-1 by inhibition of PI3K or a dominant negative (DN) Akt-1 resulted in loss of mitochondrial transmembrane potential, activation of caspases-9 and -3, and DNA fragmentation. The effects of PI3K inhibition were reversed by the ectopic expression of constitutively activated Akt-1 or Bcl-x(L). Inhibition of PI3K/Akt-1 pathway either by LY294002 or DN Akt-1 had no effect on the constitutive or inducible activation of nuclear factor (NF)-kappaB in human macrophages. However, after inhibition of the PI3K/Akt-1 pathway, a marked decrease in the expression of the antiapoptotic molecule Mcl-1, but not other Bcl-2 family members was observed, and Mcl-1 rescued macrophages from LY294002-induced cell death. Further, inhibition of Mcl-1 by antisense oligonucleotides, also resulted in macrophage apoptosis. Thus, our findings demonstrate that the constitutive activation of Akt-1 regulates macrophage survival through Mcl-1, which is independent of caspases, NF-kappaB, or Bad.

Animals↗
PubMed2006

Phosphoinositide 3-kinase binds to TRPV1 and mediates NGF-stimulated TRPV1 trafficking to the plasma membrane.

Sensitization of the pain-transducing ion channel TRPV1 underlies thermal hyperalgesia by proalgesic agents such as nerve growth factor (NGF). The currently accepted model is that the NGF-mediated increase in TRPV1 function during hyperalgesia utilizes activation of phospholipase C (PLC) to cleave PIP2, proposed to tonically inhibit TRPV1. In this study, we tested the PLC model and found two lines of evidence that directly challenge its validity: (1) polylysine, a cationic phosphoinositide sequestering agent, inhibited TRPV1 instead of potentiating it, and (2) direct application of PIP2 to inside-out excised patches dramatically potentiated TRPV1. Furthermore, we show four types of experiments indicating that PI3K is physically and functionally coupled to TRPV1: (1) the p85beta subunit of PI3K interacted with the N-terminal region of TRPV1 in yeast 2-hybrid experiments, (2) PI3K-p85beta coimmunoprecipitated with TRPV1 from both HEK293 cells and dorsal root ganglia (DRG) neurons, (3) TRPV1 interacted with recombinant PI3K-p85 in vitro, and (4) wortmannin, a specific inhibitor of PI3K, completely abolished NGF-mediated sensitization in acutely dissociated DRG neurons. Finally, simultaneous electrophysiological and total internal reflection fluorescence (TIRF) microscopy recordings demonstrate that NGF increased the number of channels in the plasma membrane. We propose a new model for NGF-mediated hyperalgesia in which physical coupling of TRPV1 and PI3K in a signal transduction complex facilitates trafficking of TRPV1 to the plasma membrane.

Androstadienes↗
PubMed2005

Phosphatidylinositol 3-kinase C2alpha is essential for ATP-dependent priming of neurosecretory granule exocytosis.

Neurotransmitter release and hormonal secretion are highly regulated processes culminating in the calcium-dependent fusion of secretory vesicles with the plasma membrane. Here, we have identified a role for phosphatidylinositol 3-kinase C2alpha (PI3K-C2alpha) and its main catalytic product, PtdIns3P, in regulated exocytosis. In neuroendocrine cells, PI3K-C2alpha is present on a subpopulation of mature secretory granules. Impairment of PI3K-C2alpha function specifically inhibits the ATP-dependent priming phase of exocytosis. Overexpression of wild-type PI3K-C2alpha enhanced secretion, whereas transfection of PC12 cells with a catalytically inactive PI3K-C2alpha mutant or a 2xFYVE domain sequestering PtdIns3P abolished secretion. Based on these results, we propose that production of PtdIns3P by PI3K-C2alpha is required for acquisition of fusion competence in neurosecretion.

Adenosine Triphosphate↗
PubMed1998

Identification and analysis of PH domain-containing targets of phosphatidylinositol 3-kinase using a novel in vivo assay in yeast.

Phosphatidylinositol 3-kinase (PI3K) mediates a variety of cellular responses by generating PtdIns(3,4)P2 and PtdIns(3,4,5)P3. These 3-phosphoinositides then function directly as second messengers to activate downstream signaling molecules by binding pleckstrin homology (PH) domains in these signaling molecules. We have established a novel assay in the yeast Saccharomyces cerevisiae to identify proteins that bind PtdIns(3,4)P2 and PtdIns(3,4,5)P3 in vivo which we have called TOPIS (Targets of PI3K Identification System). The assay uses a plasma membrane-targeted Ras to complement a temperature-sensitive CDC25 Ras exchange factor in yeast. Coexpression of PI3K and a fusion protein of activated Ras joined to a PH domain known to bind PtdIns(3,4)P2 (AKT) or PtdIns(3,4,5)P3 (BTK) rescues yeast growth at the non-permissive temperature of 37 degreesC. Using this assay, we have identified several amino acids in the beta1-beta2 region of PH domains that are critical for high affinity binding to PtdIns(3,4)P2 and/or PtdIns(3,4,5)P3, and we have proposed a structural model for how these PH domains might bind PI3K products with high affinity. From these data, we derived a consensus sequence which predicts high-affinity binding to PtdIns(3, 4)P2 and/or PtdIns(3,4,5)P3, and we have identified several new PH domain-containing proteins that bind PI3K products, including Gab1, Dos, myosinX, and Sbf1. Use of this assay to screen for novel cDNAs which rescue yeast at the non-permissive temperature should provide a powerful approach for uncovering additional targets of PI3K.

Amino Acid Sequence↗
PubMed2005

Inhibition of phosphatidylinositol-3-kinase synergizes with gemcitabine in low-passage tumor cell lines correlating with Bax translocation to the mitochondria.

Apoptotic pathways, including the phosphatidylinositol-3-kinase (PI3K)/AKT survival pathway, are altered in most cancer cells in relation to their normal counterparts and these differences may present an excellent therapeutic window. To gain insight into the relevance of the PI3K pathway as a target for drug discovery we generated tumor cell lines from different tumor samples that we maintained at low passage. The characterization of these cell lines indicates that all of them have constitutively activated the PI3K pathway through different mechanisms. All cell lines were differentially sensitive to the PI3K inhibitor LY294002. Our data also support previous work indicating that PI3K inhibition might help classical chemotherapeutic treatments such as gemcitabine and strengthen suboptimal doses that might be effective for these purposes in decreasing the risk of side-effects. Finally, the analysis of the molecular markers that might be implicated in the synergism between LY294002 and gemcitabine suggests that PI3K inhibition might aid chemotherapeutic treatment, leading to changes in the balance between anti- and pro-apoptotic molecules of the Bcl-2 family, Bcl-XL and Bax. These results facilitate the exploration of potential synergism between chemotherapeutic treatment and the search for others that can account for similar molecular mechanisms of cooperation.

Carcinoma↗
PubMed2006

The targeting of phosphoinositide-3 kinase attenuates pulmonary metastatic tumor growth following laparotomy.

OBJECTIVE: We aimed to characterize a potential role for phosphatidylinositol 3-kinase (PI3k) in leading to accelerated postoperative metastatic tumor growth. BACKGROUND: PI3k enhances tumor cell survival in part by phosphorylating Akt and reducing apoptosis. Postoperatively, apoptosis is reduced within local recurrences and distant metastases. This reduction is associated with the phenomenon of accelerated postoperative tumor growth. METHODS: Balb/c mice underwent a tail vein injection of 1x10 metastatic murine mammary adenocarcinoma 4T1 cells. Animals were divided into the following treatment groups (n=10/group): group A, controls; group B, DMSO intraperitoneally (IP) daily from days 14 to 21; group C, IP LY294002 daily from days 14 to 21; group D, laparotomy only; group E, laparotomy followed by IP DMSO for 7 days; and group F, laparotomy followed by LY294002 IP for 7 days. All laparotomies were performed on day 14. Animals were killed at day 28. Metastatic tumor burden was assessed using the lung/body weight ratio and a histologic metastatic index. Mitotic counts and apoptotic indices were established using a combination of hematoxylin and eosin histology and TUNEL immunohistochemistry. A parallel survival study was performed, and PI3k activity was assessed using western blots for phospho-Akt. RESULTS: Laparotomy was associated with increased systemic tumor burden (P=0.001). Postoperatively, LY294002 significantly attenuated metastatic tumor growth (P<0.001). Effective PI3k inhibition was confirmed by demonstrating a reduced Akt phosphorylation. Moreover, PI3k inhibition led to reduced proliferation, increased apoptosis (P<0.001), and enhanced postoperative survival (P<0.001). CONCLUSIONS: Targeting PI3k with postoperative LY294002 significantly attenuates the acceleration in postoperative metastatic tumor growth seen following laparotomy.

Adenocarcinoma↗
PubMed2005

Hyposmolarity-induced ErbB4 phosphorylation and its influence on the non-receptor tyrosine kinase network response in cultured cerebellar granule neurons.

Exposure of cultured cerebellar granule neurons (24 h serum-starved) during 3 min to 30% hyposmotic medium activated the tyrosine kinase receptor ErbB4 in the absence of its ligand. Hyposmolarity also activated the non-receptor tyrosine kinases, Src, focal adhesion kinase (FAK), extracellular signal-regulated protein kinase (ERK)1/2, and the tyrosine kinase target phosphatidyl-inositol-3-kinase (PI3K). The hyposmotic-induced activation of these kinases required the prior phosphorylation of ErbB4 as shown by the effect of ErbB4 blockade with AG213 reducing by 85-95% the phosphorylation of FAK and ERK1/2, by 74% and 36% that of PI3K and Src, respectively. These results suggest a key role of ErbB4 as a signal integrator of events associated with hyposmolarity. PI3K seems to be an important connecting element in the signaling network evoked by the hyposmolarity/ErbB4 activation as: (i) the p85 regulatory subunit of PI3K co-immunoprecipitates with ErbB4 and with FAK; (ii) PI3K blockade with wortmannin reduced the hyposmotic activation of FAK (90%) and ERK1/2 (84-91%). Inhibition of Src with PP2 reduced ErbB4 phosphorylation and inhibited the subsequent cytosolic kinase activation with the same potency as ErbB4 blockade. These results point to Src and ErbB4 and as early targets of the hyposmotic stimulus and osmosignaling. The functional significance for cell volume regulation of the ErbB4-Src-PI3K signaling cascade is indicated by the 48-66% decrease of the hyposmotic taurine efflux observed by inhibition of these kinases.

Animals↗
PubMed2005

Opposing roles of endothelial and smooth muscle phosphatidylinositol 3-kinase in vasoconstriction: effects of rho-kinase and hypertension.

Phosphatidylinositol 3-kinase (PI3K) can activate endothelial nitric oxide synthase (eNOS), leading to production of the vasodilator NO. In contrast, vascular smooth muscle (VSM) PI3K may partially mediate vascular contraction, particularly during hypertension. We tested whether endothelial and VSM PI3K may have opposing functional roles in regulating vascular contraction. Secondly, we tested whether the procontractile protein rho-kinase can suppress endothelial PI3K/eNOS activity in intact arteries, thus contributing to vasoconstriction by G protein-coupled receptor (GPCR) agonists. We studied contractile responses to the GPCR agonist phenylephrine, and the receptor-independent vasoconstrictor KCl, in aortic rings from Sprague-Dawley rats. In endothelium-intact rings, the PI3K inhibitor wortmannin (0.1 microM) markedly augmented responses to phenylephrine (P < 0.05) by approximately 50% but not to KCl. However, in endothelium-denuded or N(G)-nitro-L-arginine methyl ester (L-NAME) (100 microM)-treated rings, wortmannin reduced responses to phenylephrine and KCl (P < 0.05). Furthermore, the rhokinase inhibitor Y-27632 (R-[+]-trans-N-[4-pyridyl]-4-[1-aminoethyl]-cycloheaxanecarboxamide; 1 microM) abolished responses to phenylephrine, and this effect was partially reversed by wortmannin or L-NAME. The ability of wortmannin to oppose the effect of rho-kinase inhibition on contractions to phenylephrine was L-NAME-sensitive. In aortas from angiotensin II-induced hypertensive rats, relaxation to acetylcholine (10 microM) was impaired (P < 0.05), and vasoconstriction by phenylephrine was markedly enhanced and not further augmented by wortmannin. These data suggest that endothelial PI3K-induced NO production can modulate GPCR agonist-induced vascular contraction and that this effect is impaired in hypertension in association with endothelial dysfunction. In addition, endothelial rho-kinase may act to suppress PI3K activity and, hence, attenuate NO-mediated relaxation and augment GPCR-dependent contraction.

Animals↗
PubMed2006

Ca2+-independent, inhibitory effects of cyclic adenosine 5'-monophosphate on Ca2+ regulation of phosphoinositide 3-kinase C2alpha, Rho, and myosin phosphatase in vascular smooth muscle.

We have recently demonstrated in vascular smooth muscle (VSM) that membrane depolarization by high KCl induces Ca(2+)-dependent Rho activation and myosin phosphatase (MLCP) inhibition (Ca(2+)-induced Ca(2+)-sensitization) through the mechanisms involving phosphorylation of myosin-targeting protein 1 (MYPT1) and 17-kDa protein kinase C (PKC)-potentiated inhibitory protein of PP1 (CPI-17). In the present study, we investigated whether and how cAMP affected Ca(2+)-dependent MLCP inhibition by examining the effects of forskolin, cell-permeable dibutyryl cAMP (dbcAMP), and isoproterenol. Forskolin, but not its inactive analog 1,9-dideoxyforskolin, inhibited KCl-induced contraction and the 20-kDa myosin light chain (MLC) phosphorylation without inhibiting Ca(2+) mobilization in rabbit aortic VSM. dbcAMP mimicked these forskolin effects. We recently suggested that Ca(2+)-mediated Rho activation is dependent on class II alpha-isoform of phosphoinositide 3-kinase (PI3K-C2alpha). Forskolin inhibited KCl-induced stimulation of PI3K-C2alpha activity. KCl-induced membrane depolarization stimulated Rho in a manner dependent on a PI3K but not PKC and stimulated phosphorylation of MYPT1 at Thr(850) and CPI-17 at Thr(38) in manners dependent on both PI3K and Rho kinase, but not PKC. Forskolin, dbcAMP, and isoproterenol inhibited KCl-induced Rho activation and phosphorylation of MYPT1 and CPI-17. Consistent with these data, forskolin, isoproterenol, a PI3K inhibitor, or a Rho kinase inhibitor, but not a PKC inhibitor, abolished KCl-induced diphosphorylation of MLC. These observations indicate that cAMP inhibits Ca(2+)-mediated activation of the MLCP-regulating signaling pathway comprising PI3K-C2alpha, Rho, and Rho kinase in a manner independent of Ca(2+) and point to the novel mechanism of the cAMP actions in the regulation of vascular smooth muscle contraction.

Animals↗
PubMed2002

Subversion of cell signaling pathways by hepatitis C virus nonstructural 5A protein via interaction with Grb2 and P85 phosphatidylinositol 3-kinase.

Hepatitis C virus (HCV) sets up a persistent infection in patients that likely involves a complex virus-host interaction. We previously found that the HCV nonstructural 5A (NS5A) protein interacts with growth factor receptor-binding protein 2 (Grb2) adaptor protein and inhibits the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) by epidermal growth factor (EGF). In the present study, we extended this analysis and investigated the specificity of the Grb2-NS5A interaction and whether the subversion of mitogenic signaling involves additional pathways. NS5A containing mutations within the C-terminal proline-rich motif neither bound Grb2 nor inhibited ERK1/2 activation by EGF, demonstrating that NS5A-Grb2 binding and downstream effects were due to direct interactions. Interestingly, NS5A could also form a complex with the Grb2-associated binder 1 (Gab1) protein in an EGF treatment-dependent manner. However, the NS5A-Gab1 association, which appeared indirect, was not mediated by direct NS5A-Grb2 interaction but was likely dependent on direct NS5A interaction with the p85 subunit of phosphatidylinositol 3-kinase (PI3K). The in vivo association of NS5A with p85 PI3K required the N-terminal, but not the C-terminal, region of NS5A. The downstream effects of the NS5A-p85 PI3K interaction included increased tyrosine phosphorylation of p85 PI3K in response to EGF. Consistent with this observation and the antiapoptotic properties of NS5A, we also detected enhanced tyrosine phosphorylation of the downstream AKT protein kinase and increased serine phosphorylation of BAD, a proapoptotic factor and an AKT substrate, in the presence of NS5A. These results collectively suggest a model in which NS5A interacts with Grb2 to inhibit mitogenic signaling while simultaneously promoting the PI3K-AKT cell survival pathway by interaction with p85 PI3K, which may represent a crucial step in HCV persistence and pathogenesis.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Corneal cell survival in adenovirus type 19 infection requires phosphoinositide 3-kinase/Akt activation.

Adenovirus type 19 is a major cause of epidemic keratoconjunctivitis, the only ocular adenoviral infection associated with prolonged corneal inflammation. In this study, we investigated the role of phosphoinositide 3-kinase (PI3K) and Akt and their downstream targets in adenovirus infection, and here we report the novel finding that adenovirus type 19 utilizes the PI3K/Akt pathway to maintain corneal fibroblast viability in acute infection. We demonstrate phosphorylation of GSK-3beta and nuclear translocation of the p65 subunit of NF-kappaB, both downstream targets of the PI3K/Akt pathway, in adenovirus-infected corneal fibroblasts in a PI3K-dependent manner. Inhibition of PI3K had no effect on early viral gene expression, suggesting normal viral internalization, but pretreatment with the PI3K inhibitor LY294002 or overexpression of dominant negative Akt induced early cytopathic effect and caspase-mediated cell death in adenovirus-infected cells. Early cell death could be circumvented despite LY294002 by overexpression of constitutively active Akt. Furthermore, we show an interaction between cSrc and the p85 regulatory subunit of PI3K in infected cells through a phosphorylation-dependent mechanism. The results presented in this paper provide the first direct evidence that PI3K-mediated Akt activation in adenovirus-infected corneal cells may contribute to viral pathogenesis by the prolongation of cell viability.

Adenoviruses, Human↗
PubMed2006

Phosphoinositide 3-kinase activation in late G1 is required for c-Myc stabilization and S phase entry.

Phosphoinositide 3-kinase (PI3K) is one of the early-signaling molecules induced by growth factor (GF) receptor stimulation that are necessary for cell growth and cell cycle entry. PI3K activation occurs at two distinct time points during G(1) phase. The first peak is observed immediately following GF addition and the second in late G(1), before S phase entry. This second activity peak is essential for transition from G(1) to S phase; nonetheless, the mechanism by which this peak is induced and regulates S phase entry is poorly understood. Here, we show that activation of Ras and Tyr kinases is required for late-G(1) PI3K activation. Inhibition of late-G(1) PI3K activity results in low c-Myc and cyclin A expression, impaired Cdk2 activity, and reduced loading of MCM2 (minichromosome maintenance protein) onto chromatin. The primary consequence of inhibiting late-G(1) PI3K was c-Myc destabilization, as conditional activation of c-Myc in advanced G(1) as well as expression of a stable c-Myc mutant rescued all of these defects, restoring S phase entry. These results show that Tyr kinases and Ras cooperate to induce the second PI3K activity peak in G(1), which mediates initiation of DNA synthesis by inducing c-Myc stabilization.

Animals↗
PubMed1999

Protein kinase A-dependent and -independent signaling pathways contribute to cyclic AMP-stimulated proliferation.

The effects of cyclic AMP (cAMP) on cell proliferation are cell type specific. Although the growth-inhibitory effects of cAMP have been well studied, much less is known regarding how cAMP stimulates proliferation. We report that cAMP stimulates proliferation through both protein kinase A (PKA)-dependent and PKA-independent signaling pathways and that phosphatidylinositol 3-kinase (PI3K) is required for cAMP-stimulated mitogenesis. In cells where cAMP is a mitogen, cAMP-elevating agents stimulate membrane ruffling, Akt phosphorylation, and p70 ribosomal S6 protein kinase (p70s6k) activity. cAMP effects on ruffle formation and Akt were PKA independent but sensitive to wortmannin. In contrast, cAMP-stimulated p70s6k activity was repressed by PKA inhibitors but not by wortmannin or microinjection of the N-terminal SH2 domain of the p85 regulatory subunit of PI3K, indicating that p70s6k and Akt can be regulated independently. Microinjection of highly specific inhibitors of PI3K or Rac1, or treatment with the p70s6k inhibitor rapamycin, impaired cAMP-stimulated DNA synthesis, demonstrating that PKA-dependent and -independent pathways contribute to cAMP-mediated mitogenesis. Direct elevation of PI3K activity through microinjection of an antibody that stimulates PI3K activity or stable expression of membrane-localized p110 was sufficient to confer hormone-independent DNA synthesis when accompanied by elevations in p70s6k activity. These findings indicate that multiple pathways contribute to cAMP-stimulated mitogenesis, only some of which are PKA dependent. Furthermore, they demonstrate that the ability of cAMP to stimulate both p70s6k- and PI3K-dependent pathways is an important facet of cAMP-regulated cell cycle progression.

3T3 Cells↗
PubMed2001

Identification of insulin receptor substrate 1 (IRS-1) and IRS-2 as signaling intermediates in the alpha6beta4 integrin-dependent activation of phosphoinositide 3-OH kinase and promotion of invasion.

Expression of the alpha6beta4 integrin increases the invasive potential of carcinoma cells by a mechanism that involves activation of phosphoinositide 3-OH kinase (PI3K). In the present study, we investigated the signaling pathway by which the alpha6beta4 integrin activates PI3K. Neither the alpha6 nor the beta4 cytoplasmic domain contains the consensus binding motif for PI3K, pYMXM, indicating that additional proteins are likely to be involved in the activation of this lipid kinase by the alpha6beta4 integrin. We identified insulin receptor substrate 1 (IRS-1) and IRS-2 as signaling intermediates in the activation of PI3K by the alpha6beta4 integrin. IRS-1 and IRS-2 are cytoplasmic adapter proteins that do not contain intrinsic kinase activity but rather function by recruiting proteins to surface receptors, where they organize signaling complexes. Ligation of the alpha6beta4 receptor promotes tyrosine phosphorylation of IRS-1 and IRS-2 and increases their association with PI3K, as determined by coimmunoprecipitation. Moreover, we identified a tyrosine residue in the cytoplasmic domain of the beta4 subunit, Y1494, that is required for alpha6beta4-dependent phosphorylation of IRS-2 and activation of PI3K in response to receptor ligation. Most importantly, Y1494 is essential for the ability of the alpha6beta4 integrin to promote carcinoma invasion. Taken together, these results imply a key role for the IRS proteins in the alpha6beta4-dependent promotion of carcinoma invasion.

Amino Acid Motifs↗