Cloning and characterization of Lnk, a signal transduction protein that links T-cell receptor activation signal to phospholipase C gamma 1, Grb2, and phosphatidylinositol 3-kinase.

A cDNA encoding a signal transduction protein with a Src homology 2 (SH2) domain and a tyrosine phosphorylation site was cloned from a rat lymph node cDNA library. This protein, which we designate Lnk, has a calculated molecular weight of 33,988. When T lymphocytes were activated by antibody-mediated crosslinking of the T-cell receptor and CD4, Lnk became tyrosine phosphorylated. In activated T lymphocytes, phospholipase C gamma 1, phosphatidylinositol 3-kinase, and Grb-2 coimmunoprecipitated with Lnk. Our results suggest that Lnk becomes tyrosine phosphorylated and links the immediate tyrosine phosphorylation signals of the TCR to the distal phosphatidylinositol 3-kinase, phospholipase C gamma 1 and Ras signaling pathways through its multifunctional tyrosine phosphorylation site.

Grb2-associated binder-1 mediates phosphatidylinositol 3-kinase activation and the promotion of cell survival by nerve growth factor

Nerve growth factor (NGF) prevents apoptosis through stimulation of the TrkA receptor protein tyrosine kinase. The downstream activation of phosphatidylinositol 3-kinase (PI 3-kinase) is essential for the inhibition of apoptosis, although this enzyme does not bind to and is not directly activated by TrkA. We have found that the addition of NGF to PC-12 cells resulted in the phosphorylation of the Grb2-associated binder-1 (Gab1) docking protein and induced the association of several SH2 domain-containing proteins, including PI 3-kinase. A substantial fraction of the total cellular PI 3-kinase activity was associated with Gab1. PC-12 cells that overexpressed Gab1 show a decreased requirement for the amount of NGF necessary to inhibit apoptosis. The expression of a Gab1 mutant that lacked the binding sites for PI 3-kinase enhanced apoptosis and diminished the protective effect of NGF. Hence, Gab1 has a major role in connecting TrkA with PI 3-kinase activation and for the promotion of cell survival by NGF.

A phosphatidylinositol 3-kinase/Akt/mTOR pathway mediates and PTEN antagonizes tumor necrosis factor inhibition of insulin signaling through insulin receptor substrate-1

Tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) by the insulin receptor permits this docking protein to interact with signaling proteins that promote insulin action. Serine phosphorylation uncouples IRS-1 from the insulin receptor, thereby inhibiting its tyrosine phosphorylation and insulin signaling. For this reason, there is great interest in identifying serine/threonine kinases for which IRS-1 is a substrate. Tumor necrosis factor (TNF) inhibited insulin-promoted tyrosine phosphorylation of IRS-1 and activated the Akt/protein kinase B serine-threonine kinase, a downstream target for phosphatidylinositol 3-kinase (PI 3-kinase). The effect of TNF on insulin-promoted tyrosine phosphorylation of IRS-1 was blocked by inhibition of PI 3-kinase and the PTEN tumor suppessor, which dephosphorylates the lipids that mediate PI 3-kinase functions, whereas constitutively active Akt impaired insulin-promoted IRS-1 tyrosine phosphorylation. Conversely, TNF inhibition of IRS-1 tyrosine phosphorylation was blocked by kinase dead Akt. Inhibition of IRS-1 tyrosine phosphorylation by TNF was blocked by rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), a downstream target of Akt. mTOR induced the serine phosphorylation of IRS-1 (Ser-636/639), and such phosphorylation was inhibited by rapamycin. These results suggest that TNF impairs insulin signaling through IRS-1 by activation of a PI 3-kinase/Akt/mTOR pathway, which is antagonized by PTEN.

Identification of an early endosomal protein regulated by phosphatidylinositol 3-kinase

Phosphatidylinositol 3-kinases (PI 3-kinases) have been implicated in membrane trafficking in the secretory and endocytic pathways of yeast and mammalian cells, but the molecular mechanisms by which these lipid kinases operate are not known. Here we identify a protein of 170 kDa that is rapidly released from cell membranes in response to wortmannin, a potent inhibitor of mammalian PI 3-kinases. The amino acid sequence of peptides from p170 reveal its identity to early endosomal antigen (EEA) 1, an endosomal antigen with homology to several yeast proteins genetically implicated in membrane trafficking. Immunofluorescence analysis of 3T3-L1 adipocytes with antisera against p170/EEA1 reveal a punctate peripheral pattern that becomes diffuse in response to wortmannin. In vitro, p170/EEA1 binds specifically to liposomes containing PIns(3)P, suggesting that the effect of wortmannin on cells is due to inhibition of PIns(3)P production. Thus, p170/EEA1 may define a family of proteins that mediate the regulatory effects of 3′-phosphoinositides on membrane trafficking in yeast and mammalian cells.

The E5 gene product of rhesus papillomavirus is an activator of endogenous Ras and phosphatidylinositol-3′-kinase in NIH 3T3 cells

We examined the effect of two rhesus papillomavirus 1 (RhPV) oncogenes on cytokine-induced signal transduction pathways leading to the possible activation of Ras protein (p21ras) and phosphatidylinositol kinase. p21ras in both the activated (GTP-bound) and inactivated (GDP-bound) states were quantitated. NIH 3T3 cell lines expressing the RhPV 1 E5 gene or epidermal growth factor receptor cDNA had about a sixfold higher ratio of p21ras-bound GTP to p21ras-bound GDP as compared with parental NIH 3T3 cells or a cell line expressing the RhPV 1 E7 gene under normal culture conditions, yet expressed similar levels of p21ras. Quiescent cells had dramatically reduced levels of activated p21ras, except those containing RhPV 1 E7. Levels were restored by stimulation with epidermal growth factor or platelet-derived growth factor. Both epidermal growth factor and platelet-derived growth factor receptor of RhPV 1 E5- and E7-containing cells responded to cytokine stimulation. Endogenous phosphatidylinositol-3′-kinase was up-regulated in NIH 3T3 cells transformed with the E5 genes of RhPV 1 and bovine papillomavirus 1. These results suggest that E5 genes of papillomaviruses play a major role in the regulation of transduction pathways.

An essential role of phosphatidylinositol 3-kinase in myogenic differentiation

The oncogene p3k, coding for a constitutively active form of phosphatidylinositol 3-kinase (PI 3-kinase; EC 2.7.1.137), strongly enhances myogenic differentiation in cultures of chicken-embryo myoblasts. It increases the size of the myotubes and induces elevated levels of the muscle-specific proteins MyoD, myosin heavy chain, creatine kinase, and desmin. Inhibition of PI 3-kinase activity with LY294002 or with dominant-negative mutants of PI 3-kinase interferes with myogenic differentiation and with the induction of muscle-specific genes. PI 3-kinase is therefore an upstream mediator for the expression of the muscle-specific genes and is both necessary and rate-limiting for the process of myogenesis.

Regulation of G1 progression by the PTEN tumor suppressor protein is linked to inhibition of the phosphatidylinositol 3-kinase/Akt pathway

PTEN/MMAC1 is a tumor suppressor gene located on chromosome 10q23. Inherited PTEN/MMAC1 mutations are associated with a cancer predisposition syndrome known as Cowden’s disease. Somatic mutation of PTEN has been found in a number of malignancies, including glioblastoma, melanoma, and carcinoma of the prostate and endometrium. The protein product (PTEN) encodes a dual-specificity protein phosphatase and in addition can dephosphorylate certain lipid substrates. Herein, we show that PTEN protein induces a G1 block when reconstituted in PTEN-null cells. A PTEN mutant associated with Cowden’s disease (PTEN;G129E) has protein phosphatase activity yet is defective in dephosphorylating inositol 1,3,4,5-tetrakisphosphate in vitro and fails to arrest cells in G1. These data suggest a link between induction of a cell-cycle block by PTEN and its ability to dephosphorylate, in vivo, phosphatidylinositol 3,4,5-trisphosphate. In keeping with this notion, PTEN can inhibit the phosphatidylinositol 3,4,5-trisphosphate-dependent Akt kinase, a downstream target of phosphatidylinositol 3-kinase, and constitutively active, but not wild-type, Akt overrides a PTEN G1 arrest. Finally, tumor cells lacking PTEN contain high levels of activated Akt, suggesting that PTEN is necessary for the appropriate regulation of the phosphatidylinositol 3-kinase/Akt pathway.

Phosphatidylinositol 3-kinase binding to polyoma virus middle tumor antigen mediates elevation of glucose transport by increasing translocation of the GLUT1 transporter.

Elevation in the rate of glucose transport in polyoma virus-infected mouse fibroblasts was dependent upon phosphatidylinositol 3-kinase (PI 3-kinase; EC 2.7.1.137) binding to complexes of middle tumor antigen (middle T) and pp60c-src. Wild-type polyoma virus infection led to a 3-fold increase in the rate of 2-deoxyglucose (2DG) uptake, whereas a weakly transforming polyoma virus mutant that encodes a middle T capable of activating pp60c-src but unable to promote binding of PI 3-kinase induced little or no change in the rate of 2DG transport. Another transformation-defective mutant encoding a middle T that retains functional binding of both pp60c-src and PI 3-kinase but is incapable of binding Shc (a protein involved in activation of Ras) induced 2DG transport to wild-type levels. Wortmannin (< or = 100 nM), a known inhibitor of PI 3-kinase, blocked elevation of glucose transport in wild-type virus-infected cells. In contrast to serum stimulation, which led to increased levels of glucose transporter 1 (GLUT1) RNA and protein, wild-type virus infection induced no significant change in levels of either GLUT1 RNA or protein. Nevertheless, virus-infected cells did show increases in GLUT1 protein in plasma membranes. These results point to a posttranslational mechanism in the elevation of glucose transport by polyoma virus middle T involving activation of PI 3-kinase and translocation of GLUT1.

Evidence for phosphatidylinositol 3-kinase as a regulator of endocytosis via activation of Rab5.

Phosphatidylinositol (PI) 3-kinases have been implicated in several aspects of intracellular membrane trafficking, although a detailed mechanism is yet to be established. In this study we demonstrated that wortmannin, a specific inhibitor of PI 3-kinases, inhibited constitutive endocytosis of horseradish peroxidase and transferrin in BHK-21 and TRVb-1 cells. The IC50 was approximately 40 ng/ml (93 nM). In addition, wortmannin blocked the stimulation of horseradish peroxidase uptake by the small GTPase Rab5 but not the stimulation by the GTPase-defective, constitutively activated Rab5 Gln79-->Leu mutant (Rab5:Q79L), providing further evidence that PI 3-kinase activity is essential for the early endocytic process. To further investigate the mechanism, we examined the effect of wortmannin on early endosome fusion in vitro. Wortmannin decreased endosome fusion by 80% with an IC50 value similar to that in intact cells. Addition of Rab5:Q79L but not wild-type Rab5 reversed the inhibitory effect of wortmannin. Furthermore, addition of a constitutively activated PI 3-kinase but not its inactive counterpart stimulated early endosome fusion in vitro. These results strongly indicate that PI 3-kinase plays an important role in regulation of early endosome fusion, probably via activation of Rab5.

The inability of phosphatidylinositol 3-kinase activation to stimulate GLUT4 translocation indicates additional signaling pathways are required for insulin-stimulated glucose uptake.

Recent experimental evidence has focused attention to the role of two molecules, insulin receptor substrate 1 (IRS-1) and phosphatidylinositol 3-kinase (PI3-kinase), in linking the insulin receptor to glucose uptake; IRS-1 knockout mice are insulin resistant, and pharmacological inhibitors of PI3-kinase block insulin-stimulated glucose uptake. To investigate the role of PI3-kinase and IRS-1 in insulin-stimulated glucose uptake we examined whether stimulation of insulin-sensitive cells with platelet-derived growth factor (PDGF) or with interleukin 4 (IL-4) stimulates glucose uptake; the activated PDGF receptor (PDGFR) directly binds and activates PI3-kinase, whereas the IL-4 receptor (IL-4R) activates PI3-kinase via IRS-1 or the IRS-1-related molecule 4PS. We found that stimulation of 3T3-L1 adipocytes with PDGF resulted in tyrosine phosphorylation of the PDGFR and activation of PI3-kinase in these cells. To examine whether IL-4 stimulates glucose uptake, L6 myoblasts were engineered to overexpress GLUT4 as well as both chains of the IL-4R (L6/IL-4R/GLUT4); when these L6/IL-4R/GLUT4 myoblasts were stimulated with IL-4, IRS-1 became tyrosine phosphorylated and associated with PI3-kinase. Although PDGF and IL-4 can activate PI3-kinase in the respective cell lines, they do not possess insulin's ability to stimulate glucose uptake and GLUT4 translocation to the plasma membrane. These findings indicate that activation of PI3-kinase is not sufficient to stimulate GLUT4 translocation to the plasma membrane. We postulate that activation of a second signaling pathway by insulin, distinct from PI3-kinase, is necessary for the stimulation of glucose uptake in insulin-sensitive cells.

Phosphatidylinositol 3-kinase signals activation of p70 S6 kinase in situ through site-specific p70 phosphorylation.

The p70 S6 kinase is activated by insulin and mitogens through multisite phosphorylation of the enzyme. One set of activating phosphorylations occurs in a putative autoinhibitory domain in the noncatalytic carboxyl-terminal tail. Deletion of this tail yields a variant (p70 delta CT104) that nevertheless continues to be mitogen regulated. Coexpression with a recombinant constitutively active phosphatidylinositol (PI) 3-kinase (EC 2.7.1.137) gives substantial activation of both full-length p70 and p70 delta CT104 but not Rsk. Activation of p70 delta CT104 by PI 3-kinase and inhibition by wortmannin are each accompanied by parallel and selective changes in the phosphorylation of p70 Thr-252. A Thr or Ser at this site, in subdomain VIII of the catalytic domain just amino-terminal to the APE motif, is necessary for p70 40S kinase activity. The inactive ATP-binding site mutant K123M p70 delta CT104 undergoes phosphorylation of Thr-252 in situ but does not undergo direct phosphorylation by the active PI 3-kinase in vitro. PI 3-kinase provides a signal necessary for the mitogen activation of the p70 S6 kinase, which directs the site-specific phosphorylation of Thr-252 in the p70 catalytic domain, through a distinctive signal transduction pathway.

The inositol polyphosphate 4-phosphatase forms a complex with phosphatidylinositol 3-kinase in human platelet cytosol

Inositol polyphosphate 4-phosphatase (4-phosphatase) is an enzyme that catalyses the hydrolysis of the 4-position phosphate from phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2]. In human platelets the formation of this phosphatidylinositol, by the actions of phosphatidylinositol 3-kinase (PI 3-kinase), correlates with irreversible platelet aggregation. We have shown previously that a phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase forms a complex with the p85 subunit of PI 3-kinase. In this study we investigated whether PI 3-kinase also forms a complex with the 4-phosphatase in human platelets. Immunoprecipitates of the p85 subunit of PI 3-kinase from human platelet cytosol contained 4-phosphatase enzyme activity and a 104-kDa polypeptide recognized by specific 4-phosphatase antibodies. Similarly, immunoprecipitates made using 4-phosphatase-specific antibodies contained PI 3-kinase enzyme activity and an 85-kDa polypeptide recognized by antibodies to the p85 adapter subunit of PI 3-kinase. After thrombin activation, the 4-phosphatase translocated to the actin cytoskeleton along with PI 3-kinase in an integrin- and aggregation-dependent manner. The majority of the PI 3-kinase/4-phosphatase complex (75%) remained in the cytosolic fraction. We propose that the complex formed between the two enzymes serves to localize the 4-phosphatase to sites of PtdIns(3,4)P2 production.

Src-induced activation of inducible T cell kinase (ITK) requires phosphatidylinositol 3-kinase activity and the Pleckstrin homology domain of inducible T cell kinase

The Tec family of tyrosine kinases are involved in signals emanating from cytokine receptors, antigen receptors, and other lymphoid cell surface receptors. One family member, ITK (inducible T cell kinase), is involved in T cell activation and can be activated by the T cell receptor and the CD28 cell surface receptor. This stimulation of tyrosine phosphorylation and activation of ITK can be mimicked by the Src family kinase Lck. We have explored the mechanism of this requirement for Src family kinases in the activation of ITK. We found that coexpression of ITK and Src results in increased membrane association, tyrosine phosphorylation and activation of ITK, which could be blocked by inhibitors of the lipid kinase phosphatidylinositol 3-kinase (PI 3-kinase) as well as overexpression of the p85 subunit of PI 3-kinase. Removal of the Pleckstrin homology domain (PH) of ITK resulted in a kinase that could no longer be induced to localize to the membrane or be activated by Src. The PH of ITK was also able to bind inositol phosphates phosphorylated at the D3 position. Membrane targeting of ITK without the PH recovered its ability to be activated by Src. These results suggest that ITK can be activated by a combination of Src and PI 3-kinase.

Phosphatidylinositol 3-Kinase-mediated Endocytosis of Renal Na+,K+-ATPase α Subunit in Response to Dopamine

Dopamine (DA) inhibition of Na+,K+-ATPase in proximal tubule cells is associated with increased endocytosis of its α and β subunits into early and late endosomes via a clathrin vesicle-dependent pathway. In this report we evaluated intracellular signals that could trigger this mechanism, specifically the role of phosphatidylinositol 3-kinase (PI 3-K), the activation of which initiates vesicular trafficking and targeting of proteins to specific cell compartments. DA stimulated PI 3-K activity in a time- and dose-dependent manner, and this effect was markedly blunted by wortmannin and LY 294002. Endocytosis of the Na+,K+-ATPase α subunit in response to DA was also inhibited in dose-dependent manner by wortmannin and LY 294002. Activation of PI 3-K generally occurs by association with tyrosine kinase receptors. However, in this study immunoprecipitation with a phosphotyrosine antibody did not reveal PI 3-K activity. DA-stimulated endocytosis of Na+,K+-ATPase α subunits required protein kinase C, and the ability of DA to stimulate PI 3-K was blocked by specific protein kinase C inhibitors. Activation of PI 3-K is mediated via the D1 receptor subtype and the sequential activation of phospholipase A2, arachidonic acid, and protein kinase C. The results indicate a key role for activation of PI 3-K in the endocytic sequence that leads to internalization of Na+,K+-ATPase α subunits in response to DA, and suggest a mechanism for the participation of protein kinase C in this process.

Phosphatidylinositol 3-kinase signaling mediates angiogenesis and expression of vascular endothelial growth factor in endothelial cells

Phosphatidylinositol 3-kinase (PI 3-kinase) is a signaling molecule that controls numerous cellular properties and activities. The oncogene v-p3k is a homolog of the gene coding for the catalytic subunit of PI 3-kinase, p110α. P3k induces transformation of cells in culture, formation of hemangiosarcomas in young chickens, and myogenic differentiation in myoblasts. Here, we describe a role of PI 3-kinase in angiogenesis. Overexpression of the v-P3k protein or of cellular PI 3-kinase equipped with a myristylation signal, Myr-P3k, can induce angiogenesis in the chorioallantoic membrane (CAM) of the chicken embryo. This process is characterized by extensive sprouting of new blood vessels and enlargement of preexisting vessels. Overexpression of the myristylated form of the PI 3-kinase target Akt, Myr-Akt, also induces angiogenesis. Overexpression of the tumor suppressor PTEN or of dominant-negative constructs of PI 3-kinase inhibits angiogenesis in the yolk sac of chicken embryos, suggesting that PI 3-kinase and Akt signaling is required for normal embryonal angiogenesis. The levels of mRNA for vascular endothelial growth factor (VEGF) are elevated in cells expressing activated PI 3-kinase or Myr-Akt. VEGF mRNA levels are also increased by insulin treatment through the PI 3-kinase-dependent pathway. VEGF mRNA levels are decreased in cells treated with the PI 3-kinase inhibitor LY294002 and restored by overexpression of v-P3k or Myr-Akt. Overexpression of VEGF by the RCAS vector induces angiogenesis in chicken embryos. These results suggest that PI 3-kinase plays an important role in angiogenesis and regulates VEGF expression.