Assigning functional domains within the p101 regulatory subunit of phosphoinositide 3-kinase gamma

Phosphoinositide 3-Kinase (PI3K) gamma is a lipid kinase that is regulated by G-protein-coupled receptors. It plays a crucial role in inflammatory and allergic processes. Activation of PI3Kgamma is primarily mediated by Gbetagamma subunits. The regulatory p101 subunit of PI3Kgamma binds to Gbetagamma and, thereby, recruits the catalytic p110gamma subunit to the plasma membrane. Despite its crucial role in the activation of PI3Kgamma, the structural organization of p101 is still largely elusive. Employing fluorescence resonance energy transfer measurements, coimmunoprecipitation and colocalization studies with p101 deletion mutants, we show here that distinct regions within the p101 primary structure are responsible for interaction with p110gamma and Gbetagamma. The p110gamma binding site is confined to the N terminus, whereas binding to Gbetagamma is mediated by a C-terminal domain of p101. These domains appear to be highly conserved among various species ranging from Xenopus to men. In addition to establishing a domain structure for p101, our results point to the existence of a previously unknown, p101-related regulatory subunit for PI3Kgamma.     

Characterization of p87PIKAP, a novel regulatory subunit of phosphoinositide 3-kinase gamma that is highly expressed in heart and interacts with PDE3B

Phosphoinositide 3-kinase (PI3K) gamma has been implicated in a vast array of physiological settings including the activation of different leukocyte species and the regulation of myocardial contractility. Activation of PI3Kgamma is primarily mediated by Gbetagamma subunits of heterotrimeric G proteins, which are recognized by a p101 regulatory subunit. Here, we describe the identification and characterization of a novel regulatory subunit of PI3Kgamma, which we termed p87(PIKAP) (PI3Kgamma adapter protein of 87 kDa). It is homologous to p101 in areas that we have recently shown that they mediate binding to the catalytic p110gamma subunit and to Gbetagamma. Like p101, p87(PIKAP) binds to both p110gamma and Gbetagamma and mediates activation of p110gamma downstream of G protein-coupled receptors. In contrast to p101, p87(PIKAP) is highly expressed in heart and may therefore be crucial to PI3Kgamma cardiac function. Moreover, p87(PIKAP) and p101 are both expressed in dendritic cells, macrophages, and neutrophils, raising the possibility of regulatory subunit-dependent differences in PI3Kgamma signaling within the same cell type. We further provide evidence that p87(PIKAP) physically interacts with phosphodiesterase (PDE) 3B, suggesting that p87(PIKAP) is also involved in the recently described noncatalytic scaffolding interaction of p110gamma with PDE3B. However, coexpression of PDE3B and PI3Kgamma subunits was not sufficient to reconstitute the regulatory effect of PI3Kgamma on PDE3B activity observed in heart, implying further molecules to be present in the complex regulating PDE3B in heart.     

Roles of non-catalytic subunits in gbetagamma-induced activation of class I phosphoinositide 3-kinase isoforms beta and gamma.

By using purified preparations we show that nanomolar concentrations of Gbetagamma significantly stimulated lipid kinase activity of phosphatidylinositol 3-kinase (PI3K) beta and PI3Kgamma in the presence as well as in the absence of non-catalytic subunits such as p85alpha or p101. Concomitantly, Gbetagamma stimulated autophosphorylation of the catalytic subunit of PI3Kgamma (EC(50), 30 nM; stoichiometry >/=0.6 mol of P(i)/mol of p110gamma), which also occurred in the absence of p101. Surprisingly, we found that p101 affected the lipid substrate preference of PI3Kgamma in its Gbetagamma-stimulated state. With phosphatidylinositol as substrate, p110gamma but not p101/p110gamma was significantly stimulated by Gbetagamma to form PI-3-phosphate (EC(50), 20 nM). The opposite situation was found when PI-4,5-bisphosphate served as substrate. Gbetagamma efficiently and potently (EC(50), 5 nM) activated the p101/p110gamma heterodimer but negligibly stimulated the p110gamma monomer to form PI-3,4,5-trisphosphate. However, this weak stimulatory effect on p110gamma was overcome by excess concentrations of Gbetagamma (EC(50), 100 nM). This finding is in accordance with the in vivo situation, where activated PI3K catalyzes the formation of PI-3,4,5-trisphosphate but not PI-3-phosphate. We conclude that p101 is responsible for PI-4, 5-bisphosphate substrate selectivity of PI3Kgamma by sensitizing p110gamma toward Gbetagamma in the presence of PI-4,5-P(2).     

Agonists cause nuclear translocation of phosphatidylinositol 3-kinase gamma. A Gbetagamma-dependent pathway that requires the p110gamma amino terminus.

In hematopoietic cells, the signals initiated by activation of the phosphoinositide 3-kinase (PI3K) family have been implicated in cell proliferation and survival, membrane and cytoskeletal reorganization, chemotaxis, and the neutrophil respiratory burst. Of the four isoforms of human PI3K that phosphorylate phosphatidylinositol 4, 5-bisphosphate, only p110gamma (or PI3Kgamma) is associated with the regulatory subunit, p101, and is stimulated by G protein betagamma heterodimers. We performed immunolocalization of transfected p110gamma in HepG2 cells and found that, under resting conditions, p110gamma was present in a diffuse cytoplasmic pattern, but translocated to the cell nucleus after serum stimulation. Serum-stimulated p110gamma translocation was inhibited by pertussis toxin and could also be induced by overexpression of Gbetagamma in the absence of serum. In addition, we found that deletion of the amino-terminal 33 residues of p110gamma had no effect on association with p101 or on its agonist-regulated translocation, but truncation of the amino-terminal 82 residues yielded a p110gamma variant that did not associate with p101 and was constitutively localized in the nucleus. This finding implies that the intracellular localization of p110gamma is regulated by p101 as well as Gbetagamma. The effect of PI3Kgamma in the nucleus is an area of active investigation.     

Differential regulation of lipid and protein kinase activities of phosphoinositide 3-kinase gamma in vitro

G protein sensitive phosphoinositide 3-kinase gamma (PI3Kgamma) has been characterised as a pleiotropic signalling protein expressing lipid kinase and protein kinase activities. Whereas the regulation of the lipid kinase activity has been investigated in detail, the regulatory features of PI3Kgamma protein kinase activity are unknown. Here we report that Gbetagamma subunits of heterotrimeric G proteins induce a biphasic response of PI3Kgamma autophosphorylation in vitro, which contrasts the regulatory effects of the G proteins on PI3Kgamma lipid kinase activity. In addition to autophosphorylation PI3Kgamma is able to catalyse transphosphorylation of the adapter protein p101 and the protein kinase MEK-1. In the presence of the p101, Gbetagamma affects PI3Kgamma protein kinase activities in a complex manner. In summary, the differential regulatory effects of heterotrimeric G proteins on PI3Kgamma lipid and protein kinase activities in vitro reflect the functional diversity of the enzyme observed in vivo.     

Roles of G beta gamma in membrane recruitment and activation of p110 gamma/p101 phosphoinositide 3-kinase gamma

Receptor-regulated class I phosphoinositide 3-kinases (PI3K) phosphorylate the membrane lipid phosphatidylinositol (PtdIns)-4,5-P2 to PtdIns-3,4,5-P3. This, in turn, recruits and activates cytosolic effectors with PtdIns-3,4,5-P3-binding pleckstrin homology (PH) domains, thereby controlling important cellular functions such as proliferation, survival, or chemotaxis. The class IB p110 gamma/p101 PI3K gamma is activated by G beta gamma on stimulation of G protein-coupled receptors. It is currently unknown whether in living cells G beta gamma acts as a membrane anchor or an allosteric activator of PI3K gamma, and which role its noncatalytic p101 subunit plays in its activation by G beta gamma. Using GFP-tagged PI3K gamma subunits expressed in HEK cells, we show that G beta gamma recruits the enzyme from the cytosol to the membrane by interaction with its p101 subunit. Accordingly, p101 was found to be required for G protein-mediated activation of PI3K gamma in living cells, as assessed by use of GFP-tagged PtdIns-3,4,5-P3-binding PH domains. Furthermore, membrane-targeted p110 gamma displayed basal enzymatic activity, but was further stimulated by G beta gamma, even in the absence of p101. Therefore, we conclude that in vivo, G beta gamma activates PI3K gamma by a mechanism assigning specific roles for both PI3K gamma subunits, i.e., membrane recruitment is mediated via the noncatalytic p101 subunit, and direct stimulation of G beta gamma with p110 gamma contributes to activation of PI3K gamma.     

Signal amplification between Gbetagamma release and PI3Kgamma-mediated PI(3,4,5)P3 formation monitored by a fluorescent Gbetagamma biosensor protein and repetitive two component total internal reflection/fluorescence redistribution after photobleaching analysis

Phosphoinositide 3-kinase gamma (PI3Kgamma) is activated by Gbetagamma release after stimulation of Galpha i -coupled receptors, involving a recruitment of the enzyme to the plasma membrane via interaction of the regulatory subunit p101 or p87 with Gbetagamma. The receptor-mediated release of Gbetagamma was, however, insufficient to elicit a translocation of p101 observable by classical fluorescence microscopy approaches. Since the mobilities of plasma membrane-associated and cytosolic proteins differ strongly, small changes in the amount of plasma membrane association should be detectable by an altered diffusional behavior. Here, changes in mobility were monitored by fluorescence redistribution after photobleaching (FRAP) which was repetitively applied before and after stimulation of cells. To combine the advantages of total internal reflection (TIR) illumination, which preferentially excites fluorophors located at or near the plasma membrane, with that provided by the mobility information, we developed a combined TIR/FRAP setup which enabled us to point bleach parts of an image that was observed under TIR illumination. For FRAP data analysis, we introduce a convolution-based method and a global two component model. Using this TIR/FRAP approach, an increased plasma membrane association of the fluorescent Gbetagamma-binding domain of p101 after Gbetagamma release by G protein-coupled receptor stimulation could be detected and quantified. By comparing the translocation efficiency of this domain with that of YFP-GRP1(PH), a biosensor for the PI3Kgamma product PI(3,4,5)P3, we evaluate the signal amplification between Gbetagamma release and PI(3,4,5)P3 formation after activation of Galpha i -coupled receptors.     

Characterizing the interactions between the two subunits of the p101/p110gamma phosphoinositide 3-kinase and their role in the activation of this enzyme by G beta gamma subunits.

Recently, we have reported the purification and cloning of a novel G protein betagamma subunit-activated phosphoinositide 3-kinase from pig neutrophils. The enzyme comprises a p110gamma catalytic subunit and a p101 regulatory subunit. Now we have cloned the human ortholog of p101 and generated panels of p101 and p110gamma truncations and deletions and used these in in vitro and in vivo assays to determine the protein domains responsible for subunit interaction and activation by betagamma subunits. Our results suggest large areas of p101 including both N- and C-terminal portions interact with the N-terminal half of p110gamma. While modifications of the N terminus of p110gamma could modulate its intrinsic catalytic activity, binding to the N-terminal region of p101 was found to be indispensable for activation of heterodimers with Gbetagamma.     

Gbetagammas and the Ras binding domain of p110gamma are both important regulators of PI(3)Kgamma signalling in neutrophils

Through their ability to regulate production of the key lipid messenger PtdIns(3,4,5)P(3), the class I phosphatidylinositol-3-OH kinases (PI(3)Ks) support many critical cell responses. They, in turn, can be regulated by cell-surface receptors through signals acting on either their adaptor subunits (for example, through phosphotyrosine or Gbetagammas) or their catalytic subunits (for example, through GTP-Ras). The relative significance of these controlling inputs is undefined in vivo. Here, we have studied the roles of Gbetagammas, the adaptor p101, Ras and the Ras binding domain (RBD) in the control of the class I PI(3)K, PI(3)Kgamma, in mouse neutrophils. Loss of p101 leads to major reductions in the accumulation of PtdIns(3,4,5)P(3), activation of protein kinase B (PKB) and in migration towards G-protein activating ligands in vitro, and to an aseptically inflamed peritoneum in vivo. Loss of sensitivity of PI(3)Kgamma to Ras unexpectedly caused similar reductions, but additionally caused a substantial loss in production of reactive oxygen species (ROS). We conclude that Gbetagammas, p101 and the Ras-RBD interaction all have important roles in the regulation of PI(3)Kgamma in vivo and that they can simultaneously, but differentially, control distinct PI(3)Kgamma effectors.     

Use of the GRP1 PH domain as a tool to measure the relative levels of PtdIns(3,4,5)P3 through a protein-lipid overlay approach

We describe a novel approach to the relative quantification of phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P(3)] and its application to measure, in neutrophils, the activation of phosphoinositide 3-kinase (PI3K). This protein-lipid overlay-based assay allowed us to confirm and extend the observations, first, that N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation of primed human neutrophils leads to a transient and biphasic increase in PtdIns(3,4,5)P(3) levels and, second, that the ability of fMLP to stimulate PtdIns(3,4,5)P(3) accumulation in neutrophils isolated from mice carrying a Ras-insensitive ('DASAA') knock-in of PI3Kgamma (p110gamma(DASAA/DASAA)) is substantially dependent on the Ras binding domain of PI3Kgamma.     

Phosphatidylinositol 3-kinase gamma signaling through protein kinase Czeta induces NADPH oxidase-mediated oxidant generation and NF-kappaB activation in endothelial cells

We addressed the role of class 1B phosphatidylinositol 3-kinase (PI3K) isoform PI3Kgamma in mediating NADPH oxidase activation and reactive oxidant species (ROS) generation in endothelial cells (ECs) and of PI3Kgamma-mediated oxidant signaling in the mechanism of NF-kappaB activation and intercellular adhesion molecule (ICAM)-1 expression. We used lung microvascular ECs isolated from mice with targeted deletion of the p110gamma catalytic subunit of PI3Kgamma. Tumor necrosis factor (TNF) alpha challenge of wild type ECs caused p110gamma translocation to the plasma membrane and phosphatidylinositol 1,4,5-trisphosphate production coupled to ROS production; however, this response was blocked in p110gamma-/- ECs. ROS production was the result of TNFalpha activation of Ser phosphorylation of NADPH oxidase subunit p47(phox) and its translocation to EC membranes. NADPH oxidase activation failed to occur in p110gamma-/- ECs. Additionally, the TNFalpha-activated NF-kappaB binding to the ICAM-1 promoter, ICAM-1 protein expression, and PMN adhesion to ECs required functional PI3Kgamma. TNFalpha challenge of p110gamma-/- ECs failed to induce phosphorylation of PDK1 and activation of the atypical PKC isoform, PKCzeta. Thus, PI3Kgamma lies upstream of PKCzeta in the endothelium, and its activation is crucial in signaling NADPH oxidase-dependent oxidant production and subsequent NF-kappaB activation and ICAM-1 expression.     

Identification and characterization of the autophosphorylation sites of phosphoinositide 3-kinase isoforms beta and gamma

Class I phosphoinositide 3-kinases (PI3Ks) are bifunctional enzymes possessing lipid kinase activity and the capacity to phosphorylate their catalytic and/or regulatory subunits. In this study, in vitro autophosphorylation of the G protein-sensitive p85-coupled class I(A) PI3K beta and p101-coupled class I(B) PI3K gamma was examined. Autophosphorylation sites of both PI3K isoforms were mapped to C-terminal serine residues of the catalytic p110 subunit (i.e. serine 1070 of p110 beta and serine 1101 of p110 gamma). Like other class I(A) PI3K isoforms, autophosphorylation of p110 beta resulted in down-regulated PI3K beta lipid kinase activity. However, no inhibitory effect of p110 gamma autophosphorylation on PI3K gamma lipid kinase activity was observed. Moreover, PI3K beta and PI3K gamma differed in the regulation of their autophosphorylation. Whereas p110 beta autophosphorylation was stimulated neither by G beta gamma complexes nor by a phosphotyrosyl peptide derived from the platelet-derived growth factor receptor, autophosphorylation of p110 gamma was significantly enhanced by G beta gamma in a time- and concentration-dependent manner. In summary, we show that autophosphorylation of both PI3K beta and PI3K gamma occurs in a C-terminal region of the catalytic p110 subunit but differs in its regulation and possible functional consequences, suggesting distinct roles of autophosphorylation of PI3K beta and PI3K gamma.     

Phosphoinositide 3-kinases in lysophosphatidic acid signaling: regulation and cross-talk with the Ras/mitogen-activated protein kinase pathway

Recent reports have shown that phosphoinositide 3-kinases (PI3Ks) mediate various biological activities of lysophosphatidic acid (LPA), including cell proliferation or survival. In addition, these enzymes have been proposed to be early intermediates of mitogen-activated protein kinase (MAPK) activation. Here we summarize our current knowledge of the mechanisms underlying these observations. p110gamma is an isoform of PI3K that can be activated in vitro by Gbetagamma subunits and was therefore considered as the logical candidate to mediate responses induced by G protein-coupled receptor (GPCR) agonists. In agreement with this, p110gamma has been involved in different biochemical models linking Gbetagamma to MAPK activation. Nevertheless, its apparent tissue-specific distribution has raised questions regarding the physiological relevance of these models. In addition, LPA can activate p110beta, a member of the phosphotyrosine-dependent PI3K subfamily that participates in the mitogenic effect of LPA. Its activation is thought to involve a synergistic effect of Gbetagamma and phosphotyrosine motifs provided by a transactivated EGF receptor/Gab1 pathway. We are currently studying a possible role of p110beta upstream from Ras, suggesting that this protein could provide a novel connection between betagamma and the MAPK pathway.     

p87 and p101 Subunits Are Distinct Regulators Determining Class IB Phosphoinositide 3-Kinase (PI3K) Specificity

Class I_B phosphoinositide 3-kinase γ (PI3Kγ) comprises a single catalytic p110γ subunit, which binds to two non-catalytic subunits, p87 or p101, and controls a plethora of fundamental cellular responses. The non-catalytic subunits are assumed to be redundant adaptors for Gβγ enabling G-protein-coupled receptor-mediated regulation of PI3Kγ. Growing experimental data provide contradictory evidence. To elucidate the roles of the non-catalytic subunits in determining the specificity of PI3Kγ, we tested the impact of p87 and p101 in heterodimeric p87-p110γ and p101-p110γ complexes on the modulation of PI3Kγ activity in vitro and in living cells. RT-PCR, biochemical, and imaging data provide four lines of evidence: (i) specific expression patterns of p87 and p101, (ii) up-regulation of p101, providing the basis to consider p87 as a protein forming a constitutively and p101 as a protein forming an inducibly expressed PI3Kγ, (iii) differences in basal and stimulated enzymatic activities, and (iv) differences in complex stability, all indicating apparent diversity within class I_B PI3Kγ. In conclusion, expression and activities of PI3Kγ are modified differently by p87 and p101 in vitro and in living cells, arguing for specific regulatory roles of the non-catalytic subunits in the differentiation of PI3Kγ signaling pathways.     

Search for a molecular determinant in phosphatidylinositol-3-kinase molecules,involved in the interaction with the beta-gamma-G protein dimer

The heterodimer p85/p110 and p101/p120 gamma phosphatidylinositol-3-kinases (PI3K) are important effector proteins in the signal transduction in a cell. beta gamma-subunits of heterotrimetic G-proteins are some of the main regulators of PI3K functional activity. Molecular determinants in the molecules of PI3K which may be responsible for coupling with beta gamma-dimer, remain obscure. The aim of this work was to identify the determinants of the basis of a comparative analysis of primary structures of PI3K and other beta gamma-binding proteins (adenylyl cyclases of the different types, G-protein-coupled receptor kinases, phospholypase C beta). The obtained data enables us to make some conclusions. In p85/p110 PI3K, beta gamma-binding determinants are located mainly in its regulatory subunit (BCR-domain, inter-SH2-domain). However, the interaction between beta gamma and catalytic domain of the catalytic p-110 subunit is also possible. In p101/p120 gamma PI3K, beta gamma-binding regions are located only in the catalytic p120 gamma-subunit of the enzyme, i.e. in its middle part and C-terminal catalytic domain regions of 436-502, 791-822 and 911-1000). In spite of the fact that potential beta gamma-binding regions are localized in different loci of PI3K subunits, they can form a compact beta gamma-binding surface in the process of its molecule folding, similar by as in other beta gamma-binding proteins.     

Effects of tyroserleutide on gene expression of calmodulin and PI3K in hepatocellular carcinoma

Tyroserleutide (YSL) is a tripeptide compound that has exhibited inhibitory effects on hepatocellular carcinoma in our previous research. The mechanism of this antitumor activity involves the second messenger, Ca(2+). Ca(2+) influences cell function through the Ca(2+)/calmodulin (CaM) pathway, and abnormality of the Ca(2+)/CaM system correlates closely with the occurrence of tumors. In addition, CaM associates with phosphatidylinositol 3 kinase (PI3K), thereby enhancing the activity of PI3K, which promotes cell proliferation. In order to investigate its anti-tumor mechanism, we studied the effects of YSL on CaM protein expression and mRNA level, PI3K activity, PI3K regulatory subunit p85 protein expression and mRNA level, and the mRNA level of PI3K catalytic subunits p110alpha and p110gamma in human hepatocellular carcinoma BEL-7402 xenograft tumors in nude mice. Our results showed that YSL decreased the mRNA level and protein expression of CaM, inhibited the activity of PI3K, and reduced the mRNA level and protein expression of the PI3K regulatory subunit p85 and mRNA level of PI3K catalytic subunits p110alpha and p110gamma. Accordingly, it is suggestive that the anti-tumor effects of YSL may be mediated by down regulation of CaM and PI3K subunits p85 and p110, influencing the signal transduction pathway in the tumor cells and perhaps overcoming the dysfunctional PI3K activity in tumors.     

Synergistic activation of a family of phosphoinositide 3-kinase via G-protein coupled and tyrosine kinase-related receptors.

Phosphoinositide 3-kinase (PI 3-kinase) is a key signaling enzyme implicated in a variety of receptor-stimulated cell responses. Stimulation of receptors possessing (or coupling to) protein-tyrosine kinase activates heterodimeric PI 3-kinases, which consist of an 85-kDa regulatory subunit (p85) containing Src-homology 2 (SH2) domains and a 110-kDa catalytic subunit (p110 alpha or p110 beta). Thus, this form of PI 3-kinases could be activated in vitro by a phosphotyrosyl peptide containing a YMXM motif that binds to the SH2 domains of p85. Receptors coupling to alpha beta gamma-trimeric G proteins also stimulate the lipid kinase activity of a novel p110 gamma isoform, which is not associated with p85, and thereby is not activated by tyrosine kinase receptors. The activation of p110 gamma PI 3-kinase appears to be mediated through the beta gamma subunits of the G protein (G beta gamma). In addition, rat liver heterodimeric PI 3-kinases containing the p110 beta catalytic subunit are synergistically activated by the phosphotyrosyl peptide plus G beta gamma. Such enzymatic properties were also observed with a recombinant p110 beta/p85 alpha expressed in COS-7 cells. In contrast, another heterodimeric PI 3-kinase consisting of p110 alpha and p85 in the same rat liver, together with a recombinant p110 alpha/p85 alpha, was not activated by G beta gamma, though their activities were stimulated by the phosphotyrosyl peptide. Synergistic activation of PI 3-kinase by the stimulation of the two major receptor types was indeed observed in intact cells, such as chemotactic peptide (N-formyl-Met-Leu-Phe) plus insulin (or Fc gamma II) receptors in differentiated THP-1 and CHO cells and adenosine (A1) plus insulin receptors in rat adipocytes. Thus, PI 3-kinase isoforms consisting of p110 beta catalytic and SH2-containing (p85 or its related) regulatory subunits appeared to function as a 'cross-talk' enzyme between the two signal transduction pathways mediated through tyrosine kinase and G protein-coupled receptors.     

Molecular Balance between the Regulatory and Catalytic Subunits of Phosphoinositide 3-Kinase Regulates Cell Signaling and Survival

Class Ia phosphoinositide (PI) 3-kinase is a central component in growth factor signaling and is comprised of a p110 catalytic subunit and a regulatory subunit, the most common family of which is derived from the p85alpha gene (Pik3r1). Optimal signaling through the PI 3-kinase pathway depends on a critical molecular balance between the regulatory and catalytic subunits. In wild-type cells, the p85 subunit is more abundant than p110, leading to competition between the p85 monomer and the p85-p110 dimer and ineffective signaling. Heterozygous disruption of Pik3r1 results in increased Akt activity and decreased apoptosis by insulin-like growth factor 1 (IGF-1) through up-regulated phosphatidylinositol (3,4,5)-triphosphate production. Complete depletion of p85alpha, on the other hand, results in significantly increased apoptosis due to reduced PI 3-kinase-dependent signaling. Thus, a reduction in p85alpha represents a novel therapeutic target for enhancing IGF-1/insulin signaling, prolongation of cell survival, and protection against apoptosis.