Phosphotidylinositol 3 kinase 92E


Table of contents

PI3Ks of Drosophila

Mammalian phosphoinositide 3-kinases (PI 3-kinases) are involved in receptor-mediated signal transduction and have been implicated in processes such as transformation and mitogenesis through their role in elevating cellular phosphatidylinositol (3,4,5)-trisphosphate. Additionally, a PI 3-kinase activity that generates phosphatidylinositol 3-phosphate has been shown to be required for protein trafficking in yeast. A family of three distinct PI 3-kinases has been identified in Drosophila, using an approach based on the polymerase chain reaction to amplify a region corresponding to the conserved catalytic domain of PI 3-kinases. One of these family members, PI3K_92E, is closely related to the prototypical PI 3-kinase, p110 alpha; PI3K_59F is homologous to Vps34p, whereas the third, PI3K_68D, is a novel PI 3-kinase that is widely expressed throughout the Drosophila life cycle. The PI3K_68D cDNA encodes a protein of 210 kDa that lacks sequences implicated in linking p110 PI 3-kinases to p85 adaptor proteins, but contains an amino-terminal proline-rich sequence, which could bind to SH3 domains, and a carboxy-terminal C2 domain. Biochemical analyses demonstrate that PI3K_68D has a novel substrate specificity in vitro, restricted to phosphatidylinositol and phosphatidylinositol 4-phosphate, and is unable to phosphorylate phosphatidylinositol (4,5)-bisphosphate, the implied in vivo substrate for p110. PI3K_68D, representing a novel PI3K class in the Drosophila family of PI 3-kinases, has the potential to bind to signaling molecules containing SH3 domains, lacks p85-adaptor-binding sequences, has a Ca(2+)-independent phospholipid-binding domain and displays a restricted in vitro substrate specificity -- therefore, it could define a novel signal transduction pathway (MacDougall, 1995).

Molecular, biochemical and genetic characterization of phosphoinositide 3-kinases (PI3Ks) have identified distinct classes of enzymes involved in processes mediated by activation of cell-surface receptors and in constitutive intracellular protein trafficking events. The latter process appears to involve a PtdIns-specific PI3K first described in yeast as a mutant, vps34, defective in the sorting of newly synthesized proteins from the Golgi to the vacuole. A representative member of each class of PI3Ks has been identified in Drosophila using a PCR-based approach. Described in this paper is the the molecular cloning of a PI3K from Drosophila (P13K_59F) that shows sequence similarity to Vps34. PI3K_59F encodes a protein of 108 kDa co-linear with Vps34 homologs, and with three regions of sequence similarity to other PI3Ks. Biochemical characterization of the enzyme, by expression of the complete coding sequence as a glutathione S-transferase fusion protein in Sf9 cells, demonstrates that PI3K_59F is a PtdIns-specific PI3K that can utilize either Mg2+ or Mn2+. This activity is sensitive to inhibition both by non-ionic detergent (Nonidet P40) and by wortmannin (IC50 10 nM). PI3K_59F, therefore, conserves both the structural and biochemical properties of the Vps34 class of enzymes (Linassier, 1997).

Dictyostelium PI3K, cell polarity and cell-cell communication

Starving amoebae of Dictyostelium discoideum communicate by relaying extracellular cAMP signals, which direct chemotactic movement, resulting in the aggregation of thousands of cells into multicellular aggregates. Both cAMP relay and chemotaxis require the activation of PI3 kinase signaling. The spatiotemporal dynamics of PI3 kinase signaling can be followed in individual cells via the cAMP-induced membrane recruitment of a GFP-tagged PH domain-containing protein, CRAC, which is required for the activation of adenylylcyclase. This study shows that polarized periodic CRAC-GFP translocation occurs during the aggregation and mound stages of development in response to periodic cAMP signals. Membrane localization is the highest at the anterior leading edge of the cells but is often more widespread along the plasma membrane, as compared to aggregating cells. The duration of CRAC translocation to the membrane is determined by the duration of the rising phase of the cAMP signal. The system shows rapid adaptation and responds to the rate of change of the extracellular cAMP concentration. When the cells are in close contact, it takes 10 s for the signal to propagate from one cell to the next. In slugs, all cells show a permanent polarized PI3 kinase signaling in their leading edge that is dependent on cell-cell contact. Measuring the redistribution of GFP-tagged CRAC has enabled a study of the dynamics of PI3 kinase-mediated cell-cell communication at the individual cell level in the multicellular stages of Dictyostelium development. This approach should also be useful to study the interactions between cell-cell signaling, cell polarization, and movement in the development of other organisms (Dormann, 2002).

The mechanisms of leading edge formation in chemotaxing Dictyostelium cells have been investigated. While phosphatidylinositol 3-kinase (PI3K) transiently translocates to the plasma membrane in response to chemoattractant stimulation and to the leading edge in chemotaxing cells, PTEN, a negative regulator of PI3K pathways, exhibits a reciprocal pattern of localization. By uniformly localizing PI3K along the plasma membrane, it has been shown that chemotaxis pathways are activated along the lateral sides of cells and PI3K can initiate pseudopod formation, providing evidence for a direct instructional role of PI3K in leading edge formation. These findings provide evidence that differential subcellular localization and activation of PI3K and PTEN is required for proper chemotaxis (Funamoto, 2002).

When presented with a gradient of chemoattractant, many eukaryotic cells respond with polarized accumulation of the phospholipid PtdIns(3,4,5)P3. This lipid asymmetry is one of the earliest readouts of polarity in neutrophils, Dictyostelium and fibroblasts. However, the mechanisms that regulate PtdInsP3 polarization are not well understood. Using a cationic lipid shuttling system, exogenous PtdInsP3 was delivered to neutrophils. Exogenous PtdInsP3 elicits accumulation of endogenous PtdInsP3 in a positive feedback loop that requires endogenous phosphatidylinositol-3-OH kinases (PI3Ks) and Rho family GTPases. This feedback loop is important for establishing PtdInsP3 polarity in response to both chemoattractant and to exogenous PtdInsP3; it may function through a self-organizing pattern formation system. Emergent properties of positive and negative regulatory links between PtdInsP3 and Rho family GTPases may constitute a broadly conserved module for the establishment of cell polarity during eukaryotic chemotaxis (Weiner, 2003).

Mutation of PI3K

Phosphatidylinositol 3,4,5-trisphosphate is a phospholipid signaling molecule involved in many cellular functions including growth factor receptor signaling, cytoskeletal organization, chemotaxis, apoptosis, and protein trafficking. Phosphorylation at the 3 position of the inositol ring is catalyzed by many different 3-kinases (classified as types IA, IB, II, and III), but the physiological roles played by each of the different 3-kinase isozymes during embryonic development and in homeostasis in animals is incompletely understood. Mammalian type IA kinase isozymes are heterodimers that are active at 37 degrees C when the catalytic 110-kDa subunit interacts through an amino-terminal binding domain with a regulatory 85- or 55-kDa subunit. Using gene targeting in embryonic stem cells, this binding domain was deleted in the gene encoding the alpha isoform of the 110-kDa catalytic subunit (Pik3ca) of the alpha isozyme of the type IA kinases, leading to loss of expression of the p110 catalytic subunit. Pik3cadel/del embryos are developmentally delayed at embryonic day (E) 9.5 and die between E9.5 and E10.5. E9. 5 Pik3cadel/del embryos have a profound proliferative defect but no increase in apoptosis. A proliferative defect is supported by the observation that fibroblasts from Pik3cadel/del embryos fail to replicate in Dulbecco's modified Eagle's medium and fetal calf serum, even with supplemental growth factors (Bi, 1999).

Mice with a targeted gene disruption of p85alpha, a regulatory subunit of phosphoinositide 3-kinase, have impaired B cell development at the pro-B cell stage, reduced numbers of mature B cells and peritoneal CD5+ Ly-1 B cells, reduced B cell proliferative responses, and no T cell-independent antibody production. These phenotypes are nearly identical to those of Btk-/- or xid (X-linked immunodeficiency) mice. These results provide evidence that p85alpha is functionally linked to the Btk pathway in antigen receptor-mediated signal transduction and is pivotal in B cell development and functions (Suzuki, 1999).

Regulatory subunits of PI3K and their interactions

Phosphatidylinositol (PI) 3-kinase has an 85 kDa subunit (p85 alpha) that mediates its association with activated protein tyrosine kinase receptors through SH2 domains, and an 110 kDa subunit (p110) which has intrinsic catalytic activity. p85 alpha and a related protein, p85 beta, are shown to form stable complexes with recombinant p110 in vivo and in vitro. Using a panel of glutathione S-transferase (GST) fusion proteins of the inter-SH2 region of p85, 104 amino acids were found to directly bind the p110 protein, while deletion mutants within this region further define the binding site to a sequence of 35 amino acids. Transient expression of the mutant p85 alpha protein in mouse L cells shows it was unable to bind PI 3-kinase activity in vivo. Mapping of the complementary site of interaction on the p110 protein defines 88 amino acids in the N-terminal region of p110 that mediate the binding of this subunit to either the p85 alpha or the p85 beta proteins. The inter-SH2 region of p85 is predicted to be an independently folded module of a coiled-coil of two long anti-parallel alpha-helices. The predicted structure of p85 suggests a basis for the intersubunit interaction and the relevance of this interaction with respect to the regulation of the PI 3-kinase complex is discussed (Dhand, 1994).

Phosphatidylinositol 3-kinase (PI 3-kinase) is stimulated by association with a variety of tyrosine kinase receptors and intracellular tyrosine-phosphorylated substrates. A cDNA was isolated that encodes a 50-kDa regulatory subunit of PI 3-kinase with an expression cloning method using 32P-labeled insulin receptor substrate-1 (IRS-1). This 50-kDa protein contains two SH2 domains and an inter-SH2 domain of p85alpha, but the SH3 and bcr homology domains of p85alpha were replaced by a unique 6-amino acid sequence. Thus, this protein appears to be generated by alternative splicing of the p85alpha gene product. It is suggested that this protein be called p50alpha. Northern blotting using a specific DNA probe corresponding to p50alpha reveals 6.0- and 2.8-kb bands in hepatic, brain, and renal tissues. The expression of p50alpha protein and its associated PI 3-kinase are detected in lysates prepared from the liver, brain, and muscle using a specific antibody against p50alpha. Taken together, these observations indicate that the p85alpha gene actually generates three protein products of 85, 55, and 50 kDa. The distributions of the three proteins (p85alpha, p55alpha, and p50alpha), in various rat tissues and also in various brain compartments, are found to be different. Interestingly, p50alpha forms a heterodimer with p110. Two subfractions of p50alpha differ: one can be labeled with wortmannin, while the other cannot. However, p85alpha and p55alpha associate only with p110 that can be wortmannin-labeled. Furthermore, p50alpha exhibits a markedly higher capacity for activation of associated PI 3-kinase via insulin stimulation and has a higher affinity for tyrosine-phosphorylated IRS-1 than the other isoforms. Considering the high level of p50alpha expression in the liver and its marked responsiveness to insulin, p50alpha appears to play an important role in the activation of hepatic PI 3-kinase. Each of the three alpha isoforms has a different function and may have specific roles in various tissues (Inukai, 1997).

A novel model is proposed for the regulation of the p85/pl10alpha phosphatidylinositol 3'-kinase. In insect cells, the p110alpha catalytic subunit is active as a monomer but its activity is decreased by coexpression with the p85 regulatory subunit. Similarly, the lipid kinase activity of recombinant glutathione S-transferase (GST)-p110alpha is reduced by 65% to 85% upon in vitro reconstitution with p85. Incubation of p110alpha/p85 dimers with phosphotyrosyl peptides restores activity, but only to the level of monomeric p110alpha. These data show that the binding of phosphoproteins to the SH2 domains of p85 activates the p85/p110alpha dimers by inducing a transition from an inhibited to a disinhibited state. In contrast, monomeric p110 has little activity in HEK 293T cells, and its activity is increased 15- to 20-fold by coexpression with p85. However, this apparent requirement for p85 is eliminated by the addition of a bulky tag to the N terminus of p110alpha or by the growth of the HEK 293T cells at 30 degrees C. These nonspecific interventions mimic the effects of p85 on p110alpha, suggesting that the regulatory subunit acts by stabilizing the overall conformation of the catalytic subunit rather than by inducing a specific activated conformation. This stabilization has been directly demonstrated in metabolically labeled HEK 293T cells, in which p85 increases the half-life of p110. Furthermore, p85 protects p110 from thermal inactivation in vitro. Importantly, when the effect of p85 on GST-p110alpha was examined in mammalian cells at 30 degrees C, in culture conditions that stabilize the catalytic subunit and that are similar to the conditions used for insect cells, p85 inhibits p110alpha. Thus, two effects of p85 on p110alpha have been experimentally distinguished: conformational stabilization of the catalytic subunit and inhibition of its lipid kinase activity. These data reconcile the apparent conflict between previous studies of insect versus mammalian cells and show that p110alpha is both stabilized and inhibited by dimerization with p85 (Yu, 1999).

Two novel alternatively spliced forms of the p85alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase have been identified by expression screening of a human skeletal muscle library with phosphorylated baculovirus- produced human insulin receptor substrate 1. One form that has been found is identical to p85alpha throughout the region that encodes both Src homology 2 (SH2) domains and the inter-SH2 domain/p110 binding region but diverges in sequence from p85alpha on the 5' side of nucleotide 953, where the entire break point cluster gene and SH3 regions are replaced by a unique 34-amino-acid N terminus. This form has an estimated molecular mass of approximately 53 kDa and has been termed p85/AS53. The second form is identical to p85 and p85/AS53 except for a 24-nucleotide insert between the SH2 domains that results in a replacement of aspartic acid 605 with nine amino acids, adding two potential serine phosphorylation sites in the vicinity of the known serine autophosphorylation site (Ser-608). Northern (RNA) analyses reveal a wide tissue distribution of p85alpha, whereas p85/AS53 is dominant in skeletal muscle and brain, and the insert isoforms are restricted to cardiac muscle and skeletal muscle. Western blot (immunoblot) analyses using an anti-p85 polyclonal antibody and a specific anti-p85/AS53 antibody confirm the tissue distribution of p85/AS53 protein and indicate an approximately 7-fold higher expression of p85/AS53 protein than that of p85 in skeletal muscle. Both p85 and p85/AS53 bind to p110 in coprecipitation experiments, but p85alpha itself appears to have preferential binding to insulin receptor substrate 1 following insulin stimulation. These data indicate that the gene for the p85alpha regulatory subunit of PI 3-kinase can undergo tissue-specific alternative splicing. Two novel splice variants of the regulatory subunit of PI 3-kinase are present in skeletal muscle, cardiac muscle, and brain; these variants may have important functional differences in activity and may play a role in tissue-specific signals such as insulin-stimulated glucose transport or control of neurotransmitter secretion or action (Antonetti, 1996).

The regulatory subunit of phosphatidylinositol 3-kinase, p85, contains a number of well defined domains involved in protein-protein interactions, including an SH3 domain and two SH2 domains. In order to investigate in detail the nature of the interactions of these domains with each other and with other binding partners, a series of deletion and point mutants was constructed, and their binding characteristics and apparent molecular masses under native conditions were analyzed. The SH3 domain and the first proline-rich motif bind each other, and variants of p85 containing the SH3 and BH domains and the first proline-rich motif are dimeric. Analysis of the apparent molecular mass of the deletion mutants indicates that each of these domains contribute residues to the dimerization interface, and competition experiments reveal that there are intermolecular SH3 domain-proline-rich motif interactions and BH-BH domain interactions mediating dimerization of p85alpha both in vitro and in vivo. Binding of SH2 domain ligands does not affect the dimeric state of p85alpha. Recently, roles for the p85 subunit have been postulated that do not involve the catalytic subunit, and if p85 exists on its own it is likely to be dimeric (Harpur, 1999).

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. A phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase has been shown to form a complex with the p85 subunit of PI 3-kinase. Whether PI 3-kinase also forms a complex with the 4-phosphatase in human platelets was investigated. Immunoprecipitates of the p85 subunit of PI 3-kinase from human platelet cytosol contain 4-phosphatase enzyme activity and a 104-kDa polypeptide recognized by specific 4-phosphatase antibodies. Similarly, immunoprecipitates made using 4-phosphatase-specific antibodies contain 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 translocates 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%) remains in the cytosolic fraction. It is proposed that the complex formed between the two enzymes serves to localize the 4-phosphatase to sites of PtdIns(3,4)P2 production (Munday, 1999).

The focal adhesion kinase (FAK) has been implicated in signal transduction pathways initiated by cell adhesion receptor integrins and by neuropeptide growth factors. To gain insight into FAK function, the potential interaction of FAK with intracellular signaling molecules containing the Src homology 2 domains was examined. FAK stabily associates with phosphatidylinositol 3-kinase in NIH 3T3 mouse fibroblasts. This interaction is stimulated by cell adhesion concomitant with FAK activation. Recombinant FAK binds to the p85 subunit of PI 3-kinase directly in vitro, and autophosphorylation of recombinant FAK in vitro increases its binding to PI 3-kinase. Increased tyrosine phosphorylation of the p85 subunit of PI 3-kinase is observed during cell adhesion and observed direct phosphorylation of p85 by FAK in vitro. Together, these results suggest that PI 3-kinase may be a FAK substrate in vivo and serve as an effector of FAK (Chen, 1994).

ERM (Ezrin-Radixin-Moesin) proteins function as plasma membrane-actin cytoskeleton linkers and participate in the formation of specialized domains of the plasma membrane. Ezrin function in tubulogenesis of a kidney-derived epithelial cell line, LLC-PK1, has been investigated. Cells overproducing a mutant form of ezrin in which Tyr-353 is changed to a phenylalanine (Y353F) undergoes apoptosis when assayed for tubulogenesis. While investigating the mechanism responsible for this apoptosis, it was found that ezrin interacts with p85, the regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase). Two distinct sites of ezrin are involved in this interaction, the amino-terminal domain containing the first 309 aa, and the phosphorylated Tyr-353 residue, which binds to the carboxyl-terminal SH2 domain of p85. Cells producing Y353F ezrin are defective in activation of the protein kinase Akt, a downstream target of PI 3-kinase that protects cells against apoptosis. Furthermore, the apoptotic phenotype of these cells is rescued by production of a constitutively activated form of PI 3-kinase. Taken together, these results establish a novel function for ezrin in determining survival of epithelial cells by activating the PI 3-kinase/Akt pathway (Gautreau, 1999).

SH2 domain proteins transmit intracellular signals initiated by activated tyrosine kinase-linked receptors. Recent three-dimensional structures suggest mechanisms by which tandem SH2 domains might confer higher specificity than individual SH2 domains. To test this, binding studies were conducted with tandem domains from the five signaling enzymes: phosphatidylinositol 3-kinase p85, ZAP-70, Syk, SHP-2, and phospholipase C-gamma1. Bisphosphorylated TAMs (tyrosine-based activation motifs) were derived from biologically relevant sites in platelet-derived growth factor, T cell, B cell, and high affinity IgE receptors and the receptor substrates IRS-1 (insulin receptor substrate-1) and SHPS-1/SIRP. Each tandem SH2 domain binds a distinct TAM corresponding to its appropriate biological partner with highest affinity (0.5-3.0 nM). Alternative TAMs bind the tandem SH2 domains with 1,000- to >10,000-fold lower affinity than biologically relevant TAMs. This level of specificity is significantly greater than the approximately 20-50-fold typically seen for individual SH2 domains. It is concluded that high biological specificity is conferred by the simultaneous interaction of two SH2 domains in a signaling enzyme with bisphosphorylated TAMs in activated receptors and substrates (Ottinger, 1998).

Insulin-like growth factor I (IGF-I) stimulates smooth muscle cell (SMC) migration and the PI-3 kinase pathway plays an important role in mediating the IGF-I induced migratory response. Prior studies have shown that the tyrosine phosphatase SHP-2 is necessary to activate PI-3 kinase in response to growth factors and expression of a phosphatase inactive form of SHP-2 (SHP-2/C459S) impairs IGF-I stimulated cell migration. However, the mechanism by which SHP-2 phosphatase activity or the recruitment of SHP-2 (see Drosophila Corkscrew) to other signaling molecules contributes to IGF-I stimulated PI-3 kinase activation has not been determined. SMCs that have stable expression of SHP-2/C459S, have reduced cell migration and Akt activation in response to IGF-I compared with SMC expressing native SHP-2. Similarly in cells expressing native SHP-2, IGF-I induces SHP-2 binding to p85, whereas in cells expressing SHP-2/C459S there is no increase. Since the C459S substitution results in loss of the ability of SHP-2 to disassociate from its substrates, making it inaccessible not only to p85 but also the other proteins, a p85 mutant in which tyrosines 528 and 556 were changed to phenylalanines was prepared to determine if this would disrupt the p85/SHP-2 interaction and if the loss of this specific interaction would alter IGF-I stimulated the cell migration. Substitution for these tyrosines in p85 resulted in loss of SHP-2 recruitment and was associated with a reduction in association of the p85/p110 complex with IRS-1. Cells stably expressing this p85 mutant also showed a decrease in IGF-I stimulated PI-3 kinase activity and cell migration. Pre-incubation of cells with a cell permeable peptide that contains the tyrosine556 motif of p85 also disrupts SHP-2 binding to p85 and inhibits the IGF-I induced increase in cell migration. The findings indicate that tyrosines 528 and 556 in p85 are required for SHP-2 association. SHP-2 recruitment to p85 is required for IGF-I stimulated association of the p85/p110 complex with IRS-1 and for the subsequent activation of the PI-3 kinase pathway leading to increased cell migration (Kwon, 2005).

Signaling downstream of the insulin receptor involving IRS and PI3K

For more information on signaling downstream of the insulin receptor see Chico and the following review:

Shepherd, P. R., Withers, D. J. and Siddle, K (1998). Phosphoinositide 3-kinase: the key switch mechanism in insulin signalling. Biochem. J. 333: 471-490

let-502 rho-binding kinase and mel-11 myosin phosphatase regulate Caenorhabditis elegans embryonic morphogenesis. Genetic analysis presented here establishes the following modes of let-502 action: (1) loss of only maternal let-502 results in abnormal early cleavages, (2) loss of both zygotic and maternal let-502 causes elongation defects, and (3) loss of only zygotic let-502 results in sterility. The morphogenetic function of let-502 and mel-11 is apparently redundant with another pathway since elimination of these two genes results in progeny that undergo near-normal elongation. Triple mutant analysis indicates that unc-73 (Rho/Rac guanine exchange factor) and mlc-4 (myosin light chain) act in parallel to or downstream of let-502/mel-11. In contrast mig-2 (Rho/Rac), daf-2 (insulin receptor), and age-1 (PI3 kinase) act within the let-502/mel-11 pathway. Mutations in the sex-determination gene fem-2, which encodes a PP2c phosphatase (unrelated to the MEL-11 phosphatase), enhances mutations of let-502 and suppressed those of mel-11. fem-2's elongation function appears to be independent of its role in sexual identity since the sex-determination genes fem-1, fem-3, tra-1, and tra-3 have no effect on mel-11 or let-502. By itself, fem-2 affects morphogenesis with low penetrance. fem-2 blocks the near-normal elongation of let-502; mel-11, indicating that fem-2 acts in a parallel elongation pathway. The action of two redundant pathways likely ensures accurate elongation of the C. elegans embryo (Piekny, 2002).

The contribution of the insulin receptor substrate proteins (IRS-1 and IRS-2) to insulin/insulin like growth factor I (IGF-I)-signaling pathways was investigated in fetal rat brown adipocytes, a model that expresses both insulin and IGF-I receptors. Insulin/IGF-I rapidly stimulates IRS-1 and IRS-2 tyrosine phosphorylation, their association with p85alpha, and IRS-1- and IRS-2-associated phosphatidylinositol (PI) 3-kinase activation to the same extent, the effect of insulin being stronger than the effect of IGF-I at the same physiological dose (10 nM). Furthermore, insulin/IGF-I stimulates IRS-1-associated Grb-2 phosphorylation. However, IRS-2-associated Grb-2 phosphorylation is barely detected. Pull-down experiments with glutathione-S-transferase-fusion proteins containing SH2-domains of p85alpha reveal a strong association between IRS-1 and IRS-2 with p85alpha in response to insulin/IGF-I, the insulin effect being stronger than IGF-I. However, the Grb-2-SH2 domain shows functional differences. While a strong association between IRS-1/Grb-2 is found, IRS-2/Grb-2 association is virtually absent in response to insulin/IGF-I, as also demonstrated in competition studies with a phosphopeptide containing the phosphotyrosine 895 residue within the putative Grb-2-binding domain. Finally, insulin/IGF-I stimulates tyrosine phosphorylation of the three SHC proteins (46, 52, and 66 kDa). Moreover, insulin/IGF-I markedly increases the amount of Grb-2-associated SHC proteins by the same extent. These results suggest that both IRS-1 and IRS-2 are required for phosphatidylinositol 3-kinase activation, which leads to adipogenic and thermogenic differentiation of fetal brown adipose tissue; meanwhile, IRS-1 and SHC, but not IRS-2, associate with Grb-2, leading to the ras-mitogen-activated protein kinase-signaling pathway required for fetal brown adipocyte proliferation (Valverde, 1998).

Phosphatidylinositol (PI) 3-kinase plays an important role in various insulin-stimulated biological responses, including glucose transport, glycogen synthesis, and protein synthesis. However, the molecular link between PI 3-kinase and these biological responses is still unclear. Is targeting of the catalytic p110 subunit of PI 3-kinase to cellular membranes both sufficient and necessary to induce PI 3-kinase dependent signaling responses, as is characteristic of insulin action? Myc-tagged, membrane-targeted p110 [p110(CAAX)], and wild-type p110 [p110(WT)] in 3T3-L1 adipocytes were engineered by adenovirus-mediated gene transfer. Overexpressed p110(CAAX) exhibits approximately 2-fold increase in basal kinase activity in p110 immunoprecipitates; this further increases to approximately 4-fold with insulin. Even at this submaximal PI 3-kinase activity, p110(CAAX) fully stimulates p70 S6 kinase, Akt, 2-deoxyglucose uptake, and Ras, whereas, p110(WT) has little or no effect on these downstream effects. Interestingly, p110(CAAX) does not activate MAP kinase, despite its stimulation of p21(ras). Surprisingly, p110(CAAX) does not increase basal glycogen synthase activity, and inhibits insulin stimulated activity, indicative of cellular resistance to this action of insulin. p110(CAAX) also inhibits insulin stimulated, but not platelet-derived growth factor-stimulated mitogen-activated protein kinase phosphorylation, demonstrating that the p110(CAAX) induced inhibition of mitogen-activated protein kinase and insulin signaling is specific, and not due to some toxic or nonspecific effect on the cells. Moreover, p110(CAAX) stimulates IRS-1 Ser/Thr phosphorylation, and inhibits IRS-1 associated PI 3-kinase activity, without affecting insulin receptor tyrosine phosphorylation, suggesting that it may play an important role as a negative regulator for insulin signaling. In conclusion, these studies show that membrane-targeted PI 3-kinase can mimic a number of biologic effects normally induced by insulin. In addition, the persistent activation of PI 3-kinase induced by p110(CAAX) expression leads to desensitization of specific signaling pathways. Interestingly, the state of cellular insulin resistance is not global, in that some of insulin's actions are inhibited, whereas others are intact (Egawa, 1999).

The activity of the Na+-K+-pump is intricately linked to the maintenance of vascular tone. Insulin-like growth factor I (IGF-I) is shown to increase Na+-K+-pump activity in the vascular smooth muscle cell (VSMC) clone A7r5 in a time- and dose-dependent manner. This stimulatory effect of IGF-I is prevented by the tyrosine kinase inhibitor genistein (5 microM) and by the specific phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin (100 nM) and LY-294002 (25 microM). IGF-I activates a wortmannin-sensitive PI3K and its purported effector, the atypical protein kinase C (PKC)-zeta. Stimulation of PKC-zeta is prevented. A concentration of GF109203x (10 microM) that inhibits the atypical PKCs, abolishes Na+-K+-pump stimulation by IGF-I. These results suggest that IGF-I directly stimulates the Na+-K+ pump via a signaling pathway involving PI3K and atypical PKC (zeta) (Li, 1999).

Activation of phosphatidyl-inositol-3'-OH-kinase (PI3K) and the resulting production of phosphatidyl-inositol-3,4,5-trisphosphate (PIP3) are ubiquitous signaling steps that link various cell surface receptors to multiple intracellular targets. In fat and muscle cells, the same PI3K pathway that regulates metabolic enzymes, proliferation, and differentiation has also been shown to be involved in insulin-triggered insertion of glucose transporter GLUT4 into the plasma membrane. The multiple PI3K functions raise the question of how the same PI3K pathway can be selectively used for different cell functions. A dual-color evanescent wave microscopy method has been developed to simultaneously measure PIP3 production and GLUT4 insertion in individual 3T3L1 adipocytes. Activation of PI3K was found to be both necessary and sufficient for triggering GLUT4 insertion, but transporter insertion is markedly suppressed for small-amplitude, persistent PIP3 signals and for large-amplitude, short PIP3 signals. The rejection of these common PI3K signaling responses may explain the selective advantage of insulin over platelet-derived growth factor and other stimuli for inducing GLUT4 insertion. This study suggests that the same PI3K pathway can control specific cell functions by relying on effector systems that respond to particular receptor-encoded time courses and amplitudes of PIP3 signals (Tengholm, 2002).

Phosphoinositide 3-kinase activates Rac by entering in a complex with Eps8, Abi1, and Sos-1

Class I phosphoinositide 3-kinases (PI3Ks) are implicated in many cellular responses controlled by receptor tyrosine kinases (RTKs), including actin cytoskeletal remodeling. Within this pathway, Rac is a key downstream target/effector of PI3K. However, how the signal is routed from PI3K to Rac is unclear. One possible candidate for this function is the Rac-activating complex Eps8-Abi1-Sos-1, which possesses Rac-specific guanine nucleotide exchange factor (GEF) activity. Abi1 (also known as E3b1) recruits PI3K, via p85, into a multimolecular signaling complex that includes Eps8 and Sos-1. The recruitment of p85 to the Eps8-Abi1-Sos-1 complex and phosphatidylinositol 3, 4, 5 phosphate (PIP3), the catalytic product of PI3K, concur to unmask its Rac-GEF activity in vitro. Moreover, they are indispensable for the activation of Rac and Rac-dependent actin remodeling in vivo. On growth factor stimulation, endogenous p85 and Abi1 consistently colocalize into membrane ruffles, and cells lacking p85 fail to support Abi1-dependent Rac activation. These results define a mechanism whereby propagation of signals, originating from RTKs or Ras and leading to actin reorganization, is controlled by direct physical interaction between PI3K and a Rac-specific GEF complex (Innocetti, 2003).

Table of contents

Phosphotidylinositol 3 kinase 92E: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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