SH3 domain of the phosphatidylinositol 3-kinase regulatory subunit is responsible for the formation of a sequestration complex with insulin receptor substrate-1

Class IA phosphatidylinositol 3-kinase (PI 3-kinase), which is composed of a 110 kDa catalytic subunit and a regulatory subunit, plays a key role in most insulin dependent cellular responses. To date, five mammalian regulatory subunit isoforms have been identified, including two 85 kDa proteins (p85α and p85β), two 55 kDa proteins (p55γ and p55α), and one 50 kDa protein (p50α). In the present study, we overexpressed these recombinant proteins, tagged with green fluorescent proteins (GFP), in CHO-IR cells and investigated intracellular localizations in both the presence and the absence of insulin stimulation. Interestingly, in response to insulin, only p85α and p85β redistributed to isolated foci in the cells, while both were present throughout the cytoplasm in quiescent cells. In contrast, p55s accumulated in the perinuclear region irrespective of insulin stimulation, while p50α behaved similarly to control GFP. Immunofluorescent antibodies against endogenous IRS-1 revealed IRS-1 to be co-localized in the p85 foci in response to insulin. As both insulin receptors and p110α catalytic subunits were absent from these foci on immunofluorescence study, only p85 and IRS-1 were suggested to form a sequestration complex in response to insulin. To determine the domain responsible for IRS-1 complex formation, we prepared and overexpressed the SH3 domain deletion mutant of p85α in CHO-IR cells. This mutant failed to form foci, suggesting the SH3 domain of regulatory subunits to be responsible for formation of the p85-IRS-1 sequestration complex. In conclusion, our study revealed the SH3 domain of PI 3-kinase to play a critical role in intracellular localizations, including formation of foci with IRS-1 in response to insulin.     

Positive and negative regulation of phosphoinositide 3-kinase-dependent signaling pathways by three different gene products of the p85alpha regulatory subunit

Phosphoinositide (PI) 3-kinase is a key mediator of insulin-dependent metabolic actions, including stimulation of glucose transport and glycogen synthesis. The gene for the p85alpha regulatory subunit yields three splicing variants, p85alpha, AS53/p55alpha, and p50alpha. All three have (i) a C-terminal structure consisting of two Src homology 2 domains flanking the p110 catalytic subunit-binding domain and (ii) a unique N-terminal region of 304, 34, and 6 amino acids, respectively. To determine if these regulatory subunits differ in their effects on enzyme activity and signal transduction from insulin receptor substrate (IRS) proteins under physiological conditions, we expressed each regulatory subunit in fully differentiated L6 myotubes using adenovirus-mediated gene transfer with or without coexpression of the p110alpha catalytic subunit. PI 3-kinase activity associated with p50alpha was greater than that associated with p85alpha or AS53. Increasing the level of p85alpha or AS53, but not p50alpha, inhibited both phosphotyrosine-associated and p110-associated PI 3-kinase activities. Expression of a p85alpha mutant lacking the p110-binding site (Deltap85) also inhibited phosphotyrosine-associated PI 3-kinase activity but not p110-associated activity. Insulin stimulation of two kinases downstream from PI-3 kinase, Akt and p70 S6 kinase (p70(S6K)), was decreased in cells expressing p85alpha or AS53 but not in cells expressing p50alpha. Similar inhibition of PI 3-kinase, Akt, and p70(S6K) was observed, even when p110alpha was coexpressed with p85alpha or AS53. Expression of p110alpha alone dramatically increased glucose transport but decreased glycogen synthase activity. This effect was reduced when p110alpha was coexpressed with any of the three regulatory subunits. Thus, the three different isoforms of regulatory subunit can relay the signal from IRS proteins to the p110 catalytic subunit with different efficiencies. They also negatively modulate the PI 3-kinase catalytic activity but to different extents, dependent on the unique N-terminal structure of each isoform. These data also suggest the existence of a mechanism by which regulatory subunits modulate the PI 3-kinase-mediated signals, independent of the kinase activity, possibly through subcellular localization of the catalytic subunit or interaction with additional signaling molecules.     

Reduction of insulin-stimulated glucose uptake by peroxynitrite is concurrent with tyrosine nitration of insulin receptor substrate-1

Inducible nitric oxide synthetase plays an essential role in insulin resistance induced by a high-fat diet. The reaction of nitric oxide with superoxide leads to the formation of peroxynitrite (ONOO-), which can modify several proteins. In this study, we investigated whether peroxynitrite impairs insulin-signalling pathway. Our experiments showed that 3-(4-morpholinyl)sydnonimine hydrochloride (SIN-1), a constitutive producer of peroxynitrite, dose-dependently inhibited insulin-stimulated glucose uptake. While SIN-1 did not affect the insulin receptor protein level and tyrosine phosphorylation, it reduced the insulin receptor substrate-1 (IRS-1) protein level, and IRS-1 associated phosphatidylinositol-3 kinase (PI-3 kinase) activity. Although SIN-1 did not induce Ser307 phosphorylation of IRS-1, tyrosine nitration of IRS-1 was detected in SIN-1-treated-Rat1 fibroblasts expressing human insulin receptors. Mass spectrometry showed that peroxynitrite induced at least four nitrated tyrosine residues in rat IRS-1, including Tyr939, which is critical for association of IRS-1 with the p85 subunit of PI-3 kinase. Our results suggest that peroxynitrite reduces the IRS-1 protein level and decreases phosphorylation of IRS-1 concurrent with nitration of its tyrosine residues.     

Pleiotropic insulin signals are engaged by multisite phosphorylation of IRS-1.

IRS-1 (insulin receptor substrate 1) is a principal insulin receptor substrate that undergoes tyrosine phosphorylation during insulin stimulation. It contains over 20 potential tyrosine phosphorylation sites, and we suspect that multiple insulin signals are enabled when the activated insulin receptor kinase phosphorylates several of them. Tyrosine-phosphorylated IRS-1 binds specifically to various cellular proteins containing Src homology 2 (SH2) domains (SH2 proteins). We identified some of the tyrosine residues of IRS-1 that undergo insulin-stimulated phosphorylation by the purified insulin receptor and in intact cells during insulin stimulation. Automated sequencing and manual radiosequencing revealed the phosphorylation of tyrosine residues 460, 608, 628, 895, 939, 987, 1172, and 1222; additional sites remain to be identified. Immobilized SH2 domains from the 85-kDa regulatory subunit (p85 alpha) of the phosphatidylinositol 3'-kinase bind preferentially to tryptic phosphopeptides containing Tyr(P)-608 and Tyr(P)-939. By contrast, the SH2 domain in GRB2 and the amino-terminal SH2 domain in SHPTP2 (Syp) specifically bind to Tyr(P)-895 and Tyr(P)-1172, respectively. These results confirm the p85 alpha recognizes YMXM motifs and suggest that GRB2 prefers a phosphorylated YVNI motif, whereas SHPTP2 (Syp) binds to a phosphorylated YIDL motif. These results extend the notion that IRS-1 is a multisite docking protein that engages various downstream regulatory elements during insulin signal transmission.     

SHORT Syndrome with Partial Lipodystrophy Due to Impaired Phosphatidylinositol 3 Kinase Signaling

The phosphatidylinositol 3 kinase (PI3K) pathway regulates fundamental cellular processes such as metabolism, proliferation, and survival. A central component in this pathway is the p85α regulatory subunit, encoded by PIK3R1. Using whole-exome sequencing, we identified a heterozygous PIK3R1 mutation (c.1945C>T [p.Arg649Trp]) in two unrelated families affected by partial lipodystrophy, low body mass index, short stature, progeroid face, and Rieger anomaly (SHORT syndrome). This mutation led to impaired interaction between p85α and IRS-1 and reduced AKT-mediated insulin signaling in fibroblasts from affected subjects and in reconstituted Pik3r1-knockout preadipocytes. Normal PI3K activity is critical for adipose differentiation and insulin signaling; the mutated PIK3R1 therefore provides a unique link among lipodystrophy, growth, and insulin signaling.     

Five isoforms of the phosphatidylinositol 3-kinase regulatory subunit exhibit different associations with receptor tyrosine kinases and their tyrosine phosphorylations

There are five isoforms of the regulatory subunit for the heterodimeric type of phosphatidylinositol 3-kinase. These five regulatory subunit isoforms were overexpressed using an adenovirus transfection system, and their own tyrosine phosphorylations and associations with various tyrosine kinase receptors were investigated. When overexpressed in CHO-PDGFR cells, the associations of these regulatory subunit isoforms with the platelet-derived growth factor receptor were similar. However, when overexpressed in CHO-IR cells, p55gamma exhibited a significantly lower ability to bind with IRS-1 upon insulin stimulation, as compared with other regulatory subunit isoforms. Furthermore, p55alpha and p55gamma were found to be tyrosine-phosphorylated. Finally, interestingly, when overexpressed in CHO-EGFR cells or A431 cells and stimulated with epidermal growth factor (EGF), phosphorylated EGF receptor was detected in p85alpha, p85beta and p50alpha immunoprecipitates, but not in p55alpha and p55gamma immunoprecipitates. In addition, EGF-induced tyrosine phosphorylation was observed in p85alpha, p85beta, p55alpha and p55gamma, but not in p50alpha, immunoprecipitates. Thus, each regulatory subunit exhibits specific responses regarding both the association with tyrosine-phosphorylated substrates and its own tyrosine phosphorylation. These results suggest that each isoform possesses specific roles in signal transduction, based on its individual tyrosine kinase receptor.     

Insulin and insulinomimetic agents induce activation of phosphatidylinositol 3'-kinase upon its association with pp185 (IRS-1) in intact rat livers.

The major cytosolic substrate of the insulin receptor is a 185-kDa phosphoprotein (IRS-1) that contains multiple putative attachment sites for the p85 alpha regulatory subunit of phosphatidylinositol 3'-kinase (PI3K). To examine the possible interaction of pp185 with p85 alpha in vivo, we injected insulin or insulinomimetic agents (a combination of H2O2 and vanadate (H/V)) into the portal vein of anesthetized rats. IN this model system, H/V treatment and, to a lesser extent, injection of insulin resulted in rapid and sustained tyrosine phosphorylation of multiple cellular proteins, including pp185/IRS-1. The latter was found to undergo specific association with the p85 alpha regulatory subunit of PI3K but not with two other proteins that contain src homology domains. As p85 alpha was not detectably phosphorylated on tyrosine residues and did not appear to interact directly with the insulin receptor, we conclude that tyrosine phosphorylation of pp185 promotes its association with p85 alpha and the catalytic subunit of PI3K. The recruitment of the holoenzyme may also involve its enzymatic activation and thus constitute an important step in the transduction of insulin signals.     

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.     

Intracellular segregation of phosphatidylinositol-3,4,5-trisphosphate by insulin-dependent actin remodeling in L6 skeletal muscle cells

Insulin stimulates glucose uptake by recruiting glucose transporter 4 (GLUT4) from an intracellular pool to the cell surface through a mechanism that is dependent on phosphatidylinositol (PI) 3-kinase (PI3-K) and cortical actin remodeling. Here we test the hypothesis that insulin-dependent actin filament remodeling determines the location of insulin signaling molecules. It has been shown previously that insulin treatment of L6 myotubes leads to a rapid rearrangement of actin filaments into submembrane structures where the p85 regulatory subunit of PI3-K and organelles containing GLUT4, VAMP2, and the insulin-regulated aminopeptidase (IRAP) colocalize. We now report that insulin receptor substrate-1 and the p110alpha catalytic subunit of PI3-K (but not p110beta) also colocalize with the actin structures. Akt-1 was also found in the remodeled actin structures, unlike another PI3-K effector, atypical protein kinase C lambda. Transiently transfected green fluorescent protein (GFP)-tagged pleckstrin homology (PH) domains of general receptor for phosphoinositides-1 (GRP1) or Akt (ligands of phosphatidylinositol-3,4,5-trisphosphate [PI-3,4,5-P(3)]) migrated to the periphery of the live cells; in fixed cells, they were detected in the insulin-induced actin structures. These results suggest that PI-3,4,5-P(3) is generated on membranes located within the actin mesh. Actin remodeling and GLUT4 externalization were blocked in cells highly expressing GFP-PH-GRP1, suggesting that PI-3,4,5-P(3) is required for both phenomena. We propose that PI-3,4,5-P(3) leads to actin remodeling, which in turn segregates p85alpha and p110alpha, thus localizing PI-3,4,5-P(3) production on membranes trapped by the actin mesh. Insulin-stimulated actin remodeling may spatially coordinate the localized generation of PI-3,4,5-P(3) and recruitment of Akt, ultimately leading to GLUT4 insertion at the plasma membrane.     

Interferon-dependent activation of the serine kinase PI 3'-kinase requires engagement of the IRS pathway but not the Stat pathway

Several signaling pathways are activated by interferon alpha (IFNalpha) in hematopoietic cells, including the Jak-Stat and the insulin receptor substrate (IRS) pathways. It has been previously shown that IFNalpha activates the phosphatidylinositol (PI) 3'-kinase via an interaction of the p85 subunit of PI 3'-kinase with IRS proteins. Other studies have proposed that Stat-3 also functions as an adapter for p85. We sought to identify the major pathway that regulates IFNalpha activation of the PI3'-kinase in hematopoietic cells. Our data demonstrate that IFNalpha induces the interaction of p85 with IRS-1 or IRS-2, but not Stat-3, in various hematopoietic cell lines in which IRS-1 and/or IRS-2 and Stat-3 are activated by IFNalpha. In addition, inhibition of PI 3'-kinase activity by preincubation of cells with the PI 3'-kinase inhibitor LY294002 does not affect IFN-dependent formation of SIF complexes that contain Stat-3. To determine whether phosphorylation of tyrosine residues in the IFN receptor is required for activation of the PI 3'-kinase, we performed studies using mouse L929 fibroblasts transfected with mutated human IFNAR1 and/or IFNAR2 subunits of the Type I IFN receptor, lacking tyrosine phosphorylation sites. The serine kinase activity of the PI-3K was activated by human IFNalpha in these cells, suggesting that phosphorylation of the Type I IFN receptor is not essential for PI3K activation. We then determined whether IFNalpha activates the Akt kinase, a known downstream target for PI 3'-kinase that mediates anti-apoptotic signals. Akt was activated by insulin or IGF-1, but not IFNalpha, in the IFNalpha-sensitive U-266 myeloma cell line. Altogether, our data establish that the IRS pathway and not the Stat pathway, is the major pathway regulating engagement of PI 3'-kinase in hematopoietic cells. Furthermore, the selective activation of Akt by insulin/IGF-1 suggests the existence of distinct regulatory activities of PI3'-kinase in growth factor versus interferon signaling.     

Insulin-dependent formation of a complex containing an 85-kDa subunit of phosphatidylinositol 3-kinase and tyrosine-phosphorylated insulin receptor substrate 1.

Monoclonal antibodies raised against the 85-kDa subunit (p85) of bovine phosphatidylinositol (PI) 3-kinase were found to recognize uncomplexed p85 or p85 in the active PI 3-kinase. Immunoprecipitation studies of Chinese hamster ovary cells, which overexpress the human insulin receptor when treated with insulin, showed increased amounts of p85 and PI 3-kinase activity immunoprecipitable with monoclonal anti-p85 antibody and no increase in the tyrosine phosphorylation of p85. Insulin also induced an association of p85 with the tyrosine-phosphorylated insulin receptor substrate 1 (IRS-1) and other phosphorylated proteins ranging in size from 100 to 170 kDa but not with the activated insulin receptor. In vitro reconstitution studies were used to show p85 in the active PI 3-kinase associated with the tyrosine-phosphorylated IRS-1 but not with the activated insulin receptor. Competition studies using synthetic phosphopeptides corresponding to potential tyrosine phosphorylation sites of IRS-1 revealed that phosphopeptides containing YMXM motifs inhibited this association with different potencies, whereas nonphosphorylated analogues and a phosphopeptide containing the EYYE motif had no effect. Src homology region 2 domains of p85 expressed as glutathione S-transferase fusion proteins also bound to tyrosine-phosphorylated IRS-1. These results suggest that insulin causes the association of PI 3-kinase with IRS-1 via phosphorylated YMXM motifs of IRS-1 and Src homology region 2 domains of p85.     

Differential regulation of signaling pathways for insulin and insulin-like growth factor I.

The insulin receptor (IR) and the insulin-like growth factor receptor I (IGF-IR) have different functions in cell growth, apoptosis, differentation, and transformation. Although some of these differences may be explained by the relative level of receptor expression and receptor structure (alpha and beta subunits), they may also be attributed to differences in intracellular signals generated by insulin and IGF-I. The presence of hybrid receptors (IR alphabeta subunits and IGF-IR alphabeta subunits) making up the heterotetramers has added a new dimension to our understanding of the functional roles of these receptors. However, to date the results of efforts to understand the differences between these two closely related receptors have indicated mostly similarities. For example, both receptors utilize IRS-1/IRS-2 and Shc as immediate downstream adaptors, leading to activation of the Ras, Raf, ERK kinases and PI-3 kinase pathways. We have used the yeast two hybrid system to identify proteins which bind to the activated IGF-IR but not to the IR. The cytoplasmic domain of the IGF-IR was used to screen a human fetal brain library and two isoforms of the 14-3-3 family were identified. 14-3-3 proteins are a highly conserved family of proteins which have recently been shown to interact with other components of the mitogenic and apoptotic signaling pathways, including Raf, BAD, Bcr/Bcr-Abl, middle-T antigen, Ksr, PKC, PI-3 kinase, ASK1 kinase, and cdc25C phosphatase. We also identified human Grb10, an adaptor protein with SH2 domain associated with the IGF-IR beta subunit. Smith's laboratory showed that Grb10 preferentially binds to the IR in intact cells. Using the interaction trap screen (active cytoplasmic domain of the IGF-IR) 55PIK and SOCS-2 proteins were also identified. However, 55PIK and SOCS-2 also interact with the IR in the yeast two hybrid system. These studies raise the possibility that 14-3-3 and Grb10 may play a role in insulin and IGF-I signal transduction and may underlie the observed differences.     

Overexpression of Fer increases the association of tyrosine-phosphorylated IRS-1 with P85 phosphatidylinositol kinase via SH2 domain of Fer in transfected cells

We have reported that the protein-tyrosine kinase Fer is associated with signaling complexes containing insulin receptor substrate-1 (IRS-1) and phosphatidylinositol 3-kinase (PI-3 kinase) in insulin-stimulated 3T3-L1 adipocytes [J. Biol. Chem. 275 (50) (2000) 38995]. We examined the subcellular localization of this complex in 3T3-L1 adipocytes and performed transfection study to know how this complex is formed. Interestingly we have detected that this complex is formed in LDM of insulin-stimulated 3T3-L1 adipocytes, which may be important for specific biological insulin effect. Based on transfection study, we have demonstrated that overexpression of both Fer and IRS-1 can induce Fer/IRS-1/P85 complexes without insulin stimulation and SH2 domain of Fer is essential for this complex. We have also demonstrated that Fer was an efficient substrate for insulin receptor kinase. Taken together, these data suggested that Fer may play a critically important role to form Fer/IRS-1/P85 complex in LDM of insulin-stimulated adipocytes and elicit biological effect through PI-3 kinase activity in LDM.     

Phosphatidylinositol-3-OH kinase regulatory subunits are differentially expressed during development of the rat cerebellum

Recent evidence implicates a central role for PI3K signalling in mediating cell survival during the process of neuronal differentiation. Although PI3K activity is stimulated by a wide range of growth factors and cytokines in different cell lines and tissues, activation of this pathway by insulin-like growth factor I (IGF-I) most likely represents the main survival signal during neuronal differentiation. IGF-I is highly expressed during development of the central nervous system, and thus is a critical factor for the development and maturation of the cerebellum. Upon ligand binding, the IGF-I receptor phosphorylates tyrosine residues in SHC and insulin receptor substrates (IRSs) initiating two main signalling cascades, the MAP kinase and the phosphatidylinositol 3-kinase (PI3K) pathways. Activated PI3K is composed of a catalytic subunit (p110alpha or beta) associated with one of a large family of regulatory subunits (p85alpha, p85beta, p55gamma, p55alpha, and p50alpha). To evaluate the contributions of these various regulatory subunits to neuronal differentiation, we have used antibodies specific for each of the PI3K subunits. Using these antisera, we now demonstrate that PI3K subunits are differentially regulated in cerebellar development, and that the expression level of the p55gamma regulatory subunit reaches a maximum during postnatal development, decreasing thereafter to low levels in the adult cerebellum. Furthermore, our studies reveal that the distribution of the various PI3K regulatory subunits varies during development of the cerebellum. Interestingly, p55gamma is expressed in both glial and neuronal cells; moreover, in Purkinje neurones, this subunit colocalises with the IGF-IR.     

Diversification of cardiac insulin signaling involves the p85 alpha/beta subunits of phosphatidylinositol 3-kinase

Ventricular cardiomyocytes and cardiac tissue of lean and genetically obese (fa/fa) Zucker rats were used 1) to study the role of the p85 regulatory subunit isoforms p85 alpha and p85 beta for insulin signaling through the phosphatidylinositol (PI) 3-kinase pathway, and 2) to elucidate the implications of these mechanisms for cardiac insulin resistance. Western blot analysis of cardiomyocyte lysates revealed expression of p85 alpha and p85 beta but no detectable amounts of the splice variants of p85 alpha. Essentially no p85 alpha subunit of PI 3-kinase was found to be associated with insulin receptor substrate (IRS)-1 or IRS-2 in basal and insulin-stimulated (5 min) cardiomyocytes. Instead, insulin produced a twofold increase in p85 beta associated with IRS-1, leading to a three- to fourfold increase in p85 beta-associated PI 3-kinase activity. This response was significantly reduced in obese animals. Comparable results were obtained in the intact heart after in vivo stimulation. In GLUT-4-containing vesicles, an increased abundance (3.7 +/- 0.7-fold over basal) of p85 alpha was observed after insulin stimulation of lean animals, with no significant effect in the obese group. No p85 beta could be detected in GLUT-4-containing vesicles. Recruitment of the p110 catalytic subunit of PI 3-kinase and a twofold increase in enzyme activity in GLUT-4-containing vesicles by insulin was observed only in lean rats. We conclude that, in the heart, p85 alpha recruits PI 3-kinase activity to GLUT-4 vesicles, whereas p85 beta represents the main regulator of IRS-1- and IRS-2-mediated PI 3-kinase activation. Furthermore, multiple defects of PI 3-kinase activation, involving both the p85 alpha and the p85 beta adaptor subunits, may contribute to cardiac insulin resistance.     

Phosphatidylinositol‐3‐OH kinase regulatory subunits are differentially expressed during development of the rat cerebellum

Recent evidence implicates a central role for PI3K signalling in mediating cell survival during the process of neuronal differentiation. Although PI3K activity is stimulated by a wide range of growth factors and cytokines in different cell lines and tissues, activation of this pathway by insulin‐like growth factor I (IGF‐I) most likely represents the main survival signal during neuronal differentiation. IGF‐I is highly expressed during development of the central nervous system, and thus is a critical factor for the development and maturation of the cerebellum. Upon ligand binding, the IGF‐I receptor phosphorylates tyrosine residues in SHC and insulin receptor substrates (IRSs) initiating two main signalling cascades, the MAP kinase and the phosphatidylinositol 3‐kinase (PI3K) pathways. Activated PI3K is composed of a catalytic subunit (p110α or β) associated with one of a large family of regulatory subunits (p85α, p85β, p55γ, p55α, and p50α). To evaluate the contributions of these various regulatory subunits to neuronal differentiation, we have used antibodies specific for each of the PI3K subunits. Using these antisera, we now demonstrate that PI3K subunits are differentially regulated in cerebellar development, and that the expression level of the p55γ regulatory subunit reaches a maximum during postnatal development, decreasing thereafter to low levels in the adult cerebellum. Furthermore, our studies reveal that the distribution of the various PI3K regulatory subunits varies during development of the cerebellum. Interestingly, p55γ is expressed in both glial and neuronal cells; moreover, in Purkinje neurones, this subunit colocalises with the IGF‐IR. © 2001 John Wiley & Sons, Inc. J Neurobiol 47: 39–50, 2001     

The p85 regulatory subunit of phosphoinositide 3-kinase down-regulates IRS-1 signaling via the formation of a sequestration complex

Phosphoinositide (PI) 3-kinase is required for most insulin and insulin-like growth factor (IGF) 1-dependent cellular responses. The p85 regulatory subunit of PI 3-kinase is required to mediate the insulin-dependent recruitment of PI 3-kinase to the plasma membrane, yet mice with reduced p85 expression have increased insulin sensitivity. To further understand the role of p85, we examined IGF-1-dependent translocation of p85alpha by using a green fluorescence protein (GFP)-tagged p85alpha (EGFP-p85alpha). In response to IGF-1, but not to PDGF signaling, EGFP-p85alpha translocates to discrete foci in the cell. These foci contain the insulin receptor substrate (IRS) 1 adaptor molecule, and their formation requires the binding of p85 to IRS-1. Surprisingly, monomeric p85 is preferentially localized to these foci compared with the p85-p110 dimer, and these foci are not sites of phosphatidylinositol-3,4,5-trisphosphate production. Ultrastructural analysis reveals that p85-IRS-1 foci are cytosolic protein complexes devoid of membrane. These results suggest a mechanism of signal down-regulation of IRS-1 that is mediated by monomeric p85 through the formation of a sequestration complex between p85 and IRS-1.     

Phosphorylation of PDE3B by phosphatidylinositol 3-kinase associated with the insulin receptor

Phosphatidylinositol 3-kinase mediates several actions of insulin including its antilipolytic effect. This effect is elicited by the insulin-stimulated serine phosphorylation and activation of cGMP-inhibited phosphodiesterase (PDE3B). In human adipocytes, we found that insulin differentially stimulated phosphatidylinositol 3-kinase activity; the lipid kinase activity was associated with IRS-1, whereas the serine kinase activity was associated with the insulin receptor and phosphorylated a number of proteins including p85, p110, and a 135-kDa protein identified as PDE3B. PDE3B phosphorylation was associated with enzyme activation, thus initiating the antilipolytic effect of insulin. These results show a novel pathway for intracellular signaling through the insulin receptor leading to the serine phosphorylation of key proteins involved in insulin action.     

Effects of oncogenic p110alpha subunit mutations on the lipid kinase activity of phosphoinositide 3-kinase

The PIK3CA gene, encoding the p110alpha catalytic subunit of Class IA PI3Ks (phosphoinositide 3-kinases), is frequently mutated in many human tumours. The three most common tumour-derived alleles of p110alpha, H1047R, E542K and E545K, were shown to potently activate PI3K signalling in human epithelial cells. In the present study, we examine the biochemical activity of the recombinantly purified PI3K oncogenic mutants. The kinetic characterizations of the wt (wild-type) and the three 'hot spot' PI3K mutants show that the mutants all have approx. 2-fold increase in lipid kinase activities. Interestingly, the phosphorylated IRS-1 (insulin receptor substrate-1) protein shows activation of the lipid kinase activity for the wt and H1047R but not E542K and E545K PI3Kalpha, suggesting that these mutations represent different mechanisms of lipid kinase activation and hence transforming activity in cancer cells.