Intermolecular interactions of the p85alpha regulatory subunit of phosphatidylinositol 3-kinase

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 bound each other, and variants of p85 containing the SH3 and BH domains and the first proline-rich motif were dimeric. Analysis of the apparent molecular mass of the deletion mutants indicated that each of these domains contributed residues to the dimerization interface, and competition experiments revealed that there were 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 did 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 we propose that it would be dimeric.     

Inhibition of insulin signaling in endothelial cells by protein kinase C-induced phosphorylation of p85 subunit of phosphatidylinositol 3-kinase (PI3K)

The regulation of endothelial function by insulin is consistently abnormal in insulin-resistant states and diabetes. Protein kinase C (PKC) activation has been reported to inhibit insulin signaling selectively in endothelial cells via the insulin receptor substrate/PI3K/Akt pathway to reduce the activation of endothelial nitric-oxide synthase (eNOS). In this study, it was observed that PKC activation differentially inhibited insulin receptor substrate 1/2 (IRS1/2) signaling of insulin's activation of PI3K/eNOS by decreasing only tyrosine phosphorylation of IRS2. In addition, PKC activation, by general activator and specifically by angiotensin II, increased the phosphorylation of p85/PI3K, which decreases its association with IRS1 and activation. Thr-86 of p85/PI3K was identified to be phosphorylated by PKC activation and confirmed to affect IRS1-mediated activation of Akt/eNOS by insulin and VEGF using a deletion mutant of the Thr-86 region of p85/PI3K. Thus, PKC and angiotensin-induced phosphorylation of Thr-86 of p85/PI3K may partially inhibit the activation of PI3K/eNOS by multiple cytokines and contribute to endothelial dysfunction in metabolic disorders.     

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.     

Novel mechanism of interaction of p85 subunit of phosphatidylinositol 3-kinase and ErbB3 receptor-derived phosphotyrosyl peptides

Ligand-activated and tyrosine-phosphorylated ErbB3 receptor binds to the SH2 domain of the p85 subunit of phosphatidylinositol 3-kinase and initiates intracellular signaling. Here, we studied the interactions between the N- (N-SH2) and C- (C-SH2) terminal SH2 domains of the p85 subunit of the phosphatidylinositol 3-kinase and eight ErbB3 receptor-derived phosphotyrosyl peptides (P-peptides) by using molecular dynamics, free energy, and surface plasmon resonance (SPR) analyses. In SPR analysis, these P-peptides showed no binding to the C-SH2 domain, but P-peptides containing a phospho-YXXM or a non-phospho-YXXM motif did bind to the N-SH2 domain. The N-SH2 domain has two phosphotyrosine binding sites in its N- (N1) and C- (N2) terminal regions. Interestingly, we found that P-peptides of pY1180 and pY1241 favored to bind to the N2 site, although all other P-peptides showed favorable binding to the N1 site. Remarkably, two phosphotyrosines, pY1178 and pY1243, which are just 63 amino acids apart from the pY1241 and pY1180, respectively, showed favorable binding to the N1 site. These findings indicate a possibility that the pair of phosphotyrosines, pY1178-pY1241 or pY1243-pY1180, will fold into an appropriate configuration for binding to the N1 and N2 sites simultaneously. Our model structures of the cytoplasmic C-terminal domain of ErbB3 receptor also strongly supported the speculation. The calculated binding free energies between the N-SH2 domain and P-peptides showed excellent qualitative agreement with SPR data with a correlation coefficient of 0.91. The total electrostatic solvation energy between the N-SH2 domain and P-peptide was the dominant factor for its binding affinity.     

Over-expression of the p110beta but not p110alpha isoform of PI 3-kinase inhibits motility in breast cancer cells

Phosphoinositide 3-kinase (PI 3-kinase) activity is required for growth factor-induced cytoskeletal regulation and cell migration. We previously found that in MTLn3 rat adenocarcinoma cells, EGF-stimulated induction of actin barbed ends and lamellipod extension specifically requires the p85/p110alpha isoform of PI 3-kinase. To further characterize signaling by distinct PI 3-kinase isoforms, we have developed MTLn3 cells that transiently or stably overexpress either p110alpha or p110beta. Transient overexpression of p110beta inhibited EGF-stimulated lamellipod extension, whereas p110alpha-transfected cells showed normal EGF-stimulated lamellipod extension. Similar results were obtained by overexpression of kinase-dead p110beta, suggesting that effects on cytoskeletal signaling were due to competition with p85/p110alpha complexes. Stable overexpression of p110alpha appeared to be toxic, based on the difficulty in obtaining stable overexpressing clones. In contrast, cells expressing a 2-fold increase in p110beta were readily obtainable. Interestingly, cells stably expressing p110beta showed a marked inhibition of EGF-stimulated lamellipod extension. Using computer-assisted analysis of time-lapse images, we found that overexpression of p110beta caused a nearly complete inhibition of motility. Cells overexpressing p110beta showed normal activation of Akt and Erk, suggesting that overall PI 3-kinase signaling was intact. A chimeric p110 molecule containing the p85-binding and Ras-binding domains of p110alpha and the C2, helical, and kinase domains of p110beta, was catalytically active yet also inhibited EGF-stimulated lamellipod extension. These data highlight the differential signaling by distinct p110 isoforms. Identification of effectors that are differently regulated by p110alpha versus p110beta will be important for understanding cell migration and its role in metastasis.     

PKCalpha reduces the lipid kinase activity of the p110alpha/p85alpha PI3K through the phosphorylation of the catalytic subunit

The modulation of phosphoinositide 3-kinase (PI3K) activity influences the quality of cellular responses triggered by various receptor tyrosine kinases. Protein kinase C (PKC) has been reported to phosphorylate signalling molecules upstream of PI3K and thereby it may affect the activation of PI3K. Here, we provide the first evidence for a direct effect of a PKC isoenzyme on the activity of PI3K. PKCalpha but not PKCepsilon phosphorylated the catalytic subunit of the p110alpha/p85alpha PI3K in vitro in a manner inhibited by the PKC inhibitor bisindolylmaleimide I (BIM I). The incubation of PI3K with active PKCalpha resulted in a significant decrease in its lipid kinase activity and this effect was also attenuated by BIM I. We conclude that PKCalpha is able to modulate negatively the lipid kinase activity of the p110alpha/p85alpha PI3K through the phosphorylation of the catalytic subunit.     

SH2 domains of the p85 alpha subunit of phosphatidylinositol 3-kinase regulate binding to growth factor receptors.

The binding of cytoplasmic signaling proteins such as phospholipase C-gamma 1 and Ras GTPase-activating protein to autophosphorylated growth factor receptors is directed by their noncatalytic Src homology region 2 (SH2) domains. The p85 alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase, which associates with several receptor protein-tyrosine kinases, also contains two SH2 domains. Both p85 alpha SH2 domains, when expressed individually as fusion proteins in bacteria, bound stably to the activated beta receptor for platelet-derived growth factor (PDGF). Complex formation required PDGF stimulation and was dependent on receptor tyrosine kinase activity. The bacterial p85 alpha SH2 domains recognized activated beta PDGF receptor which had been immobilized on a filter, indicating that SH2 domains contact autophosphorylated receptors directly. Several receptor tyrosine kinases within the PDGF receptor subfamily, including the colony-stimulating factor 1 receptor and the Steel factor receptor (Kit), also associate with PI 3-kinase in vivo. Bacterially expressed SH2 domains derived from the p85 alpha subunit of PI 3-kinase bound in vitro to the activated colony-stimulating factor 1 receptor and to Kit. We infer that the SH2 domains of p85 alpha bind to high-affinity sites on these receptors, whose creation is dependent on receptor autophosphorylation. The SH2 domains of p85 are therefore primarily responsible for the binding of PI 3-kinase to activated growth factor receptors.     

Binding of influenza A virus NS1 protein to the inter-SH2 domain of p85 suggests a novel mechanism for phosphoinositide 3-kinase activation

Influenza A virus NS1 protein stimulates host-cell phosphoinositide 3-kinase (PI3K) signaling by binding to the p85beta regulatory subunit of PI3K. Here, in an attempt to establish a mechanism for this activation, we report further on the functional interaction between NS1 and p85beta. Complex formation was found to be independent of NS1 RNA binding activity and is mediated by the C-terminal effector domain of NS1. Intriguingly, the primary direct binding site for NS1 on p85beta is the inter-SH2 domain, a coiled-coil structure that acts as a scaffold for the p110 catalytic subunit of PI3K. In vitro kinase activity assays, together with protein binding competition studies, reveal that NS1 does not displace p110 from the inter-SH2 domain, and indicate that NS1 can form an active heterotrimeric complex with PI3K. In addition, it was established that residues at the C terminus of the inter-SH2 domain are essential for mediating the interaction between p85beta and NS1. Equivalent residues in p85alpha have previously been implicated in the basal inhibition of p110. However, such p85alpha residues were unable to substitute for those in p85beta with regards NS1 binding. Overall, these data suggest a model by which NS1 activates PI3K catalytic activity by masking a normal regulatory element specific to the p85beta inter-SH2 domain.     

Reduction of PTP1B induces differential expression of PI3-kinase (p85alpha) isoforms

Protein tyrosine phosphatase 1B (PTP1B) inhibition increases insulin sensitivity and normalizes blood glucose levels in animals. The molecular events associated with PTP1B inhibition that increase insulin sensitivity remain controversial. Insulin resistant, diabetic ob/ob mice, dosed with PTP1B antisense for 3 weeks exhibited a decrease in PTP1B protein levels and a change in the expression level of p85alpha isoforms in liver, characterized by a reduction in p85alpha and an upregulation of the p50alpha and p55alpha isoforms. Transfection of mouse hepatocytes with PTP1B antisense caused a downregulation PTP1B and p85alpha protein levels. Furthermore, transfection of mouse hepatocytes with PTP1B siRNA downregulated p85alpha protein expression and enhanced insulin-induced PKB phosphorylation. Treatment of mouse hepatocytes with p85alpha antisense oligonucleotide caused a reduction of p85alpha and an increase in p50alpha and p55alpha isoforms and enhanced insulin-stimulated PKB activation. These results demonstrate that PTP1B inhibition causes a direct differential regulation of p85alpha isoforms of PI3-kinase in liver and that reduction of p85alpha may be one mechanism by which PTP1B inhibition improves insulin sensitivity and glucose metabolism in insulin-resistant states.     

Multiple domains of Ruk/CIN85/SETA/CD2BP3 are involved in interaction with p85alpha regulatory subunit of PI 3-kinase

Ruk/CIN85/SETA/CD2BP3 and CD2AP/CMS/METS-1 comprise a new family of proteins involved in such fundamental processes as clustering of receptors and rearrangement of the cytoskeleton in regions of specialised cell-cell contacts, ligand-activated internalisation and targeting to lysosome degradation pathway of receptor tyrosine kinases, and apoptotic cell death. As typical adapter proteins they execute these functions by interacting with other signalling molecules via multiple protein-protein interaction interfaces: SH3 domains, Pro-rich region and coiled-coil domain. It has been previously demonstrated that Ruk is able to interact with the p85alpha regulatory subunit of PI 3-kinase and that the SH3 domain of p85alpha is required for this interaction. However, later observations hinted at a more complex mechanism than simple one-way SH3-Pro-rich interaction. Because interaction with p85alpha was suggested to be important for pro-apoptotic activity of the long isoform of Ruk, Ruk(l)/CIN85, we carried out detailed studies of the mechanism of this interaction and demonstrated that multiple domains are involved; SH3 domains of Ruk are required and sufficient for efficient interaction with full-length p85alpha but the SH3 domain of p85alpha is vital for their "activation" by ousting them from intramolecular interaction with the Pro-rich region of Ruk. Our data also suggest that homodimerisation via C-terminal coiled-coil domain affects both intra- and intermolecular interactions of Ruk proteins.     

Activation of phosphatidylinositol 3-kinase signaling by the nonstructural NS1 protein is not conserved among type A and B influenza viruses

Recently it has been shown by several laboratories that the influenza A virus nonstructural protein 1 (A/NS1) binds and activates phosphatidylinositol 3-kinase (PI3K). This function of the protein is likely to prevent premature apoptosis induction during viral propagation. Here we show that the B/NS1 protein completely lacks the capacity to induce PI3K signaling. Thus, PI3K activation is another unique function of A/NS1 that is different from the action of its influenza B virus counterpart.     

Disrupted RabGAP function of the p85 subunit of phosphatidylinositol 3-kinase results in cell transformation

Rab proteins regulate vesicle fusion events during the endocytosis, recycling, and degradation of activated receptor tyrosine kinases. The p85alpha subunit of phosphatidylinositol 3-kinase has GTPase-activating protein activity toward Rab5 and Rab4, an activity severely reduced by a single point mutation (p85-R274A). Expression of p85-R274A resulted in increased platelet-derived growth factor receptor (PDGFR) activation and downstream signaling (Akt and MAPK) and in decreased PDGFR degradation. We now report that the biological consequences of p85-R274A expression cause cellular transformation as determined by the following: aberrant morphological phenotype, loss of contact inhibition, growth in soft agar, and tumor formation in nude mice. Immunohistochemistry shows that the tumors contain activated PDGFR and high levels of activated Akt. Coexpression of a dominant negative Rab5-S34N mutant attenuated these transformed properties. Our results demonstrate that disruption of the RabGAP function of p85alpha due to a single point mutation (R274A) is sufficient to cause cellular transformation via a phosphatidylinositol 3-kinase-independent mechanism partially reversed by Rab5-S34N expression. This critical new role for p85 in the regulation of Rab function suggests a novel role for p85 in controlling receptor signaling and trafficking through its effects on Rab GTPases.     

Tyrosine phosphorylation of p85 relieves its inhibitory activity on phosphatidylinositol 3-kinase

Under resting conditions, the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K) serves to both stabilize and inactivate the p110 catalytic subunit. The inhibitory activity of p85 is relieved by occupancy of the NH(2)-terminal SH2 domain of p85 by phosphorylated tyrosine. Src family kinases phosphorylate tyrosine 688 in p85, a process that we have shown to be reversed by the activity of the p85-associated SH2 domain-containing phosphatase SHP1. We demonstrate that phosphorylation of the downstream PI3K target Akt is increased in cells lacking SHP1, implicating phosphorylation of p85 in the regulation of PI3K activity. Furthermore, the in vitro specific activity of PI3K associated with tyrosine- phosphorylated p85 is higher than that associated with nonphosphorylated p85. Expression of wild-type p85 inhibits PI3K enzyme activity as indicated by PI3K- dependent Akt phosphorylation. The inhibitory activity of p85 is accentuated by mutation of tyrosine 688 to alanine and reversed by mutation of tyrosine 688 to aspartic acid, changes that block and mimic tyrosine phosphorylation, respectively Strikingly, mutation of tyrosine 688 to aspartic acid completely reverses the inhibitory activity of p85 on cell viability and activation of the downstream targets Akt and NFkappaB, indicative of the physiological relevance of p85 phosphorylation. Tyrosine phosphorylation of Tyr(688) or mutation of tyrosine 688 to aspartic acid is sufficient to allow binding to the NH(2)-terminal SH2 domain of p85. Thus an intramolecular interaction between phosphorylated Tyr(688) and the NH(2)-terminal SH2 domain of p85 can relieve the inhibitory activity of p85 on p110. Taken together, the data indicate that phosphorylation of Tyr(688) in p85 leads to a novel mechanism of PI3K regulation.     

The p85alpha subunit of phosphatidylinositol 3'-kinase binds to and stimulates the GTPase activity of Rab proteins

Rab5 and Rab4 are small monomeric GTPases localized on early endosomes and function in vesicle fusion events. These Rab proteins regulate the endocytosis and recycling or degradation of activated receptor tyrosine kinases such as the platelet-derived growth factor receptor (PDGFR). The p85alpha subunit of phosphatidylinositol 3'-kinase contains a BH domain with sequence homology to GTPase activating proteins (GAPs), but has not previously been shown to possess GAP activity. In this report, we demonstrate that p85alpha has GAP activity toward Rab5, Rab4, Cdc42, Rac1 and to a lesser extent Rab6, with little GAP activity toward Rab11. Purified recombinant Rab5 and p85alpha can bind directly to each other and not surprisingly, the p85alpha-encoded GAP activity is present in the BH domain. Because p85alpha stays bound to the PDGFR during receptor endocytosis, p85alpha will also be localized to the same early endosomal compartment as Rab5 and Rab4. Taken together, the physical co-localization and the ability of p85alpha to preferentially stimulate the down-regulation of Rab5 and Rab4 GTPases suggests that p85alpha regulates how long Rab5 and Rab4 remain in their GTP-bound active state. Cells expressing BH domain mutants of p85 show a reduced rate of PDGFR degradation as compared with wild type p85 expressing cells. These cells also show sustained activation of the mitogen-activated protein kinase and Akt pathways. Thus, the p85alpha protein may play a role in the down-regulation of activated receptors through its temporal control of the GTPase cycles of Rab5 and Rab4.     

Structure of a specific peptide complex of the carboxy-terminal SH2 domain from the p85 alpha subunit of phosphatidylinositol 3-kinase.

We have determined the solution structure of the C-terminal SH2 domain of the p85 alpha subunit of human phosphatidylinositol (PI) 3-kinase (EC 2.7.1.137) in complex with a phosphorylated tyrosine pentapeptide sequence from the platelet-derived growth factor receptor using heteronuclear nuclear magnetic resonance spectroscopy. Overall, the structure is similar to other SH2 domain complexes, but displays different detail interactions within the phosphotyrosine binding site and in the recognition site for the +3 methionine residue of the peptide, the side chain of which inserts into a particularly deep and narrow pocket which is displaced relative to that of other SH2 domains. The contacts made within this +3 pocket provide the structural basis for the strong selection for methionine at this position which characterizes the SH2 domains of PI3-kinase. Comparison with spectral and structural features of the uncomplexed domain shows that the long BG loop becomes less mobile in the presence of the bound peptide. In contrast, extreme resonance broadening encountered for most residues in the beta D', beta E and beta F strands and associated connecting loops of the domain in the absence of peptide persists in the complex, implying conformational averaging in this part of the molecule on a microsecond-to-millisecond time scale.     

Protein profiling of the human epidermis from the elderly reveals up-regulation of a signature of interferon-gamma-induced polypeptides that includes manganese-superoxide dismutase and the p85beta subunit of phosphatidylinositol 3-kinase

Aging of the human skin is a complex process that consists of chronological and extrinsic aging, the latter caused mainly by exposure to ultraviolet radiation (photoaging). Here we present studies in which we have used proteomic profiling technologies and two-dimensional (2D) PAGE database resources to identify proteins whose expression is deregulated in the epidermis of the elderly. Fresh punch biopsies from the forearm of 20 pairs of young and old donors (21-30 and 75-92 years old, respectively) were dissected to yield an epidermal fraction that consisted mainly of differentiated cells. One- to two-mm3 epidermal pieces were labeled with [35S]methionine for 18 h, lysed, and subjected to 2D PAGE (isoelectric focusing and non-equilibrium pH gradient electrophoresis) and phosphorimage autoradiography. Proteins were identified by matching the gels with the master 2D gel image of human keratinocytes (proteomics.cancer.dk). In selected cases 2D PAGE immunoblotting and/or mass spectrometry confirmed the identity. Quantitative analysis of 172 well focused and abundant polypeptides showed that the level of most proteins (148) remains unaffected by the aging process. Twenty-two proteins were consistently deregulated by a factor of 1.5 or more across the 20 sample pairs. Among these we identified a group of six polypeptides (Mx-A, manganese-superoxide dismutase, tryptophanyl-tRNA synthetase, the p85beta subunit of phosphatidylinositol 3-kinase, and proteasomal proteins PA28-alpha and SSP 0107) that is induced by interferon-gamma in primary human keratinocytes and that represents a specific protein signature for the effect of this cytokine. Changes in the expression of the eukaryotic initiation factor 5A, NM23 H2, cyclophilin A, HSP60, annexin I, and plasminogen activator inhibitor 2 were also observed. Two proteins exhibited irregular behavior from individual to individual. Besides arguing for a role of interferon-gamma in the aging process, the biological activities associated with the deregulated proteins support the contention that aging is linked with increased oxidative stress that could lead to apoptosis in vivo.