The p110delta isoform of PI 3-kinase negatively controls RhoA and PTEN

Inactivation of PI 3-kinase (PI3K) signalling is critical for tumour suppression by PTEN. This is thought to be a unidirectional relationship in which PTEN degrades the lipids produced by PI3K, thus controlling cell proliferation, survival and migration. We now show that this relationship is in fact bidirectional, whereby PI3K reciprocally controls PTEN. We report that the p110delta PI3K negatively regulates PTEN, through a pathway involving inhibition of RhoA. Inactivation of p110delta in macrophages led to reduced Akt and Rac1 activation, but paradoxically to increased RhoA and PTEN activity. Partial inactivation of p190RhoGAP and a reduced binding of cytoplasmic RhoA to the cyclin-dependent kinase inhibitor p27 both contributed to the increased RhoA-GTP levels upon p110delta inactivation. Pharmacological inhibition of ROCK, a downstream effector kinase of RhoA, restored all signalling and functional defects of p110delta inactivation, including Akt phosphorylation, chemotaxis and proliferation. This work identifies the RhoA/ROCK pathway as a major target of p110delta-mediated PI3K signalling, and establishes for the first time that PI3K controls itself, via a feedback loop involving PTEN.     

Activated G alpha q inhibits p110 alpha phosphatidylinositol 3-kinase and Akt

Some Gq-coupled receptors have been shown to antagonize growth factor activation of phosphatidylinositol 3-kinase (PI3K) and its downstream effector, Akt. We used a constitutively active Galphaq(Q209L) mutant to explore the effects of Galphaq activation on signaling through the PI3K/Akt pathway. Transient expression of Galphaq(Q209L) in Rat-1 fibroblasts inhibited Akt activation induced by platelet-derived growth factor or insulin treatment. Expression of Galphaq(Q209L) also attenuated Akt activation promoted by coexpression of constitutively active PI3K in human embryonic kidney 293 cells. Galphaq(Q209L) had no effect on the activity of an Akt mutant in which the two regulatory phosphorylation sites were changed to acidic amino acids. Inducible expression of Galphaq(Q209L) in a stably transfected 293 cell line caused a decrease in PI3K activity in p110alpha (but not p110beta) immunoprecipitates. Receptor activation of Galphaq also selectively inhibited PI3K activity in p110alpha immunoprecipitates. Active Galphaq still inhibited PI3K/Akt in cells pretreated with the phospholipase C inhibitor U73122. Finally, Galphaq(Q209L) co-immunoprecipitated with the p110alpha-p85alpha PI3K heterodimer from lysates of COS-7 cells expressing these proteins, and incubation of immunoprecipitated Galphaq(Q209L) with purified recombinant p110alpha-p85alpha in vitro led to a decrease in PI3K activity. These results suggest that agonist binding to Gq-coupled receptors blocks Akt activation via the release of active Galphaq subunits that inhibit PI3K. The inhibitory mechanism seems to be independent of phospholipase C activation and might involve an inhibitory interaction between Galphaq and p110alpha PI3K.     

PI 3-kinase p110beta: a new target for antithrombotic therapy

Platelet activation at sites of vascular injury is essential for the arrest of bleeding; however, excessive platelet accumulation at regions of atherosclerotic plaque rupture can result in the development of arterial thrombi, precipitating diseases such as acute myocardial infarction and ischemic stroke. Rheological disturbances (high shear stress) have an important role in promoting arterial thrombosis by enhancing the adhesive and signaling function of platelet integrin alpha(IIb)beta(3) (GPIIb-IIIa). In this study we have defined a key role for the Type Ia phosphoinositide 3-kinase (PI3K) p110beta isoform in regulating the formation and stability of integrin alpha(IIb)beta(3) adhesion bonds, necessary for shear activation of platelets. Isoform-selective PI3K p110beta inhibitors have been developed which prevent formation of stable integrin alpha(IIb)beta(3) adhesion contacts, leading to defective platelet thrombus formation. In vivo, these inhibitors eliminate occlusive thrombus formation but do not prolong bleeding time. These studies define PI3K p110beta as an important new target for antithrombotic therapy.     

Involvement of the p110 alpha isoform of PI3K in early development of mouse embryos

Class I of phosphoinositide 3-kinases (PI3Ks) is characterized as a group of intracellular signal proteins possessing both protein and lipid kinase activities. Recent studies implicate class I of PI3Ks acts as indispensable mediators in early development of mouse embryos, but the molecular mechanisms are poorly defined. In this paper, mouse one-cell embryos were used to investigate a possible contribution of the catalytic subunit of PI3K, p110 alpha, to cell cycle progression. The expression level of p110 alpha was determined in four phases of one-cell embryos. Silencing of p110 alpha by microinjection of p110 alpha shRNA into one-cell embryos resulted in a G2/M arrest and prevented the activation of Akt and M-phase promoting factor (MPF). Further, microinjection of the synthesized mRNA coding for a constitutively active p110 alpha into one-cell embryos induced cell cleavage more effectively than microinjection of wild-type p110 alpha mRNA, whereas microinjection of mRNA of kinase-deficient p110 alpha delayed the first mitotic cleavage. Taken together, this study demonstrates that p110 alpha is significant for G2/M transition of mouse one-cell embryos and further emphasizes the importance of Akt in PI3K pathway.     

Specific requirement for the p85-p110alpha phosphatidylinositol 3-kinase during epidermal growth factor-stimulated actin nucleation in breast cancer cells

We have studied the role of phosphatidylinositol 3-kinases (PI 3-kinases) in the regulation of the actin cytoskeleton in MTLn3 rat adenocarcinoma cells. Stimulation of MTLn3 cells with epidermal growth factor (EGF) induced a rapid increase in actin polymerization, with production of lamellipodia within 3 min. EGF-stimulated lamellipodia were blocked by 100 nM wortmannin, suggesting the involvement of a class Ia PI 3-kinase. MTLn3 cells contain equal amounts of p110alpha and p110beta, and do not contain p110delta. Injection of specific inhibitory antibodies to p110alpha induced cell rounding and blocked EGF-stimulated lamellipod extension, whereas control or anti-p110beta antibodies had no effect. In contrast, both antibodies inhibited EGF-stimulated DNA synthesis. An in situ assay for actin nucleation showed that EGF-stimulated formation of new barbed ends was blocked by injection of anti-p110alpha antibodies. In summary, the p110alpha isoform of PI 3-kinase is specifically required for EGF-stimulated actin nucleation during lamellipod extension in breast cancer cells.     

The p110alpha isoform of phosphatidylinositol 3-kinase is essential for polyomavirus middle T antigen-mediated transformation

Middle T antigen (MT) of polyomavirus is known to play an important role in virus-mediated cellular transformation. While MT has been extensively examined in spontaneously immortalized rodent fibroblasts, its interactions with cells of other types and species are less well understood. We have undertaken a cross-species and cross-cell-type comparison of MT-induced transformation in cells with genetically defined backgrounds. We tested the transforming abilities of a panel of MT mutants, Y250F, Y315F, and Y322F, that are selectively mutated in the binding sites for the principal effectors of MT--Src homology 2 domain-containing transforming protein, phosphatidylinositol 3-kinase (PI3K), and phospholipase C-gamma, respectively--in fibroblasts and epithelial cells of murine or human origin. We found that the Y315F mutation disabled the ability of MT to induce transformation in all cell types and species tested. While Y315F also failed to activate the PI3K pathway in these cells, genetic evidence has indicated Y315 may make other contributions to transformation. To confirm the role of PI3K, the PIK3CA gene, encoding p110alpha, the prime effector of PI3K signaling downstream from activated growth factor receptors, was genetically ablated. This abolished the transforming activity of MT, demonstrating the essential role for this PI3K isoform in MT-mediated transformation. The Y250F mutant was able to transform the human, but not the murine, cells that were examined. Interestingly, this mutant fully activates the PI3K pathway in human cells but activated PI3K signaling poorly in the murine cells used in the study. This again points to the importance of PI3K activation for transformation and suggests that the mechanism by which MT activates the PI3K pathway differs in different species.     

Phosphatidylinositol (PI) 3-kinase and PI 4-kinase binding to the CD4-p56lck complex: the p56lck SH3 domain binds to PI 3-kinase but not PI 4-kinase.

CD4 serves as a receptor for major histocompatibility complex class II antigens and as a receptor for the human immunodeficiency virus type 1 (HIV-1) viral coat protein gp120. It is coupled to the protein-tyrosine kinase p56lck, an interaction necessary for an optimal response of certain T cells to antigen. In addition to the protein-tyrosine kinase domain, p56lck possesses Src homology 2 and 3 (SH2 and SH3) domains as well as a unique N-terminal region. The mechanism by which p56lck generates intracellular signals is unclear, although it has the potential to interact with various downstream molecules. One such downstream target is the lipid kinase phosphatidylinositol 3-kinase (PI 3-kinase), which has been found to bind to activated pp60src and receptor-tyrosine kinases. In this study, we verified that PI 3-kinase associates with the CD4:p56lck complex as judged by the presence of PI 3-phosphate generated from anti-CD4 immunoprecipitates and detected by high-pressure liquid chromatographic analysis. However, surprisingly, CD4-p56lck was also found to associate with another lipid kinase, phosphatidylinositol 4-kinase (PI 4-kinase). The level of associated PI 4-kinase was generally higher than PI 3-kinase activity. HIV-1 gp120 and antibody-mediated cross-linking induced a 5- to 10-fold increase in the level of CD4-associated PI 4- and PI 3-kinases. The use of glutathione S-transferase fusion proteins carrying Lck-SH2, Lck-SH3, and Lck-SH2/SH3 domains showed PI 3-kinase binding to the SH3 domain of p56lck, an interaction facilitated by the presence of an adjacent SH2 domain. PI 4-kinase bound to neither the SH2 nor the SH3 domain of p56lck. CD4-p56lck contributes PI 3- and PI 4-kinase to the activation process of T cells and may play a role in HIV-1-induced immune defects.     

Mechanism of influenza A virus NS1 protein interaction with the p85beta, but not the p85alpha, subunit of phosphatidylinositol 3-kinase (PI3K) and up-regulation of PI3K activity

Influenza A virus infection activates the phosphatidylinositol 3-kinase (PI3K)/Akt pathway by binding influenza A virus NS1 protein to the p85beta regulatory subunit of PI3K. In this study, we report that NS1 binds to the inter-SH2 (iSH2) domain of p85beta. Mutational analyses on p85beta iSH2 domain defined that Val-573 is the critical amino acid (AA) that mediates NS1 and p85beta interaction. In reciprocal gain of function experiments with p85alpha, we demonstrated that mutation to Val at Met-582 leads to NS1 binding and increased PI3K activity. Molecular modeling based on our experimental results suggested that, in addition to the interaction interface between the NS1 SH3 binding motif 1 (AA 164-167) and p85beta Val-573, AA 137-142 in NS1 might interact with p85beta. Indeed, mutations of AA 141 and 142 in NS1 disrupted the interaction between NS1 and p85beta. Mutant virus PR8-NS1-141/142 was not able to activate Akt phosphorylation. Furthermore, PI3K assays demonstrated that, in wild-type virus-infected cells, p85beta-associated PI3K activity was increased significantly. In contrast, in the mutant virus-infected cells containing mutant NS1 unable to interact with p85beta, the p85beta-associated PI3K activity up-regulation was not seen, suggesting that PI3K up-regulation is dependent upon the interaction between NS1 and p85beta. Competition experiments and the immunoprecipitation studies demonstrated that NS1, p85beta, and p110 form a complex in cells. Finally, the mechanism by which binding of NS1 to p85beta regulates PI3K activity was discussed based on a predicted structural model of NS1-p85-p110 complex.     

The p110delta isoform of phosphoinositide 3-kinase controls clonal expansion and differentiation of Th cells

The role of PI3K in T cell activation and costimulation has been controversial. We previously reported that a kinase-inactivating mutation (D910A) in the p110delta isoform of PI3K results in normal T cell development, but impaired TCR-stimulated cell proliferation in vitro. This proliferative defect can be overcome by providing CD28 costimulation, which raises the question as to whether p110delta activity plays a role in T cell activation in vivo, which occurs primarily in the context of costimulation. In this study, we show that the PI3K signaling pathway in CD28-costimulated p110delta D910A/D910A T cells is impaired, but that ERK phosphorylation and NF-kappaB nuclear translocation are unaffected. Under in vitro conditions of physiological Ag presentation and costimulation, p110delta D910A/D910A T cells showed normal survival, but underwent fewer divisions. Differentiation along the Th1 and Th2 lineages was impaired in p110delta D910A/D910A T cells and could not be rescued by exogenous cytokines in vitro. Adoptive transfer and immunization experiments in mice revealed that clonal expansion and differentiation in response to Ag and physiological costimulation were also compromised. Thus, p110delta contributes significantly to Th cell expansion and differentiation in vitro and in vivo, also in the context of CD28 costimulation.     

Direct association of p110 beta phosphatidylinositol 3-kinase with p85 is mediated by an N-terminal fragment of p110 beta.

Phosphatidylinositol (PI) 3-kinase is a heterodimeric enzyme of 85-kDa (p85) and 110-kDa (p110) subunits implicated in mitogenic signal transduction by virtue of its activation in cells transformed by diverse viral oncoproteins and treated with various growth factors. We have identified a domain in p110 that mediates association with p85 in vitro and in intact cells. A glutathione S-transferase fusion protein containing the N-terminal 171 amino-acids of p110 beta bound to free p85 in cell lysates. This fusion protein also bound directly to p85 immobilized on nitrocellulose filters. An epitope-tagged fragment containing amino acids 31 to 150 of p110 beta associated with p85 upon expression in intact cells. Expression of either an N-terminal fragment of p110 beta or the p85 inter-SH2 domain, which mediates association with p110, reduced the association of endogenous PI 3-kinase activity with the activated platelet-derived growth factor receptor in intact cells. Hence, these defined regions of p85 and p110 mediate the interaction between the two subunits of PI 3-kinase.     

A p85 subunit-independent p110alpha PI 3-kinase colocalizes with p70 S6 kinase on actin stress fibers and regulates thrombin-stimulated stress fiber formation in swiss 3T3 cells

The signaling pathways linking receptor activation to actin stress fiber rearrangements during growth factor-induced cell shape change are still to be determined. Recently our laboratory demonstrated the involvement of p70 S6 kinase (p70(s6k)) activation in thrombin-induced stress fiber formation in Swiss 3T3 cells. The present work shows that thrombin-induced p70(s6k) activation is inhibited by the PI 3-kinase inhibitors wortmannin and LY-294002. These inhibitors also significantly reduced thrombin-induced stress fiber formation, demonstrating a role for PI 3-kinase activity in this process, most likely upstream of p70(s6k). Furthermore, the p110alpha form of PI 3-kinase was localized to actin stress fibers, as was previously shown for p70(s6k), as well as to a golgi-like distribution. In contrast, PI 3-kinase p110gamma colocalized with microtubules. The PI 3-kinase p85 subunit, known to be capable of association with p110alpha, was present in a predominantly golgi-like distribution with no presence on actin filaments, suggesting the existence of distinctly localized PI 3-kinase pools. Immunodepletion of p85 from cell lysates resulted in only partial depletion of p110alpha and p110alpha-associated PI 3-kinase activity, confirming the presence of a p85-free p110alpha pool located on the actin stress fibers. Our data, therefore, point to the importance of subcellular localization of PI 3-kinase in signal transduction and to a novel action of p85 subunit-independent PI 3-kinase p110alpha in the stimulation by thrombin of p70(s6k) activation and actin stress fiber formation.     

Impaired kit- but not FcepsilonRI-initiated mast cell activation in the absence of phosphoinositide 3-kinase p85alpha gene products

The class I(A) phosphoinositide 3-kinases (PI3Ks) consist of a 110-kDa catalytic domain and a regulatory subunit encoded by the p85alpha, p85beta, or p55gamma genes. We have determined the effects of disrupting the p85alpha gene on the responses of mast cells stimulated by the cross-linking of Kit and FcepsilonRI, receptors that reflect innate and adaptive responses, respectively. The absence of p85alpha gene products partially inhibited Kit ligand/stem cell factor-induced secretory granule exocytosis, proliferation, and phosphorylation of the serine/threonine kinase Akt. In contrast, p85alpha gene products were not required for FcepsilonRI-initiated exocytosis and phosphorylation of Akt. LY294002, which inhibits all classes of PI3Ks, strongly suppressed Kit- and FcepsilonRI-induced responses in p85alpha -/- mast cells, revealing the contribution of another PI3K family member(s). In contrast to B lymphocytes, mast cell proliferation was not dependent on Bruton's tyrosine kinase, a downstream effector of PI3K, revealing a distinct pathway of PI3K-dependent proliferation in mast cells. Our findings represent the first example of receptor-specific usage of different PI3K family members in a single cell type. In addition, because Kit- but not FcepsilonRI-initiated signaling is associated with mast cell proliferation, the results provide evidence that distinct biologic functions signaled by these two receptors may reflect differential usage of PI3Ks.     

Control of axonal growth and regeneration of sensory neurons by the p110delta PI 3-kinase

The expression and function of the 8 distinct catalytic isoforms of PI 3-kinase (PI3K) in the nervous system are unknown. Whereas most PI3Ks have a broad tissue distribution, the tyrosine kinase-linked p110delta isoform has previously been shown to be enriched in leukocytes. Here we report that p110delta is also highly expressed in the nervous system. Inactivation of p110delta in mice did not affect gross neuronal development but led to an increased vulnerability of dorsal root ganglia neurons to exhibit growth cone collapse and decreases in axonal extension. Loss of p110delta activity also dampened axonal regeneration following peripheral nerve injury in adult mice and impaired functional recovery of locomotion. p110delta inactivation resulted in reduced neuronal signaling through the Akt protein kinase, and increased activity of the small GTPase RhoA. Pharmacological inhibition of ROCK, a downstream effector of RhoA, restored axonal extension defects in neurons with inactive p110delta, suggesting a key role of RhoA in p110delta signaling in neurons. Our data identify p110delta as an important signaling component for efficient axonal elongation in the developing and regenerating nervous system.     

Human Tumor Mutants in PI3K p110α Subunit

The PI3K-Akt pathway is frequently upregulated in human tumors. Recently, somatic mutations of PIK3CA, encoding p110α catalytic subunit of Class IA PI3Ks, have been found in various cancers. The two most common types of p110α mutants, those in the helical and kinase domains, have been shown to be very potent in Akt activation and oncogenic transformation by several groups. Notably these common mutations may not enhance recruitment of p110α to the plasma membrane where its substrates are located. We have investigated the effect of membrane localization on common PIK3CA tumor mutants via myristoylation. In addition we have studied a third class of less frequent mutants in the p85-binding domain, in an attempt to gain insight into p85’s inhibitory effect on p110α. This article briefly reviews and extends the literature on mutant forms of p110α.     

Differential regulation of class IA phosphoinositide 3-kinase catalytic subunits p110 alpha and beta by protease-activated receptor 2 and beta-arrestins

PAR-2 (protease-activated receptor 2) is a GPCR (G-protein-coupled receptor) that can elicit both G-protein-dependent and -independent signals. We have shown previously that PAR-2 simultaneously promotes Galphaq/Ca2+-dependent activation and beta-arrestin-1-dependent inhibition of class IA PI3K (phosphoinositide 3-kinase), and we sought to characterize further the role of beta-arrestins in the regulation of PI3K activity. Whereas the ability of beta-arrestin-1 to inhibit p110alpha (PI3K catalytic subunit alpha) has been demonstrated, the role of beta-arrestin-2 in PI3K regulation and possible differences in the regulation of the two catalytic subunits (p110alpha and p110beta) associated with p85alpha (PI3K regulatory subunit) have not been examined. In the present study we have demonstrated that: (i) PAR-2 increases p110alpha- and p110beta-associated lipid kinase activities, and both p110alpha and p110beta are inhibited by over-expression of either beta-arrestin-1 or -2; (ii) both beta-arrestin-1 and -2 directly inhibit the p110alpha catalytic subunit in vitro, whereas only beta-arrestin-2 directly inhibited p110beta; (iii) examination of upstream pathways revealed that PAR-2-induced PI3K activity required the small GTPase Cdc (cell-division cycle)42, but not tyrosine phosphorylation of p85; and (iv) beta-arrestins inhibit PAR-2-induced Cdc42 activation. Taken together, these results indicated that beta-arrestins could inhibit PAR-2-stimulated PI3K activity, both directly and through interference with upstream pathways, and that the two beta-arrestins differ in their ability to inhibit the p110alpha and p110beta catalytic subunits. These results are particularly important in light of the growing interest in PAR-2 as a pharmacological target, as commonly used biochemical assays that monitor G-protein coupling would not screen for beta-arrestin-dependent signalling events.     

Acetylcholine but not adenosine triggers preconditioning through PI3-kinase and a tyrosine kinase

Acrolein is a highly reactive aldehyde pollutant and an endogenous product of lipid peroxidation. Increased generation of, or exposures to, acrolein incites pulmonary and vascular injury. The effects of acrolein on the vasomotor responses of rat aortic rings were studied to understand its mechanism of action. Incubation with acrolein (10-100 microM) alone did not affect the resting tone of aortic vessels; however, a dose-dependent relaxation of phenylephrine-precontracted aortic rings was observed. Acrolein-induced relaxation was slow and time dependent and the extent of relaxation after 100 min of application was 44.7 +/- 4.1% (10 microM), 56.0 +/- 5.6% (20 microM), 61.0 +/- 7.9% (40 microM), and 96.1 +/- 2.1 (80 microM), respectively, versus 14.2 +/- 3.3% relaxation in the absence of acrolein. Acrolein-induced vasorelaxation was prevented by endothelial denudation and was abolished on pretreatment with the nitric oxide synthase inhibitor Nomega-nitro-L-arginine methyl ester, the guanylyl cyclase inhibitor 1H-[1,2,4]oxidazolo[4,3-a]quinoxaline-1-one, or the cyclooxygenase inhibitor indomethacin. Inhibition of K+ channels (by tetrabutylammonium) or Na+-K+-ATPase (by ouabain) did not significantly prevent acrolein-mediated vasorelaxation. Exposure to acrolein in the presence or absence of other compounds elicited slow wave vasomotor effect in 77% of aortic vessels versus 1.4% in control. Vasomotor responses were also studied on aortic rings prepared from rats fed 2 mg. kg-1. day-1 acrolein for 3 alternate days by oral gavage. These vessels developed a significantly lower contractile response to phenylephrine compared with controls. Together, these results indicate that acute acrolein exposure evokes delayed vasorelaxation due to a nitric oxide- and prostacyclin-dependent mechanism, whereas in vivo acrolein exposure compromises vessel contractility.     

Development of isoform selective PI3-kinase inhibitors as pharmacological tools for elucidating the PI3K pathway

Using a parallel synthesis approach to target a non-conserved region of the PI3K catalytic domain a pan-PI3K inhibitor 1 was elaborated to provide alpha, delta and gamma isoform selective Class I PI3K inhibitors 21, 24, 26 and 27. The compounds had good cellular activity and were selective against protein kinases and other members of the PI3K superfamily including mTOR and DNA-PK.     

Selective regulation of neurite extension and synapse formation by the beta but not the alpha isoform of CaMKII

Neurite extension and branching are important neuronal plasticity mechanisms that can lead to the addition of synaptic contacts in developing neurons and changes in the number of synapses in mature neurons. Here we show that Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates movement, extension, and branching of filopodia and fine dendrites as well as the number of synapses in hippocampal neurons. Only CaMKIIbeta, which peaks in expression early in development, but not CaMKIIalpha, has this morphogenic activity. A small insert in CaMKIIbeta, which is absent in CaMKIIalpha, confers regulated F-actin localization to the enzyme and enables selective upregulation of dendritic motility. These results show that the two main neuronal CaMKII isoforms have markedly different roles in neuronal plasticity, with CaMKIIalpha regulating synaptic strength and CaMKIIbeta controlling the dendritic morphology and number of synapses.