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.     

Synthesis, biological evaluation and molecular modelling of sulfonohydrazides as selective PI3K p110alpha inhibitors

A series of 2-methyl-5-nitrobenzenesulfonohydrazides were prepared and evaluated as inhibitors of PI3K. An isoquinoline derivative shows good selectivity for the p110α isoform over p110β and p110δ, and also demonstrates good in vitro activity in a cell proliferation assay. Molecular modelling provides a rationalisation for the observed SAR.     

Insights into the oncogenic effects of /PIK3CA/ mutations from the structure of p110α/p85α

Phosphatidylinositide-3-kinases (PI3K) initiate a number of signaling pathways by recruiting other kinases, such as Akt, to the plasma membrane. One of the isoforms, PI3Kα, is an oncogene frequently mutated in several cancer types. These mutations increase PI3K kinase activity, leading to increased cell survival, cell motility, cell metabolism, and cell cycle progression. The structure of the complex between the catalytic subunit of PI3Kα, p110α, and a portion of its regulatory subunit, p85α reveals that the majority of the oncogenic mutations occur at the interfaces between p110 domains and between p110 and p85 domains. At these positions, mutations disrupt interactions resulting in changes in the kinase domain that may increase enzymatic activity. The structure also suggests that interaction with the membrane is mediated by one of the p85 domains (iSH2). These findings may provide novel structural loci for the design of new anti-cancer drugs.     

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.     

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.     

Prostaglandin F2α stimulates PI3K/ERK/mTOR signaling and skeletal myotube hypertrophy

Cyclooxygenase (COX) enzymes mediate the synthesis of proinflammatory prostaglandin (PG) species from cellular arachidonic acid. COX/PGs have been implicated in skeletal muscle growth/regeneration; however, the mechanisms by which PGs influence skeletal muscle adaptation are poorly understood. The present study aimed to investigate PGF(2α) signaling and its role in skeletal myotube hypertrophy. PGF(2α) or the FP receptor agonist fluprostenol increased C2C12 myotube diameter. This effect was abolished by the FP receptor antagonist AL8810 and mammalian target of rapamycin (mTOR) inhibition. PGF(2α) stimulated time- and dose-dependent increases in the phosphorylation of extracellular receptor kinase (ERK)1/2 (Thr202/Tyr204), p70S6 kinase (p70S6K) (Thr389 and Thr421/Ser424), and eukaryotic initiation factor 4G (eIF4G) (Ser1108) without influencing Akt (Ser473). Pretreatment with the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 and the ERK inhibitor PD98059 blocked F prostanoid receptor signaling responses, whereas rapamycin blocked heightened p70S6K/eIF4G phosphorylation without influencing ERK1/2 phosphorylation. These data suggest that activation of the F prostanoid receptor is coupled to C2C12 myotube growth and intracellular signaling via a PI3K/ERK/mTOR-dependent pathway.     

Regulation of the p85/p110 Phosphatidylinositol 3′-Kinase: Stabilization and Inhibition of the p110α Catalytic Subunit by the p85 Regulatory Subunit

We propose a novel model for the regulation of the p85/p110α phosphatidylinositol 3′-kinase. In insect cells, the p110α 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)-p110α is reduced by 65 to 85% upon in vitro reconstitution with p85. Incubation of p110α/p85 dimers with phosphotyrosyl peptides restored activity, but only to the level of monomeric p110α. These data show that the binding of phosphoproteins to the SH2 domains of p85 activates the p85/p110α dimers by inducing a transition from an inhibited to a disinhibited state. In contrast, monomeric p110 had little activity in HEK 293T cells, and its activity was increased 15- to 20-fold by coexpression with p85. However, this apparent requirement for p85 was eliminated by the addition of a bulky tag to the N terminus of p110α or by the growth of the HEK 293T cells at 30°C. These nonspecific interventions mimicked the effects of p85 on p110α, 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 was directly demonstrated in metabolically labeled HEK 293T cells, in which p85 increased the half-life of p110. Furthermore, p85 protected p110 from thermal inactivation in vitro. Importantly, when we examined the effect of p85 on GST-p110α in mammalian cells at 30°C, culture conditions that stabilize the catalytic subunit and that are similar to the conditions used for insect cells, we found that p85 inhibited p110α. Thus, we have experimentally distinguished two effects of p85 on p110α: conformational stabilization of the catalytic subunit and inhibition of its lipid kinase activity. Our data reconcile the apparent conflict between previous studies of insect versus mammalian cells and show that p110α is both stabilized and inhibited by dimerization with p85.     

PI3K is negatively regulated by PIK3IP1, a novel p110 interacting protein

Signaling initiated by Class Ia phosphatidylinositol-3-kinases (PI3Ks) is essential for cell proliferation and survival. We discovered a novel protein we call PI3K interacting protein 1 (PIK3IP1) that shares homology with the p85 regulatory PI3K subunit. Using a variety of in vitro and cell based assays, we demonstrate that PIK3IP1 directly binds to the p110 catalytic subunit and down modulates PI3K activity. Our studies suggest that PIK3IP1 is a new type of PI3K regulator.     

The PI3K p110delta is required for down-regulation of RAG expression in immature B cells

At the immature B cell stage the BCR signals the down-regulation of the RAG genes and Ig L chain (LC) allelic and isotype exclusion. The signaling pathway that regulates these events is poorly characterized. We demonstrate that immature B cells from mice deficient in the PI3K catalytic subunit p110delta fail to suppress RAG expression and inappropriately recombine kappa and lambda LC loci. In addition, in the presence of the autoantigen, clonal deletion and receptor editing still takes place, demonstrating that these processes are independent of p110delta. These results demonstrate a role for p110delta in the regulation of RAG gene expression and thereby LC allelic/isotype exclusion.     

A pharmacological map of the PI3-K family defines a role for p110alpha in insulin signaling

Phosphoinositide 3-kinases (PI3-Ks) are an important emerging class of drug targets, but the unique roles of PI3-K isoforms remain poorly defined. We describe here an approach to pharmacologically interrogate the PI3-K family. A chemically diverse panel of PI3-K inhibitors was synthesized, and their target selectivity was biochemically enumerated, revealing cryptic homologies across targets and chemotypes. Crystal structures of three inhibitors bound to p110gamma identify a conformationally mobile region that is uniquely exploited by selective compounds. This chemical array was then used to define the PI3-K isoforms required for insulin signaling. We find that p110alpha is the primary insulin-responsive PI3-K in cultured cells, whereas p110beta is dispensable but sets a phenotypic threshold for p110alpha activity. Compounds targeting p110alpha block the acute effects of insulin treatment in vivo, whereas a p110beta inhibitor has no effect. These results illustrate systematic target validation using a matrix of inhibitors that span a protein family.     

ICOS ligation recruits the p50alpha PI3K regulatory subunit to the immunological synapse

ICOS ligation in concert with TCR stimulation results in strong PI3K activation in T lymphocytes. The ICOS cytoplasmic tail contains an YMFM motif that binds the p85alpha subunit of class IA PI3K, similar to the YMNM motif of CD28, suggesting a redundant function of the two receptors in PI3K signaling. However, ICOS costimulation shows greater PI3K activity than CD28 in T cells. We show in this report that ICOS expression in activated T cells triggers the participation of p50alpha, one of the regulatory subunits of class IA PI3Ks. Using different T-APC cell conjugate systems, we report that p50alpha accumulates at the immunological synapse in activated but not in resting T cells. Our results demonstrate that ICOS membrane expression is involved in this process and that p50alpha plasma membrane accumulation requires a functional YMFM Src homology 2 domain-binding motif in ICOS. We also show that ICOS triggering with its ligand, ICOSL, induces the recruitment of p50alpha at the synapse of T cell/APC conjugates. In association with the p110 catalytic subunit, p50alpha is known to carry a stronger lipid kinase activity compared with p85alpha. Accordingly, we observed that ICOS engagement results in a stronger activation of PI3K. Together, these findings provide evidence that p50alpha is likely a determining factor in ICOS-mediated PI3K activity in T cells. These results also suggest that a differential recruitment and activity of class IA PI3K subunits represents a novel mechanism in the control of PI3K signaling by costimulatory molecules.     

The PI3K p110α isoform regulates endothelial adherens junctions via Pyk2 and Rac1

Endothelial cell–cell junctions control efflux of small molecules and leukocyte transendothelial migration (TEM) between blood and tissues. Inhibitors of phosphoinositide 3-kinases (PI3Ks) increase endothelial barrier function, but the roles of different PI3K isoforms have not been addressed. In this study, we determine the contribution of each of the four class I PI3K isoforms (p110α, -β, -γ, and -δ) to endothelial permeability and leukocyte TEM. We find that depletion of p110α but not other p110 isoforms decreases TNF-induced endothelial permeability, Tyr phosphorylation of the adherens junction protein vascular endothelial cadherin (VE-cadherin), and leukocyte TEM. p110α selectively mediates activation of the Tyr kinase Pyk2 and GTPase Rac1 to regulate barrier function. Additionally, p110α mediates the association of VE-cadherin with Pyk2, the Rac guanine nucleotide exchange factor Tiam-1 and the p85 regulatory subunit of PI3K. We propose that p110α regulates endothelial barrier function by inducing the formation of a VE-cadherin–associated protein complex that coordinates changes to adherens junctions with the actin cytoskeleton.     

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.     

Human platelets contain p110delta phosphoinositide 3-kinase

The phosphoinositide 3-kinase (PI3K) catalytic subunit p110delta, the most recently discovered member of the heterodimeric Class IA PI3K family, has been detected uniquely in leukocytes, but not in one member of the leukocyte family: platelets. We have examined freshly prepared isolates of human platelets for the presence of this enzyme, realizing that p110delta is highly susceptible to proteolytic degradation. We have utilized p110delta-directed Western blotting, RT-PCR, PI3K activity assays, and immunoprecipitations of PI3K Class IA subunits p85alpha, p85beta, and p110delta from lysed human platelets, as well as Triton X-100-insoluble cytoskeletal preparations from resting and thrombin receptor-activated platelets. We report that p110delta is present in association with p85alpha and p85beta in platelets, both in cytosolic and cytoskeletal fractions. The latter finding is consistent with the proposed role of p110delta in cytoskeletal function.     

Knockout of PKC alpha enhances insulin signaling through PI3K

Insulin stimulates glucose transport and certain other metabolic processes by activating atypical PKC isoforms (lambda, zeta, iota) and protein kinase B (PKB) through increases in D3-polyphosphoinositides derived from the action of PI3K. The role of diacylglycerol-sensitive PKC isoforms is less clear as they have been suggested to be both activated by insulin and yet inhibit insulin signaling to PI3K. Presently, we found that insulin signaling to insulin receptor substrate 1-dependent PI3K, PKB, and PKC lambda, and downstream processes, glucose transport and activation of ERK, were enhanced in skeletal muscles and adipocytes of mice in which the ubiquitous conventional diacylglycerol-sensitive PKC isoform, PKC alpha, was knocked out by homologous recombination. On the other hand, insulin provoked wortmannin-insensitive increases in immunoprecipitable PKC alpha activity in adipocytes and skeletal muscles of wild-type mice and rats. We conclude that 1) PKC alpha is not required for insulin-stimulated glucose transport, and 2) PKC alpha is activated by insulin at least partly independently of PI3K, and largely serves as a physiological feedback inhibitor of insulin signaling to the insulin receptor substrate 1/PI3K/PKB/PKC lambda/zeta/iota complex and dependent metabolic processes.     

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.     

Synthesis and biological evaluation of sulfonylhydrazone-substituted imidazo[1,2-a]pyridines as novel PI3 kinase p110alpha inhibitors

We have previously reported the imidazo[1,2-a]pyridine derivative 4 as a novel p110alpha inhibitor; however, although 4 is a potent inhibitor of p110alpha enzymatic activity and tumor cell proliferation in vitro, it is unstable in solution and ineffective in vivo. To increase stability the pyrazole of 4 was replaced with a hydrazone and a moderately potent p110alpha inhibitor 7a was obtained. Subsequent optimization of 7a afforded exceptionally potent p110alpha inhibitors, including 8c and 8h, with IC(50) values of 0.30 nM and 0.26 nM, respectively; to the best of our knowledge, these compounds are the most potent PI3K p110alpha inhibitors reported to date. Compound 8c was also stable in solution and exhibited significant anti-tumor effectiveness in vivo.