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.     

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.     

Involvement of insulin in early development of mouse one-cell stage embryos

Recent studies have suggested that growth factors and hormones play important roles in cell prolif-eration and differentiation during early embryonic development. In the present study, we examined the expression and localization of insulin in the mouse oocytes and one-cell stage embryos by quantitative ELISA, RT-PCR, Western blot and immunofluorescence. In the mouse oocytes and one-cell stage em-bryos, expression of insulin was uniformly distributed in the cytoplasm. We also examined the expres-sion, activity and localization of mTOR (mammalian target of rapamycin) and p70S6K. The expression of mTOR and p70S6K was not significantly different at the cell cycle of mouse one-cell stage embryos. mTOR and S6K were distributed evenly in the cytoplasm at G1, G2 and M phase phase, but at S phase, the distribution of mTOR and S6K was around the pronucleus. At different phases, the activity of mTOR fluctuated. We also used the PI3K specific inhibitor-Wortmannin to investigate the cleavage rate of eggs. The result showed that the rate obviously decreased. When the mTOR specific inhibitor Rapa-mycin was used, the first mitotic division of the mouse one-cell stage embryo was delayed. These re-sults suggested that insulin was expressed both in mouse oocytes and one-cell stage embryos, and may play functional roles in regulation of mouse early embryogenesis by activating the signal pathway of PI3K/PKB/mTOR/S6K.     

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.     

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.     

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.     

Involvement of PI3K in HCV-related lymphoproliferative disorders

We have investigated the role of NAADP-mediated Ca(2+) mobilization in endothelin (ET) signaling via endothelin receptor subtype A (ETA) and endothelin receptor subtype B (ETB) in rat peritubular smooth muscle cells. Microinjection and extracellular application of NAADP were both able to elicit Ca(2+) release which was blocked by inhibitory concentrations of NAADP, by impairing Ca(2+) uptake in acidic stores with bafilomycin, and by thapsigargin. Ca(2+) release in response to selective ETB stimulation was abolished by inhibition of NAADP signaling through the same strategies, while these treatments only partially impaired ETA-dependent Ca(2+) signaling, showing that transduction of the ETB signal is dependent on NAADP. In addition, we show that lipid rafts/caveolae contain ETA, ETB, and NAADP/cADPR generating enzyme CD38 and that stimulation of ETB receptors results in increased CD38 activity; interestingly, ETB- (but not ETA-) mediated Ca(2+) responses were antagonized by disruption of lipid rafts/caveolae with methyl-beta-cyclodextrin. These data demonstrate a primary role of NAADP in ETB-mediated Ca(2+) signaling and strongly suggest a novel role of lipid rafts/caveolae in triggering ET-induced NAADP signaling.     

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.     

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.     

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.     

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.     

Effects of PI3K catalytic subunit and Akt isoform deficiency on mTOR and p70S6K activation in myoblasts

The PI3K/Akt/mTOR signaling pathway is critical for cellular growth and survival in skeletal muscle, and is activated in response to growth factors such as insulin-like growth factor-I (IGF-I). We found that in C2C12 myoblasts, deficiency of PI3K p110 catalytic subunits or Akt isoforms had distinct effects on phosphorylation of mTOR and p70S6K. siRNA-mediated knockdown of PI3K p110α, p110β, and simultaneous knockdown of p110α and p110β resulted in increased basal and IGF-I-stimulated phosphorylation of mTOR S2448 and p70S6K T389; however, phosphorylation of S6 was reduced in p110β-deficient cells, possibly due to reductions in total S6 protein. We found that IGF-I-stimulated Akt1 activity was enhanced in Akt2- or Akt3-deficient cells, and that knockdown of individual Akt isoforms increased mTOR/p70S6K activation in an isoform-specific fashion. Conversely, levels of IGF-I-stimulated p70S6K phosphorylation in cells simultaneously deficient in both Akt1 and Akt3 were increased beyond those seen with loss of any single Akt isoform, suggesting an alternate, Akt-independent mechanism that activates mTOR/p70S6K. Our results collectively suggest that mTOR/p70S6K is activated in a PI3K/Akt-dependent manner, but that in the absence of p110α or Akt, alternate pathway(s) may mediate activation of mTOR/p70S6K in C2C12 myoblasts.     

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.     

The early development of the lymphatic system in mouse embryos.

The early development of the lymphatic system was studied in embryos of an inbred strain of the laboratory mouse. During the first stage of its development the system is represented by a more or less regular series of small and blind-ending outgrowths of the major embryonic veins which develop in a cranio-caudalward direction from the jugular to the pelvic region. As a result of differences in growth rates of adjacent anatomical structures this series of early lymphatic primordia becomes subdivided into 4 singular primordia and 12 groups of primordia. After the constituents of each group of early primordia have fused, 16 isolated lymphatic plexuses (sacs) are formed of which 14 are in bilaterally symmetric and 2 are in a median line position: i.e. bilaterally: (1) the jugulo-axillary lymph sac situated lateral to the anterior cardinal vein and dorsal to the primitive ulnar vein and its major branch, the external mammary vein, (2) the paratracheal lymph plexus situated medial to the anterior cardinal vein, (3) the internal thoracic lymph plexus situated lateral to the thoracic part of the posterior cardinal vein, (4) the thoracic ducts situated medial to the thoracic part of the posterior cardinal vein, (5) the lumbar lymph plexus situated dorso-lateral to the abdominal part of the posterior cardinal vein, (6) the subcardinal lymph plexus and (7) the iliac lymph plexus situated ventro-lateral to the abdominal part of the posterior cardinal vein; and in the median line: (8) the subtracheal lymph plexus situated at the confluence of the pulmonary veins and (9) the mesenteric lymph plexus situated near the confluence of the splenic and the superior mesenteric veins. Except for some openings at the jugulo-subclavian confluence all connections with the veins disappear. From the primordia extensions grow out centrifugally. They invade the surrounding tissues and, in part, fuse with similar sprouts of adjacent primordia. In this way a continuous system of lymph truncs is formed that opens into the venous system at the jugulo-subclavian confluence.     

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.