A new evaluation method for quantifying PI3K activity by HTRF assay

PIK3CA, coding a catalytic subunit of PI3K p110α, is frequently mutated in cancer. In previous studies, p110α with hotspot mutations such as E545K and H1047R were shown to be gain-of-function mutations. However, quantitative evaluation of these mutants was not well established. Recently, a new method for measuring PI3K activity using homogeneous time-resolved fluorescence (HTRF) has been developed. Using this method, we constructed a quantitative evaluation system for PI3K activity. Serial dilutions of standard PIP3 were subjected to the PI3K-HTRF assay in order to establish a regression line for calibration. The recombinant FLAG-tagged p110α proteins were engineered together with a regulatory subunit p85α in human embryonic kidney 293T cells. Anti-FLAG-Ig immunoprecipitates were then subjected to the assay, which enabled us to quantitatively evaluate the activities of hotspot mutants of p110α. We believe this method will also be applicable to the evaluation of p110α having uncharacterized mutations found in cancer.     

Structural and mechanistic insights provided by single particle cryo-EM analysis of phosphoinositide 3-kinase (PI3Kα)

Recent cryo-electron microscopic (cryo-EM) investigations have succeeded in the analysis of various structural conformations and functional states of PI3Kα, a dimer consisting of the catalytic subunit p110α and the regulatory subunit p85α of class IA of phosphoinositide 3-kinase. High resolution structures have been obtained of the unliganded and of BYL-719-bound PI3Kα. The latter provides information on excessively flexible domains of p85α that are then further analyzed with nanobodies and CXMS (chemical cross-linking, digestion and mass spectrometry). Analysis of p110α helical and kinase domain mutations reveals mutant-specific features that can be linked to the gain of function in enzymatic and signaling activities.     

Characterization of PI3K class IA isoforms with regulatory subunit p55alpha using a scintillation proximity assay

Differential activation of the phosphoinositide 3-kinase (PI3K)/AKT pathway has been linked to cancer. Activation occurs through gene amplification and activating mutations. High-frequency mutations in the gene encoding the p110α catalytic subunit of PI3K (PIK3CA) have been observed in a variety of tumors including colon, brain, breast, ovarian, and gastric. Inhibition of PI3K kinase activity may provide a specific way to treat multiple types of human cancer. A scintillation proximity assay (SPA) was developed to detect phosphatidylinositol 3-kinase catalytic activity. Using this assay format, steady-state kinetic parameters were compared for the PI3K class IA enzymes p110α, p110β, and p110δ, each coexpressed with the regulatory subunit p85α or splice variant p55α. Inhibition by the natural product wortmannin and LY294002 was detected with potencies consistent with alternate assay formats. Other biochemical assay formats have been described for phosphoinositide 3-kinases but each has its unique limitations. The simple, inexpensive, sensitive high-throughput nature of the SPA format has advanced our knowledge of isoform-specific enzymology and will facilitate the discovery of novel PI3K inhibitors.     

Activation of PI3K/Akt signaling by n-terminal SH2 domain mutants of the p85α regulatory subunit of PI3K is enhanced by deletion of its c-terminal SH2 domain

The phosphoinositide 3-kinase (PI3K) is frequently activated in human cancer cells due to gain of function mutations in the catalytic (p110) and the regulatory (p85) subunits. The regulatory subunit consists of an SH3 domain and two SH2 domains. An oncogenic form of p85α named p65 lacking the c-terminal SH2 domain (cSH2) has been cloned from an irradiation-induced murine thymic lymphoma and transgenic mice expressing p65 in T lymphocytes develop a lymphoproliferative disorder. We have recently detected a c-terminal truncated form of p85α named p76α in a human lymphoma cell line lacking most of the cSH2 domain due to a frame shift mutation. Here, we report that the deletion of the cSH2 domain enhances the activating effects of the n-terminal SH2 domain (nSH2) mutants K379E and R340E on the PI3K/Akt pathway and micro tumor formation in a focus assay. Further analysis revealed that this transforming effect is mediated by activation of the catalytic PI3K isoform p110α and downstream signaling through mTOR. Our data further support a mechanistic model in which mutations of the cSH2 domain of p85α can abrogate its negative regulatory function on PI3K activity via the nSH2 domain of p85α.     

Multiple roles for the p85α isoform in the regulation and function of PI3K signalling and receptor trafficking

The p85α protein is best known as the regulatory subunit of class 1A PI3Ks (phosphoinositide 3-kinases) through its interaction, stabilization and repression of p110-PI3K catalytic subunits. PI3Ks play multiple roles in the regulation of cell survival, signalling, proliferation, migration and vesicle trafficking. The present review will focus on p85α, with special emphasis on its important roles in the regulation of PTEN (phosphatase and tensin homologue deleted on chromosome 10) and Rab5 functions. The phosphatidylinositol-3-phosphatase PTEN directly counteracts PI3K signalling through dephosphorylation of PI3K lipid products. Thus the balance of p85α-p110 and p85α-PTEN complexes determines the signalling output of the PI3K/PTEN pathway, and under conditions of reduced p85α levels, the p85α-PTEN complex is selectively reduced, promoting PI3K signalling. Rab5 GTPases are important during the endocytosis, intracellular trafficking and degradation of activated receptor complexes. The p85α protein helps switch off Rab5, and if defective in this p85α function, results in sustained activated receptor tyrosine kinase signalling and cell transformation through disrupted receptor trafficking. The central role for p85α in the regulation of PTEN and Rab5 has widened the scope of p85α functions to include integration of PI3K activation (p110-mediated), deactivation (PTEN-mediated) and receptor trafficking/signalling (Rab5-mediated) functions, all with key roles in maintaining cellular homoeostasis.     

Nuclear but not cytosolic phosphoinositide 3-kinase beta has an essential function in cell survival

Class I(A) phosphoinositide 3-kinases (PI3Ks) are heterodimeric enzymes composed of a p85 regulatory and a p110 catalytic subunit that induce the formation of 3-polyphosphoinositides, which mediate cell survival, division, and migration. There are two ubiquitous PI3K isoforms p110α and p110β that have nonredundant functions in embryonic development and cell division. However, whereas p110α concentrates in the cytoplasm, p110β localizes to the nucleus and modulates nuclear processes such as DNA replication and repair. At present, the structural features that determine p110β nuclear localization remain unknown. We describe here that association with the p85β regulatory subunit controls p110β nuclear localization. We identified a nuclear localization signal (NLS) in p110β C2 domain that mediates its nuclear entry, as well as a nuclear export sequence (NES) in p85β. Deletion of p110β induced apoptosis, and complementation with the cytoplasmic C2-NLS p110β mutant was unable to restore cell survival. These studies show that p110β NLS and p85β NES regulate p85β/p110β nuclear localization, supporting the idea that nuclear, but not cytoplasmic, p110β controls cell survival.     

The SH3 and BH Domains of the p85α Adapter Subunit Play a Critical Role in Regulating Class Ia Phosphoinositide 3-Kinase Function

We have investigated the role of the SH3 and BH domains in the function of the p85α adapter/regulatory subunit of PI 3-kinase. In these studies epitope-tagged adapter subunit constructs containing wild-type p85α, p85α lacking the SH3 domain (ΔSH3-p85α), or p85α lacking the Rac-GAP/BCR homology (BH) domain (ΔBH-p85α) were coexpressed with either the p110α or p110β PI 3-kinase catalytic subunit in HEK293 cells. The deletion of either BH or SH3 domains had no effect on the intrinsic activity of the PI 3-kinase heterodimers. However, the ability of activated Rac to stimulate PI 3-kinase activity was only observed in heterodimers containing the p85α and ΔSH3-p85α, indicating that rac binding to the BH domain is responsible for rac-induced stimulation of class Ia PI 3-kinase. We also investigated the effect of SH3 and BH domain deletion on the ability of insulin to induce recruitment of these constructs into phosphotyrosine-containing signaling complexes. We find that p85α expressed alone is poorly recruited into such signaling complexes. However, when coexpressed with catalytic subunit, the p85α adapter subunit is recruited to an extent similar to that of endogenous p85α. Maximal insulin stimulation caused a similar level of recruitment of p85α, ΔSH3-p85α, and ΔBH-p85α to signaling complexes when these adapter subunits were coexpressed with catalytic subunit. However, there was a higher level of basal association of the ΔSH3-p85α and ΔBH-p85α with tyrosine-phosphorylated proteins, meaning that the insulin-induced fold increase in recruitment was lower for these forms of the adapter. These results indicate that the N-terminal domains of p85α play a critical role in the way the adapter subunit responds to growth factor stimulation.     

Activation of PI3Kα by physiological effectors and by oncogenic mutations: structural and dynamic effects

PI3Kα, a heterodimeric lipid kinase, catalyzes the conversion of phosphoinositide-4,5-bisphosphate (PIP₂) to phosphoinositide-3,4,5-trisphosphate (PIP₃), a lipid that recruits to the plasma membrane proteins that regulate signaling cascades that control key cellular processes such as cell proliferation, carbohydrate metabolism, cell motility, and apoptosis. PI3Kα is composed of two subunits, p110α and p85, that are activated by binding to phosphorylated receptor tyrosine kinases (RTKs) or their substrates. The gene coding for p110α, PIK3CA, has been found to be mutated in a large number of tumors; these mutations result in increased PI3Kα kinase activity. The structure of the complex of p110α with a fragment of p85 containing the nSH2 and the iSH2 domains has provided valuable information about the mechanisms underlying the physiological activation of PI3Kα and its pathological activation by oncogenic mutations. This review discusses information derived from x-ray diffraction and theoretical calculations regarding the structural and dynamic effects of mutations in four highly mutated regions of PI3K p110α, as well as the proposed mechanisms by which these mutations increase kinase activity. During the physiological activation of PI3Kα, the phosphorylated tyrosine of RTKs binds to the nSH2 domain of p85, dislodging an inhibitory interaction between the p85 nSH2 and a loop of the helical domain of p110α. Several of the oncogenic mutations in p110α activate the enzyme by weakening this autoinhibitory interaction. These effects involve structural changes as well as changes in the dynamics of the enzyme. One of the most common p110α mutations, H1047R, activates PI3Kα by a different mechanism: it increases the interaction of the enzyme with the membrane, maximizing the access of the PI3Kα to its substrate PIP₂, a membrane lipid.     

Hot-spot mutations in p110α of phosphatidylinositol 3-kinase (PI3K): Differential interactions with the regulatory subunit p85 and with RAS

The phosphatidylinositol 3-kinase (PI3K) signaling pathway is frequently upregulated in cancer. PIK3CA, the gene coding for the catalytic subunit p110α of PI3K, is mutated in about 12% of all human cancers. Most of these mutants are single amino acid substitutions that map to three positions (hot spots) in the helical or kinase domains of the enzyme. The mutant proteins show gain of enzymatic function, constitutively activate AKT signaling and induce oncogenic transformation in vitro and in animal model systems. We have shown previously that hot-spot mutations in the helical domain and kinase domain of the avian p110α have different requirements for interaction with the regulatory subunit p85 and with RAS-GTP. Here, we have carried out a genetic and biochemical analysis of these "hot-spot" mutations in human p110α. The present studies add support to the proposal that helical and kinase domain mutations in p110α trigger a gain of function by different molecular mechanisms. The gain of function induced by helical domain mutations requires interaction with RAS-GTP. In contrast, the kinase domain mutation is active in the absence of RAS-GTP binding, but depends on the interaction with p85.     

Effects of tyroserleutide on gene expression of calmodulin and PI3K in hepatocellular carcinoma

Tyroserleutide (YSL) is a tripeptide compound that has exhibited inhibitory effects on hepatocellular carcinoma in our previous research. The mechanism of this antitumor activity involves the second messenger, Ca(2+). Ca(2+) influences cell function through the Ca(2+)/calmodulin (CaM) pathway, and abnormality of the Ca(2+)/CaM system correlates closely with the occurrence of tumors. In addition, CaM associates with phosphatidylinositol 3 kinase (PI3K), thereby enhancing the activity of PI3K, which promotes cell proliferation. In order to investigate its anti-tumor mechanism, we studied the effects of YSL on CaM protein expression and mRNA level, PI3K activity, PI3K regulatory subunit p85 protein expression and mRNA level, and the mRNA level of PI3K catalytic subunits p110alpha and p110gamma in human hepatocellular carcinoma BEL-7402 xenograft tumors in nude mice. Our results showed that YSL decreased the mRNA level and protein expression of CaM, inhibited the activity of PI3K, and reduced the mRNA level and protein expression of the PI3K regulatory subunit p85 and mRNA level of PI3K catalytic subunits p110alpha and p110gamma. Accordingly, it is suggestive that the anti-tumor effects of YSL may be mediated by down regulation of CaM and PI3K subunits p85 and p110, influencing the signal transduction pathway in the tumor cells and perhaps overcoming the dysfunctional PI3K activity in tumors.     

CoA Synthase is in complex with p85αPI3K and affects PI3K signaling pathway

The complex interplay between cellular signaling and metabolism in eukaryotic cells just start to emerge. Coenzyme A (CoA) and its derivatives play a key role in cell metabolism and also participate in regulatory processes. CoA Synthase (CoASy) is a mitochondria-associated enzyme which mediates two final stages of de novo CoA biosynthesis. Here, we report that CoASy is involved in signaling events in the cell and forms a functional complex with p85αPI3K in vivo. Importantly, observed interaction of endogenous CoASy and p85αPI3K is regulated in a growth factor dependent manner. Surprisingly, both catalytic p110α and regulatory p85α subunits of PI3K were detected in mitochondrial fraction where mitochondria-localized p85αPI3K was found in complex with CoASy. Unexpectedly, significant changes of PI3K signaling pathway activity were observed in experiments with siRNA-mediated CoASy knockdown pointing on the role of CoA biosynthetic pathway in signal transduction.     

Then Negative Regulation of Phosphoinositide 3-Kinase Signaling by p85 and Its Implication in Cancer

The phosphoinositide 3-kinase (PI3K) signaling pathway critically regulates cell growth and cell survival. Mutations that lead to aberrant activation of this pathway are frequent events in human cancers. Here we discuss some recent studies identifying the mechanisms by which p85, the regulatory subunit of PI3K, negatively regulates PI3K signaling. While necessary for the stability and membrane recruitment of the p110 catalytic subunit of PI3K. p85 represses the basal activity of p110 in the absence of growth factor stimulation. In its unbound, free form, p85 sequesters the adaptor protein IRS-1 and therefore limits the extent of PI3K signaling downstream of the insulin and IGF-1 receptors. These findings lend new insight to how changes in p85 gene dosage or mutations in p85 could lead to the hyper-activation of PI3K and thus contribute towards tumorigenesis.     

The p85 regulatory subunit controls sequential activation of phosphoinositide 3-kinase by Tyr kinases and Ras

Class IA phosphoinositide 3-kinase (PI3K) is a heterodimer composed of a p85 regulatory and a p110 catalytic subunit that regulates a variety of cell responses, including cell division and survival. PI3K is activated following Tyr kinase stimulation and by Ras. We found that the C-terminal region of p85, including the C-Src homology 2 (C-SH2) domain and part of the inter-SH2 region, protects the p110 catalytic subunit from Ras-induced activation. Although the p110 activity associated with a C-terminal p85 deletion mutant increased significantly in the presence of an active form of Ras, purified wild type p85-p110 was only slightly stimulated by active Ras. Nonetheless, incubation of purified p85-p110 with Tyr-phosphorylated peptides, which mimic the activated platelet-derived growth factor receptor, restored Ras-induced p85-p110 activation. In conclusion, p85 inhibits p110 activation by Ras; this blockage is released by Tyr kinase stimulation, showing that the classical mechanism of class IA PI3K stimulation mediated by Tyr kinases also regulates Ras-induced PI3K activation.     

A Role for phosphoinositide 3-kinase in the control of cell division and survival during retinal development

Neurogenesis in the retina requires the concerted action of three different cellular processes: proliferation, differentiation, and apoptosis. Class IA phosphoinositide 3-kinase (PI3K) is a heterodimer composed of a p85 regulatory and a p110 catalytic subunit. p110alpha has been shown to regulate cell division and survival. Little is known of its function in development, however, as p110alpha knockout mice exhibit CNS defects, but death at early embryonic stages impairs further study. Here, we examine the role of PI3K in mouse retina development by expressing an activating form of PI3K regulatory subunit, p65(PI3K), as a transgene in the retina. Mice expressing p65(PI3K) showed severely disrupted retina morphogenesis, with ectopic cell masses in the neuroepithelium that evolved into infoldings of adult retinal cell layers. These changes correlated with an altered cell proliferation/cell death balance at early developmental stages. Nonetheless, the most affected cell layer in adult retina was that of photoreceptors, which correlated with selectively increased survival of these cells at developmental stages at which cell division has ceased. These results demonstrate the relevance of accurate PI3K regulation for normal retinal development, supporting class IA PI3K involvement in induction of cell division at early stages of neurogenesis. These data also show that, even after cell division decline, PI3K activation mediates survival of differentiated neurons in vivo.     

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.     

Ras‐dependent and Ras‐independent effects of PI3K in Drosophila motor neurons

The lipid kinase PI3K plays key roles in cellular responses to activation of receptor tyrosine kinases or G protein coupled receptors such as the metabotropic glutamate receptor (mGluR). Activation of the PI3K catalytic subunit p110 occurs when the PI3K regulatory subunit p85 binds to phosphotyrosine residues present in upstream activating proteins. In addition, Ras is uniquely capable of activating PI3K in a p85‐independent manner by binding to p110 at amino acids distinct from those recognized by p85. Because Ras, like p85, is activated by phosphotyrosines in upstream activators, it can be difficult to determine if particular PI3K‐dependent processes require p85 or Ras. Here, we ask if PI3K requires Ras activity for either of two different PI3K‐regulated processes within Drosophila larval motor neurons. To address this question, we determined the effects on each process of transgenes and chromosomal mutations that decrease Ras activity, or mutations that eliminate the ability of PI3K to respond to activated Ras. We found that PI3K requires Ras activity to decrease motor neuron excitability, an effect mediated by ligand activation of the single Drosophila mGluR DmGluRA. In contrast, the ability of PI3K to increase nerve terminal growth is Ras‐independent. These results suggest that distinct regulatory mechanisms underlie the effects of PI3K on distinct phenotypic outputs.     

PI3K p110α/Akt signaling negatively regulates secretion of the intestinal peptide neurotensin through interference of granule transport

Neurotensin (NT), an intestinal peptide secreted from N cells in the small bowel, regulates a variety of physiological functions of the gastrointestinal tract, including secretion, gut motility, and intestinal growth. The class IA phosphatidylinositol 3-kinase (PI3K) family, which comprised of p110 catalytic (α, β and δ) and p85 regulatory subunits, has been implicated in the regulation of hormone secretion from endocrine cells. However, the underlying mechanisms remain poorly understood. In particular, the role of PI3K in intestinal peptide secretion is not known. Here, we show that PI3K catalytic subunit, p110α, negatively regulates NT secretion in vitro and in vivo. We demonstrate that inhibition of p110α, but not p110β, induces NT release in BON, a human endocrine cell line, which expresses NT mRNA and produces NT peptide in a manner analogous to N cells, and QGP-1, a pancreatic endocrine cell line that produces NT peptide. In contrast, overexpression of p110α decreases NT secretion. Consistently, p110α-inhibition increases plasma NT levels in mice. To further delineate the mechanisms contributing to this effect, we demonstrate that inhibition of p110α increases NT granule trafficking by up-regulating α-tubulin acetylation; NT secretion is prevented by overexpression of HDAC6, an α-tubulin deacetylase. Moreover, ras-related protein Rab27A (a small G protein) and kinase D-interacting substrate of 220 kDa (Kidins220), which are associated with NT granules, play a negative and positive role, respectively, in p110α-inhibition-induced NT secretion. Our findings identify the critical role and novel mechanisms for the PI3K signaling pathway in the control of intestinal hormone granule transport and release.     

The Unique Non-Catalytic C-Terminus of P37delta-PI3K Adds Proliferative Properties In Vitro and In Vivo

The PI3K/Akt pathway is central for numerous cellular functions and is frequently deregulated in human cancers. The catalytic subunits of PI3K, p110, are thought to have a potential oncogenic function, and the regulatory subunit p85 exerts tumor suppressor properties. The fruit fly, Drosophila melanogaster, is a highly suitable system to investigate PI3K signaling, expressing one catalytic, Dp110, and one regulatory subunit, Dp60, and both show strong homology with the human PI3K proteins p110 and p85. We recently showed that p37δ, an alternatively spliced product of human PI3K p110δ, displayed strong proliferation-promoting properties despite lacking the catalytic domain completely. Here we functionally evaluate the different domains of human p37δ in Drosophila. The N-terminal region of Dp110 alone promotes cell proliferation, and we show that the unique C-terminal region of human p37δ further enhances these proliferative properties, both when expressed in Drosophila, and in human HEK-293 cells. Surprisingly, although the N-terminal region of Dp110 and the C-terminal region of p37δ both display proliferative effects, over-expression of full length Dp110 or the N-terminal part of Dp110 decreases survival in Drosophila, whereas the unique C-terminal region of p37δ prevents this effect. Furthermore, we found that the N-terminal region of the catalytic subunit of PI3K p110, including only the Dp60 (p85)-binding domain and a minor part of the Ras binding domain, rescues phenotypes with severely impaired development caused by Dp60 over-expression in Drosophila, possibly by regulating the levels of Dp60, and also by increasing the levels of phosphorylated Akt. Our results indicate a novel kinase-independent function of the PI3K catalytic subunit.     

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.