P85α Regulatory Subunit Of Phosphoinositide 3-kinase Research Articles

The WW domain of IQGAP1 binds directly to the p110α catalytic subunit of PI 3-kinase.

IQGAP1 is a multi-domain cancer-associated protein that serves as a scaffold protein for multiple signaling pathways. Numerous binding partners have been found for the calponin homology, IQ and GAP-related domains in IQGAP1. Identification of a binding partner for its WW domain has proven elusive, however, even though a cell-penetrating peptide derived from this domain has marked anti-tumor activity. Here, using in vitrobinding assays with human proteins and co-precipitation from human cells, we show that the WW domain of human IQGAP1 binds directly to the p110α catalytic subunit of phosphoinositide 3-kinase (PI3K). In contrast, the WW domain does not bind to ERK1/2, MEK1/2, or the p85α regulatory subunit of PI3K when p85α is expressed alone. However, the WW domain is able to bind to the p110α/p85α heterodimer when both subunits are co-expressed, as well as to the mutationally activated p110α/p65α heterodimer. We present a model of the structure of the IQGAP1 WW domain, and experimentally identify key residues in the hydrophobic core and beta strands of the WW domain that are required for binding to p110α. These findings contribute to a more precise understanding of IQGAP1-mediated scaffolding, and of how IQGAP1-derived therapeutic peptides might inhibit tumorigenesis.

Targeting Truncated Retinoid X Receptor-α by CF31 Induces TNF-α–Dependent Apoptosis

A truncated version of retinoid X receptor-α, tRXR-α, promotes cancer cell survival by activating the phosphoinositide 3-kinase (PI3K)/AKT pathway. However, targeting the tRXR-α-mediated survival pathway for cancer treatment remains to be explored. We report here our identification of a new natural product molecule, CF31, a xanthone isolated from Cratoxylum formosum ssp. pruniflorum, and the biologic evaluation of its regulation of the tRXR-α-mediated PI3K/AKT pathway. CF31 binds RXR-α and its binding results in inhibition of RXR-α transactivation. Through RXR-α mutational analysis and computational studies, we show that Arg316 of RXR-α, known to form salt bridges with certain RXR-α ligands, such as 9-cis-retinoic acid (9-cis-RA), is not required for the antagonist effect of CF31, showing a distinct binding mode. Evaluation of several CF31 analogs suggests that the antagonist effect is mainly attributed to an interference with Leu451 of helix H12 in RXR-α. CF31 is a potent inhibitor of AKT activation in various cancer cell lines. When combined with TNF-α, it suppresses TNF-α activation of AKT by inhibiting TNF-α-induced tRXR-α interaction with the p85α regulatory subunit of PI3K. CF31 inhibition of TNF-α activation of AKT also results in TNF-α-dependent activation of caspase-8 and apoptosis. Together, our results show that CF31 is an effective converter of TNF-α signaling from survival to death by targeting tRXR-α in a unique mode and suggest that identification of a natural product that targets an RXR-mediated cell survival pathway that regulates PI3K/AKT may offer a new therapeutic strategy to kill cancer cells.

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.

Role of phosphoinositide‐3‐kinase (PI3K) in the nucleus of the solitary tract (NTS) in the modulation of baroreceptor reflex function in the hypertensive rat

Previous in vitro studies indicate an angiotensin II (AngII)‐dependent increase in PI3K signaling in the brainstem neurones of the spontaneously hypertensive (SHRs) but not Wistar Kyoto rats (WKYs; Yang & Raizada, 1999; Veerasingham et al. 2005). Here, we chronically blocked PI3K activity in the NTS of SHR by lentiviral overexpression of a dominant negative form of the p85α regulatory subunit of PI3K (DNp85α). Perfusion pressure, heart rate, and activities of the phrenic nerve and lower thoracic sympathetic chain were measured in an in situ arterially perfused rat preparation. Baroreceptors were stimulated by increasing perfusion pressure by 30–50 mmHg before and after NTS microinjection of AngII. Transgene expression and sites of AngII injections were confirmed post hoc. PI3K blockade alone reduced both the cardiac (from −3.1±0.1 to −1.4±0.5 bpm/mmHg) and sympathetic (from −5.7±3.4 to −1.0±0.6 %baseline/mmHg) baroreflex components when compared to the control rats overexpressing GFP in the NTS. AngII (500 fmol) in the NTS reduced the gain of the cardiac baroreflex in both groups to a similar extent. In contrast, the AngII‐mediated depression of the sympathetic component of baroreflex seen in controls (by 65±17%) was abolished in the DNp85α group (15±57%). We conclude that in SHR, AngII via the PI3K signalling modulates the sympathetic but not the cardiac component of the baroreflex. NIH grant #HL33610.

Chronic inhibition of phosphoinositide‐3‐kinase (PI3K) in the nucleus of the solitary tract (NTS) of hypertensive rats increases blood pressure

The NTS is the site of termination of baroreceptor primary afferents and a major regulator of arterial pressure. Our previous work indicated an increase in PI3K signalling in brainstem/hypothalamic neuronal cultures from spontaneously hypertensive (SHRs) compared to normotensive Wistar Kyoto rats (WKYs; Yang & Raizada, 1999; Veerasingham et al. 2005). To investigate the functional implications of this, we chronically blocked PI3K activity in the NTS by overexpressing a dominant negative of the p85α regulatory subunit of PI3K (DNp85α) while measuring changes in mean arterial pressure (MAP) in both SHR and WKY conscious adult rats using radio telemetry. Transgene expression was confirmed immunohistochemically post hoc. In SHR, but not WKY, MAP increased from 146±3 to 169±9 mmHg three weeks post gene delivery (n=6; P<0.05) persisting over five weeks, with no change in the spontaneous baroreflex gain (sBRG) or heart rate (HR). There was no change in MAP, sBRG and HR in either WKYs or in the control animals expressing DNp85α and eGFP respectively. We conclude that in SHR, but not WKY, the endogenous PI3K signalling acts to regulate arterial pressure. NIH grant #HL33610.

Phosphoinositide 3-kinase regulatory subunit p85alpha suppresses insulin action via positive regulation of PTEN.

The phosphoinositide 3-kinase (PI3K) pathway is central to the metabolic actions of insulin on liver. Here, we show that mice with a liver-specific deletion of the p85alpha regulatory subunit of PI3K (L-Pik3r1KO) exhibit a paradoxical improvement of hepatic and peripheral insulin sensitivity. Although PI3K enzymatic activity is diminished in L-Pik3r1KO livers because of a reduced level of regulatory and catalytic subunits of PI3K, insulin-stimulated Akt activity is actually increased. This increased Akt activity correlates with increased phosphatidylinositol (3,4,5)-trisphosphate levels which are due, at least in part, to diminished activity of the (3,4,5)-trisphosphate phosphatase PTEN. Thus, the regulatory subunit p85alpha is a critical modulator of insulin sensitivity in vivo not only because of its effects on PI3K activation, but also as a regulator of PTEN activity.

Learning Deficits and Agenesis of Synapses and Myelinated Axons in Phosphoinositide-3 Kinase-Deficient Mice

Although previous studies have reported a role for phosphoinositide-3 kinase (PI3K) in axonal definition and growth in vitro, it is not clear whether PI3K regulates axonal formation and synaptogenesis in vivo. The goal of the present study was to clarify the role of PI3K in behavioral functions and some underlying neuroanatomical structures. Immunohistochemistry, an electron-microscopic analysis and behavioral tests were carried out. Knockout mice lacking the p85α regulatory subunit of PI3K (p85α–/– mice) significantly showed learning deficits, restlessness and motivation deficit. Expression of phosphorylated Akt, which indirectly shows the activity of PI3K, was high in myelinated axons, especially in axonal bundles in the striatum of wild-type mice, but was significantly low in the striatum, cerebral cortex and the hippocampal CA3 of p85α–/– mice. The axonal marker protein level decreased mainly in the striatum and cerebral cortex of p85α–/– mice. In these two regions, myelinated axons are rich in the wild-type mice. However, the density of myelinated axons and myelin thickness were significantly low in the striatum and cerebral cortex of p85α–/– mice. Synaptic protein level was clearly decreased in the striatum, cerebral cortex, and hippocampus of p85α–/– mice when compared with wild mice. The present results suggest that PI3K plays a role in the generation and/or maintenance of synapses and myelinated axons in the brain and that deficiencies in PI3K activity result in abnormalities in several neuronal functions, including learning, restlessness and motivation.

In vitro and in vivo studies of a naturally occurring variant of the human p85alpha regulatory subunit of the phosphoinositide 3-kinase: inhibition of protein kinase B and relationships with type 2 diabetes, insulin secretion, glucose disappearance constant, and insulin sensitivity.

In humans, the Met326Ile missense variant of the p85alpha regulatory subunit of the phosphoinositide 3-kinase (PI3K) has been associated with either significant reductions in glucose effectiveness and intravenous glucose tolerance in Caucasians or a significantly higher insulin secretory response in Pima Indians. In the present study, we genotyped 1,190 Caucasian males to evaluate the impact in vivo of the Met326Ile variant of the p85alpha subunit of PI3K on the acute insulin response, intravenous glucose tolerance, insulin-mediated glucose uptake, and the prevalence of type 2 diabetes after 20 years of follow-up. We also expressed the variant in vitro to evaluate the impact on insulin-stimulated activation of protein kinase B (PKB). The Met326Ile variant of p85alpha was not associated with type 2 diabetes or with alterations in insulin secretion, insulin sensitivity, or intravenous glucose tolerance in vivo. Expressed in vitro, the Ile326 and the Met326 variant acted equally as a dominant-negative and prevented (60-70% inhibition) insulin-mediated activation of PKB by inhibiting the phosphorylation of PKB at Thr308. We conclude that the Met326Ile variant of the p85alpha regulatory subunit of PI3K is likely to be as functionally normal in vivo as in vitro.

Role of the PI3K regulatory subunit in the control of actin organization and cell migration.

Cell migration represents an important cellular response that utilizes cytoskeletal reorganization as its driving force. Here, we describe a new signaling cascade linking PDGF receptor stimulation to actin rearrangements and cell migration. We demonstrate that PDGF activates Cdc42 and its downstream effector N-WASP to mediate filopodia formation, actin stress fiber disassembly, and a reduction in focal adhesion complexes. Induction of the Cdc42 pathway is independent of phosphoinositide 3-kinase (PI3K) enzymatic activity, but it is dependent on the p85alpha regulatory subunit of PI3K. Finally, data are provided showing that activation of this pathway is required for PDGF-induced cell migration on collagen. These observations show the essential role of the PI3K regulatory subunit p85alpha in controlling PDGF receptor-induced cytoskeletal changes and cell migration, illustrating a novel signaling pathway that links receptor stimulation at the cell membrane with actin dynamics.