CD148 is a transmembrane tyrosine phosphatase that has been implicated in the regulation of cell growth and transformation. However, the signalling mechanisms of CD148 are incompletely understood. To identify the specific intracellular molecules involved in CD148 signalling, we carried out a modified yeast two-hybrid screening assay. Using the substrate-trapping mutant form of CD148 (CD148 D/A) as bait, we recovered the p85 regulatory subunit of PI3K (phosphoinositide 3-kinase). CD148 D/A, but not catalytically active CD148, interacted with p85 in a phosphorylation-dependent manner in vitro and in intact cells. Growth factor receptor and PI3K activity were also trapped by CD148 D/A via p85 from pervanadate-treated cell lysates. CD148 prominently and specifically dephosphorylated p85 in vitro . Co-expression of CD148 reduced p85 phosphorylation induced by active Src, and attenuated the increases in PI3K activity, yet CD148 did not alter the basal PI3K activity. Finally, CD148 knock-down by siRNA (short interfering RNA) increased PI3K activity on serum stimulation. Taken together, these results demonstrate that CD148 may interact with and dephosphorylate p85 when it is phosphorylated and modulate the magnitude of PI3K activity.

Resveratrol mimics calorie restriction to extend lifespan of Caenorhabditis elegans , yeast and Drosophila , possibly through activation of Sir2 (silent information regulator 2), a NAD + -dependent histone deacetylase. In the present study, resveratrol is shown to inhibit the insulin signalling pathway in several cell lines and rat primary hepatocytes in addition to its broad-spectrum inhibition of several signalling pathways. Resveratrol effectively inhibits insulin-induced Akt and MAPK (mitogen-activated protein kinase) activation mainly through disruption of the interactions between insulin receptor substrates and its downstream binding proteins including p85 regulatory subunit of phosphoinositide 3-kinase and Grb2 (growth factor receptor-bound protein 2). The inhibitory effect of resveratrol on insulin signalling is also demonstrated at mRNA level, where resveratrol reverses insulin effects on phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, fatty acid synthase and glucokinase. In addition, RNA interference experiment shows that the inhibitory effect of resveratrol on insulin signalling pathway is not weakened in cells with reduced expression of SirT1, the mammalian counterpart of Sir2. These observations raise the possibility that resveratrol may additionally modulate lifespan through inhibition of insulin signalling pathway, independently of its activation of SirT1 histone deacetylase. Furthermore, the present study may help to explain a wide range of biological effects of resveratrol, and provides further insight into the molecular basis of calorie restriction.

Several studies have reported that activation of G q -coupled receptors inhibits PI3K (phosphoinositide 3-kinase) signalling. In the present study, we used purified proteins to demonstrate that Gα q directly inhibits p110α/p85α PI3K in a GTP-dependent manner. Activated Gα q binds to the p110α/p85α PI3K with an apparent affinity that is seven times stronger than that for Gα q ·GDP as measured by fluorescence spectroscopy. In contrast, Gα q did not bind to the p110γ PI3K. Fluorescence spectroscopy experiments also showed that Gα q competes with Ras, a PI3K activator, for binding to p110α/p85α. Interestingly, co-precipitation studies using deletion mutants showed that Gα q binds to the p85-binding domain of p110α and not to the Ras-binding domain. Expression of constitutively active Gα q Q209L in cells inhibited Ras activation of the PI3K/Akt pathway but had no effect on Ras/Raf/MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase] signalling. These results suggest that activation of G q -coupled receptors leads to increased binding of Gα q ·GTP to some isoforms of PI3K, which might explain why these receptors inhibit this signalling pathway in certain cell types.

Growth factors regulate a wide range of cellular processes via activation of the class-Ia phosphoinositide 3-kinases (PI 3-kinases). We directly compared kinetic properties of lipid- and protein-kinase activities of the widely expressed p110α and p110β isoforms. The lipid-kinase activity did not display Michaelis–Menten kinetics but modelling the kinetic data demonstrated that p110α has a higher V max and a 25-fold higher K m for PtdIns than p110β. A similar situation occurs with PtdIns(4,5) P 2 , because at low concentration of PtdIns(4,5) P 2 p110β is a better PtdIns(4,5) P 2 kinase than p110α, although this is reversed at high concentrations. These differences suggest different functional roles and we hypothesize that p110β functions better in areas of membranes containing low levels of substrate whereas p110α would work best in areas of high substrate density such as membrane lipid rafts. We also compared protein-kinase activities. We found that p110β phosphorylated p85 to a lower degree than did p110α. We used a novel peptide-based assay to compare the kinetics of the protein-kinase activities of p110α and p110β. These studies revealed that, like the lipid-kinase activity, the protein-kinase activity of p110α has a higher K m (550 µ M) than p110β ( K m 8 µ M). Similarly, the relative V max towards peptide substrate of p110α was three times higher than that of p110β. This implies differences in the rates of regulatory autophosphorylation in vivo , which are likely to mean differential regulation of the lipid-kinase activities of p110α and p110β in vivo .