PI 3-kinase effector molecules

Abstract

The universal role of 3-phosphorylated inositol lipids in cellular signal transduction has become apparent over the last few years. Of these lipids phosphatidylinositol 3,4,5-trisphosphate (PIP,) has aroused the most interest as a potential second messenger'. On aspect of our work is directed at identifying in vivo PIP' receptors in order to characterise the downstream pathways that become activated following PIP, production. Experimentally we have identified various proteins based initially on their ability to specifically bind in virro the inositol head group of PIP,: inositol 1,3,4,5-tetrakisphosphate (IP,). Using this approach we have characterised GAPl'P48P and GAPI? members of the GAP1 family of Ras GTPase-activating proteins', and the ARF exchange factor$, ARNO, GRPl and cytohesin-I, as high affinity isomerically specific IP, binding proteins; data that raises the issue of whether these proteins constitute PIP' binding proteins in v i~o '~~. Through the use of green fluorescent protein (GFP) technology coupled with live cell time-lapse confocal laser scanning microscopy we have shown that a GFP-ARNO chimera is rapidly (< 40 seconds) recruited from the cytosol to the plasma membrane of adipocytes following insulin stimulation'. This translocation not only requires the ability of insulin to generate plasma membrane PIP, but also the presence of the in vitro IP4 binding ARNO pleckstrin homology (PH) domain. Similar data has been obtained for GRPI4 and cytohesin-Is. We have also recently demonstrated that GAPI" is recruited to the plasma membrane of PC12 cells following EGF stimulation. As with ARNO, this recruitment requires PIP, generation and the GAPI" PH domain but interestingly does not appear to lead to an activation of the GAPl"' H-Ras GAP activity6. These results and others regarding inositol phosphate binding to PH domains and the effect of PIP, on ARF signalling will he discussed.A wide variety of growth factors, inflammatory stimuli and antigens use the phosphoinositide 30H-kinase (F' 13K) signalling pathway to regulate a range of complex cell responses. We still have an incomplete understanding however, of the molecular mechanisms by which the lipid products of agonist-stimulated PI3Ks (PtdIns(3,4)P* and PtdIns(3,4,5)P3) actually perform their regulatory roles. A number of approaches have been used to try and resolve this, including various assays for specific PtdIns(3,4,)Pz and RdIns(3,4,5)P3 binding proteins and also in vzfro assays where the effects of addition of these lipids have been measured on physiologically relevant cell responses. We are currently developing an in vifro assay in which activations of the GPy-sensitive plOllpllOy isoform of PDK abundant in leucocytes can regulate the rate of superoxide production by a collection of neutrophil proteins. The activation of the oxidase burst by G-protein coupled receptors (e.g. receptors for FMLP, PAF, histamine) on neutrophils has been identified previously as a PI3K regulated response (e.g. Arcaro & Wymann (1993) Biochem. J. 296, 297-301) ...

Inositol 1,3,4,5-tetrakisphosphate controls proapoptotic Bim gene expression and survival in B cells

“…3d and SI Movie 1). By contrast, no redistribution of Rasa3-GFP was detected in the presence of Bt 2 Ins(1,2,4,5)P 4 / PM, which has a Ϸ100-fold lower affinity for Rasa3 PH domain than Ins(1,3,4,5)P 4 (19) or in the presence of DMSO, both used as negative controls (Fig. 3d and SI Movies 2 and 3).…”

“…18 and http://symatlas.gnf.org/ SymAtlas). The protein is essentially membrane-associated through the interaction of its PH domain with phosphatidylinositol 4,5-bisphosphate (19). Because Rasa3 PH domain has also a high affinity for the water-soluble Ins(1,3,4,5)P 4 (20,21), it has been suggested that Ins(1,3,4,5)P 4 production in stimulated thymocytes could result in a substantial redistribution of Rasa3 from the plasma membrane to the cytosol, allowing unhindered Ras activation (7).…”

Regulation of acid phosphatase in reverse micellar system by lipids additives: Structural aspects

“…The mechanism of PI influence on the AP properties in the reverse micelles can be explained as a result of complex formation between inositol lipids and proteins. Indeed, some proteins have specific binding domains for inositol lipids, which might regulate their activity [35][36][37][38]. In the case of AP, the formation of such complex in aqueous solution was recently confirmed [27].…”

Epigenetic silencing of a Ca 2+ -regulated Ras GTPase-activating protein RASAL defines a new mechanism of Ras activation in human cancers

“…Although this domain structure is conserved, subtle variation in the function of each individual domain and the interaction between domains has a pronounced effect on the regulation of each protein (9,11,12,14,15). For GAP1 IP4BP and GAP1 m , the association of phosphoinositides with their pleckstrin homology domains is an important determinant in targeting these proteins to the cytosolic leaflet of the plasma membrane where they inactivate Ras (11,16,17). In the case of CAPRI and RASAL, plasma membrane targeting is driven through their tandem C2 domains, which bind phospholipids upon an elevation in the intracellular free Ca 2ϩ concentration ([Ca 2ϩ ] i ) (12,14,15).…”