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The membrane phospholipid phosphatidylinositol is the precursor of a family of lipid second-messengers, known as phosphoinositides, which differ in the phosphorylation status of their inositol group. A major advance in understanding phosphoinositide signalling has been the identification of a number of highly conserved modular protein domains whose function appears to be to bind various phosphoinositides. Such ‘cut and paste’ modules are found in a diverse array of multidomain proteins and recruit their host protein to specific regions in cells via interactions with phosphoinositides. Here, with particular reference to proteins involved in membrane traffic pathways, we discuss recent advances in our understanding of phosphoinositide-binding domains.

GAP1(m) is a member of the GAP1 family of Ras GTPase-activating proteins (GAPs) [1]. In vitro, it has been shown to bind inositol 1, 3,4,5-tetrakisphosphate (IP4), the water-soluble inositol head group of the lipid second messenger phosphatidylinositol 3,4, 5-trisphosphate (PIP3) [2] [3]. This has led to the suggestion that GAP1(m) might function as a PIP3 receptor in vivo [4]. Here, using rat pheochromocytoma PC12 cells transiently transfected with a plasmid expressing a chimera of green fluorescent protein fused to GAP1(m) (GFP-GAP1(m)), we show that epidermal growth factor (EGF) induces a rapid (less than 60 seconds) recruitment of GFP-GAP1(m) from the cytosol to the plasma membrane. This recruitment required a functional GAP1(m) pleckstrin homology (PH) domain, because a specific point mutation (R629C) in the PH domain that inhibits IP4 binding in vitro [5] totally blocked EGF-induced GAP1(m) translocation. Furthermore, the membrane translocation was dependent on PI 3-kinase, and the time course of translocation paralleled the rate by which EGF stimulates the generation of plasma membrane PIP3 [6]. Significantly, the PIP3-induced recruitment of GAP1(m) did not appear to result in any detectable enhancement in its basal Ras GAP activity. From these results, we conclude that GAP1(m) binds PIP3 in vivo, and it is recruited to the plasma membrane, but does not appear to be activated, following agonist stimulation of PI 3-kinase.

New work on the ability of IRSp53/MIM domains to induce negative membrane curvature sheds light on the mechanisms used to generate actin-rich cell protrusions.

Many proteins are being found to bind inositol phosphates with varying degrees of specificity; the variety of domains that can bind inositol phosphates suggests convergent evolution, but the functions of most of the binding sites are not yet clear.

The debate over whether activated Ras can regulate phosphoinositide-specific phospholipase C (PLC) has been contentious and at times heated. The argument may be resolved by the recent identification of a novel Ras-regulated PLC, but some unexpected properties of this protein are sure to stimulate further controversy.

Pleckstrin homology domains are modular domains that direct membrane targeting of their host proteins by binding to polyphosphoinositides; recent results have increased our appreciation of how some of these domains actually bind 3-phosphoinositides, and along the way thrown up some unexpected observations.

Ca2+ is a universal second messenger that is critical for cell growth and is intimately associated with many Ras-dependent cellular processes such as proliferation and differentiation [1]. Ras is a small GTP binding protein that operates as a molecular switch regulating the control of gene expression, cell growth, and differentiation through a pathway from receptors to mitogen-activated protein kinases (MAPKs) [2]. A role for intracellular Ca2+ in the activation of Ras has been previously demonstrated, e.g., via the nonreceptor tyrosine kinase PYK2 [3] and by Ca2+/calmodulin-dependent guanine nucleotide exchange factors (GEFs) such as Ras-GRF [4]; however, there is no Ca2+-dependent mechanism for direct inactivation. An important advance toward greater understanding of the complex coordination within the Ras-signaling network is the spatio-temporal analysis of signaling events in vivo. Here, we describe the identification of CAPRI (Ca2+-promoted Ras inactivator), a Ca2+-dependent Ras GTPase-activating protein (GAP) that switches off the Ras-MAPK pathway following a stimulus that elevates intracellular Ca2+. Analysis of the spatio-temporal dynamics of CAPRI indicates that Ca2+ regulates the GAP by a fast C2 domain-dependent translocation mechanism.