Membrane-induced allosteric control of phospholipase C-ß isozymes.

All peripheral membrane proteins must negotiate unique constraints intrinsic to the biological interface of lipid bilayers and the cytosol. Phospholipase C-ß (PLC-ß) isozymes hydrolyze the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) to propagate diverse intracellular responses that underlie the physiological action of many hormones, neurotransmitters, and growth factors. PLC-ß isozymes are autoinhibited, and several proteins, including Gαq, Gßγ, and Rac1, directly engage distinct regions of these phospholipases to release autoinhibition. To understand this process, we used a novel, soluble analog of PIP2 that increases in fluorescence upon cleavage to monitor phospholipase activity in real time in the absence of membranes or detergents. High concentrations of Gαq or Gß1γ2 did not activate purified PLC-ß3 under these conditions despite their robust capacity to activate PLC-ß3 at membranes. In addition, mutants of PLC-ß3 with crippled autoinhibition dramatically accelerated the hydrolysis of PIP2 in membranes without an equivalent acceleration in the hydrolysis of the soluble analog. Our results illustrate that membranes are integral for the activation of PLC-ß isozymes by diverse modulators, and we propose a model describing membrane-mediated allosterism within PLC-ß isozymes.

Phosphoinositide-specific phospholipase C beta1 signal transduction in the nucleus.

The nuclear inositol lipid cycle is a well known process, and nuclear phosphoinositide-specific phospholipase C beta1 (PLCbeta1) signalling activity has been extensively studied in the last decades. We now know that nuclear PLCbeta1 is a key player in the control of cell cycle progression; in fact it appears to be involved in the cyclin-mediated regulation of the physiological machinery. Indeed, the recent discovery of a possible involvement of the interstitial deletion of PLCbeta1 gene in the progression of myelodysplastic syndrome (MDS) to acute myeloid leukemia in humans (AML) strengthens this contention. Albeit several papers have reported the techniques used for the study of inositide-dependent signaling in the nucleus, we describe here step by step protocols, which can be followed for the preparation of highly purified nuclei and the subsequent analysis of nuclear PLCbeta1 signaling. The described techniques range from nuclear purification to enzymatic activity and to molecular biology methods.

General and versatile autoinhibition of PLC isozymes.

Phospholipase C (PLC) isozymes are directly activated by heterotrimeric G proteins and Ras-like GTPases to hydrolyze phosphatidylinositol 4,5-bisphosphate into the second messengers diacylglycerol and inositol 1,4,5-trisphosphate. Although PLCs play central roles in myriad signaling cascades, the molecular details of their activation remain poorly understood. As described here, the crystal structure of PLC-beta2 illustrates occlusion of the active site by a loop separating the two halves of the catalytic TIM barrel. Removal of this insertion constitutively activates PLC-beta2 without ablating its capacity to be further stimulated by classical G protein modulators. Similar regulation occurs in other PLC members, and a general mechanism of interfacial activation at membranes is presented that provides a unifying framework for PLC activation by diverse stimuli.

Analysis and pharmacological targeting of phospholipase C beta interactions with G proteins.

Phosphatidylinositol-specific phospholipase C enzymes (PLC) catalyze hydrolysis of phosphatidylinositol 4,5-bisphosphate generating the second messengers diacylglycerol and inositol 1,4,5-triphosphate. Mammalian phosphoinositide-specific phospholipase C beta (PLCbeta) activity is regulated by the alpha(q) family of G-protein alpha subunits and by Gbetagamma subunits. Regulation of PLCbeta enzymatic activity can be assayed by reconstituting purified G-protein subunits with purified PLCbeta in the presence of phospholipid vesicles containing the substrate phosphatidylinositol 4,5-bisphosphate. This chapter describes methods for expression and purification of PLCbeta and Gbetagamma from insect cells, assay of G-protein-dependent regulation of PLC activity, and assessment of G-protein-PLC direct binding interactions. This combination of functional and direct binding analysis provides a powerful approach to characterizing PLC and G-protein interfaces, identifying inhibitors of this interaction, and potentially uncovering new modes of PLC regulation.