The phospholipase C isozymes and their regulation

Abstract

The physiological effects of many extracellular neurotransmitters, hormones, growth factors, and other stimuli are mediated by receptor-promoted activation of phospholipase C (PLC) and consequential activation of inositol lipid signaling pathways. These signaling responses include the classically described conversion of phosphatidylinositol(4,5)P(2) to the Ca(2+)-mobilizing second messenger inositol(1,4,5)P(3) and the protein kinase C-activating second messenger diacylglycerol as well as alterations in membrane association or activity of many proteins that harbor phosphoinositide binding domains. The 13 mammalian PLCs elaborate a minimal catalytic core typified by PLC-d to confer multiple modes of regulation of lipase activity. PLC-b isozymes are activated by Gaq- and Gbg-subunits of heterotrimeric G proteins, and activation of PLC-g isozymes occurs through phosphorylation promoted by receptor and non-receptor tyrosine kinases. PLC-e and certain members of the PLC-b and PLC-g subclasses of isozymes are activated by direct binding of small G proteins of the Ras, Rho, and Rac subfamilies of GTPases. Recent high resolution three dimensional structures together with biochemical studies have illustrated that the X/Y linker region of the catalytic core mediates autoinhibition of most if not all PLC isozymes. Activation occurs as a consequence of removal of this autoinhibition.

Fig. 3.1

The enzyme activity of phospholipase C. Phospholipase C (PLC) isozymes convert membrane phosphatidylinositol (4,5)bisphosphate (PtdIns(4,5)P₂) into the Ca²⁺-mobilizing second messenger inositol(1,4,5) trisphosphate (IP₃) and the protein kinase C-activating second messenger diacylglycerol (DAG). PtdIns(4,5)P₂ also acts as a second messenger that binds to a broad range of membrane, cytoskeletal, and cytosolic proteins to change their activities

Fig. 3.2

The mammalian PLC isozymes and their modes of regulation. The human PLC isozymes were aligned based upon conservation of protein sequence, and a dendrogram that clusters similar sequences within shared branches is presented. The common core of these isozymes includes a pleckstrin homology (PH) domain (purple), a series of four EF-hands (yellow), a catalytic TIM barrel (pink), and a C2 domain (green). The four PLC-β isozymes contain a long C-terminal (CT) domain (light blue). The two PLC-γ isozymes contain conserved domains inserted within the TIM barrel that include a split PH domain, two Src-homology 2 (SH2) domains and a single Src-homology 3 (SH3) domain. PLC-ε contains a guanine nucleotide exchange domain (RasGEF) that activates Rap1 and possibly other GTPases and two C-terminal Ras-association (RA) domains that bind activated Ras GTPases. A cysteine-rich (C) domain of unestablished function occurs at the N-terminus. PLC-ζ is the only mammalian PLC that lacks a PH domain. PLC-η isozymes contain a serine/proline (S/P) rich region in the C-terminus. The PLC-like (PLC-L) proteins exhibit the common core of other PLC isozymes but are catalytically inactive due to mutations of critical residues in the active site. Established modes of regulation are indicated for each of the PLC isozyme classes

Fig. 3.3

Three-dimensional structure of PLC-β2. Left panel, A ribbon diagram is illustrated of the three dimensional structure (PDB 2ZKM) of PLC-β2 solved at 1.6 Å resolution by Hicks and coworkers (Hicks et al. 2008). The PH domain (purple), EF hands (yellow), TIM barrel (red) and C2 domain (green) are colored as in Fig. 3.2. The Ca²⁺ co-factor (orange sphere) within the active site and the X/Y linker region (cyan) that occludes the active site also are shown. The approximate membrane-binding surface is indicated. Right panel, The structure is rotated 90° with respect to the left panel. This view emphasizes occlusion of the active site within the TIM barrel by the X/Y linker

Fig. 3.4

Mechanism of PLC-catalyzed PtdIns(4,5)P₂ hydrolysis. Top panel, The catalytic site of PLC-δ1 (Essen et al. 1996) is shown. The residues that ligate the soluble head group (Ins(1,4,5)P₃) of the substrate are colored in light blue. The residues that ligate the essential Ca²⁺ cofactor (yellow sphere) are colored in magenta. The residues that are essential for the acid-base mechanism of catalysis are colored in salmon. The oxygen atoms of the side chains are colored in red, and the nitrogen atoms of the side chains are colored in blue. Bottom panel, The mechanism of PtdIns(4,5)P₂ hydrolysis as proposed by Essen et al. (1996) is presented

Fig. 3.5

General model of auto-inhibition and activation of PLC isozymes. The model presents the general mechanism whereby a G protein, e.g., Gαq or Rac1, activates a PLC-β isozyme. Left side, The G protein (green toroid) is shown in an inactive GDP-bound state, and PLC-β is presented as a gold toroid except for its C-terminal (CT) domain (light pink) and X/Y linker (red cylinder and dotted lines). The CT domain of PLC-β basally associates with membranes, and the X/Y linker blocks the active site. Right side, GTP binding activates the G protein, the active G protein forms a complex with the main portion of PLC-β, the lipase active site is anchored and oriented at the membrane surface, and the X/Y linker is repulsed by the membrane surface therein freeing the active site to hydrolyze PtdIns(4,5)P₂ into diacylglycerol (DAG) and Ins(1,4,5)P₃ (IP₃)