Limited proteolysis of phospholipase C-<i>γ</i> 1 indicates stable association of X and Y domains with enhanced catalytic activity

Fernald, Amy W.; Jones, G A; Carpenter, Graham

doi:10.1042/bj3020503

Cited by 28 publications

(16 citation statements)

References 37 publications

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“…At pH 5, this SH2-SH2-SH3 lid is "open" and PI-PLC␥1 activity is high; at pH 7, the lid is "closed" and the enzyme has much lower activity. Consistent with this model is the previous observation that protease V8 cleavage activates the enzyme at pH 7, and the cleavage occurs within the lid between the SH3 and Y domains (28). Active enzyme at pH 5 can be inhibited by excess Ca 2ϩ sequestering the substrate PI; this kinetic effect depends on the detergent matrix used to solubilize PI.…”

Section: Discussionsupporting

confidence: 65%

Action of Phosphatidylinositol-specific Phospholipase Cγ1 on Soluble and Micellar Substrates

Zhou

Horstman

Carpenter

et al. 1999

Journal of Biological Chemistry

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The kinetics of PI-PLC␥1 toward a water-soluble substrate (inositol 1,2-cyclic phosphate, cIP) and phosphatidylinositol (PI) in detergent mixed micelles were monitored by 31 P NMR spectroscopy. That cIP is also a substrate (K m ‫؍‬ ϳ15 mM) implies a two-step mechanism (intramolecular phosphotransferase reaction to form cIP followed by cyclic phosphodiesterase activity to form inositol-1-phosphate (I-1-P)). PI is cleaved by PI-PLC␥1 to form cIP and I-1-P with the enzyme specific activity and ratio of products (cIP/I-1-P) regulated by assay temperature, pH, Ca 2؉ , and other amphiphilic additives. Cleavage of both cIP and PI by the enzyme is optimal at pH 5. The effect of Ca 2؉ on PI-PLC␥1 activity is unique compared with other isozymes enzymes: Ca 2؉ is necessary for the activity and low Ca 2؉ activates the enzyme; however, high Ca 2؉ inhibits PI-PLC␥1 hydrolysis of phosphoinositides (but not cIP) with the extent of inhibition dependent on pH, substrate identity (cIP or PI), substrate presentation (e.g. detergent matrix), and substrate surface concentration. This inhibition of PI-PLC␥1 by high Ca 2؉ is proposed to derive from the divalent metal ion-inducing clustering of the PI and reducing its accessibility to the enzyme. Amphiphilic additives such as phosphatidic acid, fatty acid, and sodium dodecylsulfate enhance PI cleavage in micelles at pH 7.5 but not at pH 5.0; they have no effect on cIP hydrolysis at either pH value. These different kinetic patterns are used to propose a model for regulation of the enzyme. A key hypothesis is that there is a pH-dependent conformational change in the enzyme that controls accessibility of the active site to both water-soluble cIP and interfacially organized PI. The low activity enzyme at pH 7.5 can be activated by PA (or phosphorylation by tyrosine kinase). However, this activation requires lipophilic substrate (PI) present because cIP hydrolysis is not enhanced in the presence of PA.Mammalian phosphoinositide-specific phospholipase C (PI-PLC) 1 enzymes are key components of PI-mediated signaling cascades in vivo (1, 2). These enzymes are subdivided into three main classes, PI-PLC-␤, -␥, and -␦, that share three conserved regions (3): (i) an N-terminal pleckstrin homology (PH) domain (these domains often have a high affinity for the phosphoinositide PIP 2 ), (ii) the X domain (ϳ170 amino acids), and (iii) the Y domain (ϳ260 amino acids). A C2 or Ca 2ϩ -lipid binding domain is also present at the C-terminal end of PI-PLC-␥ and -␦. In all cases, the X and Y domains are necessary for catalysis (4) with the other domains involved in regulating activity. For example, in the case of PI-PLC-␦, the PH domain plays an allosteric role in binding the protein to a bilayer (5). PH domains also bind ␤␥ G proteins very tightly (6), and PI-PLC-␤ activity is regulated by interactions with G q proteins (7,8).PI-PLC␥ isozymes are the largest of PI-PLC isozymes and are abundant in many tissues and cell types. The biological significance of the PI-PLC␥1 enzyme has been documented (2). When PI-P...

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Section: Discussionsupporting

confidence: 65%

Action of Phosphatidylinositol-specific Phospholipase Cγ1 on Soluble and Micellar Substrates

Zhou

Horstman

Carpenter

et al. 1999

Journal of Biological Chemistry

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“…For instance, protease cleavage of PLC-δ1 (Ellis et al, 1993), -β2 (Schnabel and Camps, 1998), or -γ1 (Fernald et al, 1994) within the X/Y linker results in a concomitant increase in phospholipase activity. In a different vein, active PLC-γ1 (Horstman et al, 1996) and -β2 (Zhang and Neer, 2001) can be reassembled by coexpression of the N- and C-terminal halves of these isozymes.…”

Section: Discussionmentioning

confidence: 99%

General and Versatile Autoinhibition of PLC Isozymes

et al. 2008

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SUMMARY 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-β2 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-β2 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.

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“…Specifically, limited proteolysis within PLC-δ1 (Ellis et al 1993), PLC-β2 (Schnabel and Camps 1998), PLC-γ1 (Fernald et al 1994), and PLC-ζ (Kurokawa et al 2007) targets the X/Y-linker and resulted in PLC isozymes that exhibit higher enzymatic activity. Similarly, independently expressed polypeptide chains encompassing the N-terminus to the X-box and the Y-box to the C-terminus of PLC-β2 (Zhang and Neer 2001) or PLC-γ1 (Horstman et al 1996) reassemble in the absence of their respective X/Y-linker regions and reconstitute as functional isozymes that exhibit higher basal activity than the holoenzymes.…”

Section: 6 Auto-inhibition Of Plc Isozymes By Their X/y-linkermentioning

confidence: 99%

The Phospholipase C Isozymes and Their Regulation

Gresset

Sondek

Harden

2012

Subcellular Biochemistry

168

138

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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)P2 to the Ca2+-mobilizing second messenger inositol(1,4,5)P3 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-δ to confer multiple modes of regulation of lipase activity. PLC-β isozymes are activated by Gαq- and Gβγ-subunits of heterotrimeric G proteins, and activation of PLC-γ isozymes occurs through phosphorylation promoted by receptor and non-receptor tyrosine kinases. PLC-ε and certain members of the PLC-β and PLC-γ 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.

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Limited proteolysis of phospholipase C-γ 1 indicates stable association of X and Y domains with enhanced catalytic activity

Cited by 28 publications

References 37 publications

Action of Phosphatidylinositol-specific Phospholipase Cγ1 on Soluble and Micellar Substrates

Action of Phosphatidylinositol-specific Phospholipase Cγ1 on Soluble and Micellar Substrates

General and Versatile Autoinhibition of PLC Isozymes

The Phospholipase C Isozymes and Their Regulation

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