Activation of Phospholipase C δ1 through C2 Domain by a Ca2+-Enzyme-Phosphatidylserine Ternary Complex

Abstract

The concentration of free Ca 2؉ and the composition of nonsubstrate phospholipids profoundly affect the activity of phospholipase C ␦1 (PLC␦1). The rate of PLC␦1 hydrolysis of phosphatidylinositol 4,5-bisphosphate was stimulated 20-fold by phosphatidylserine (PS), 4-fold by phosphatidic acid (PA), and not at all by phosphatidylethanolamine or phosphatidylcholine (PC). PS reduced the Ca 2؉ concentration required for half-maximal activation of PLC␦1 from 5.4 to 0.5 M. Approximately 12 distinct isoforms of phospholipase C catalyze the Ca 2ϩ -dependent hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) 1 to yield the second messengers inositol trisphosphate (IP 3 ) and diacylglycerol (1, 2). This constitutes one of the major pathways for receptor-coupled signaling at the plasma membrane of most eucaryotic cells. Three families of PLC isoforms have been described in mammals: PLC␤, PLC␦, and PLC␥ (2). The members of each family are highly homologous to one another at the amino acid sequence level, but little identity exists between members of different families (2). Three exceptions to this divergence in structure are the catalytic domain, the C2 domain, and the N-terminal pleckstrin homology (PH) domain (1, 3).Among the initial steps in activation of PLC is a translocation to the plasma membrane. The enzyme binds to the lipidwater interface via the noncatalytic lipid binding PH domain, which is located near the N terminus of PLC␦1 and binds multiple phosphoinositides such as PIP 2 and IP 3 (4 -9). This domain allows the enzyme to catalyze the hydrolysis of many substrate molecules without falling off the interface, a process referred to as processive catalysis (5, 9). Because PH domain binds IP 3 tightly, it also could function as a feedback regulator of catalysis. Although much is known about the function of the PH domain in PLC␦1, very little is known about another lipid binding motif, the C2 domain. The C2 domain comprises approximately 130 amino acid residues and has been found in nearly 100 signaling molecules (10). The C2 domain was first identified in protein kinase C, and its function was implicated in Ca 2ϩ -dependent phospholipid interactions (11). Structural studies estimate three to four divalent metal binding sites in the C2 domain of PLC␦1 (12). The C2 domain in the C terminus of PLC␦1 is essential for catalysis, because partial deletion of the C2 domain results in an inactive enzyme (13). However, the molecular mechanism by which C2 domain functions in PLC␦1 catalysis still awaits further investigation.PLC is a prototype for enzymes that operate at an interface. As for other membrane-associated enzymes, the ability of PLC to catalyze the hydrolysis of PI or PIP 2 is influenced remarkably by the presence of nonsubstrate phospholipids in the membrane (14 -19). The molecular mechanisms by which nonsubstrate phospholipid affects PLC activity is largely unknown. Nonsubstrate phospholipids could alter the structure of the PLC-membrane interface or the net charge of the interface or could promote a ...

Stimulation of phospholipase Cβ by membrane interactions, interdomain movement, and G protein binding — How many ways can you activate an enzyme?

“…Although suppression of these binding sites does not impair the ability of PLC-δ1 to hydrolyze PI(4,5) P 2 [47]. It has been shown that PLC-δ1 exhibits a specific Ca 2+ -dependant binding to PEcontaining membranes through its C2 domain [48]. Since the C2 domain is almost integrated with the Y region, it is thought to assist in orienting the catalytic site of PLC-δ1 onto the membrane once bound through initial association by the PH domain (i.e.…”

“…This peptide was therefore defined as the signal transfer region of Gβ. Extensive studies with other peptides identified a second signal transfer region (Gβ [42][43][44][45][46][47][48][49][50][51][52][53][54] ) in the seventh blade of Gβ, and revealed segments in the second, fifth and seventh blade that hold the Gβγ/PLC-β2 complex together during activation ( Figure 5) [67,68]. In addition, it was found that the geranylgeranylated moiety attached to the C-terminal segment of the Gγ, which is proximal to the membrane and anchors Gβγ to lipids, binds to the PLC-β2 [69,70].…”

“…More striking is the identification of soluble fragments of Gβγ, Gβ [86][87][88][89][90][91][92][93][94][95][96][97][98][99][100][101][102][103][104][105] and Gβ [42][43][44][45][46][47][48][49][50][51][52][53][54] , that are able to convey full activation of PLC-β2 [66,67,79]. Additionally, Gβγ has been shown to stimulate the phosphodiesterase step of the PI(4,5)P 2 hydrolysis reaction which is not dependent on membrane surfaces, and further biochemical studies have shown that in the presence of Gβγ subunits, product inhibition does not occur suggesting that activation is through an enhanced release of product [19].…”

“…For example, the effect of membrane components on PLC activity other than PI lipids appear to be specific. Anionic lipids such as PS will increase PLC-δ1 activity [32,48] through specific interactions between PS and the C2 domain of the phospholipase [48], and interactions between the EF hands and free fatty acids also stimulate PLC-δ1 activity region [44]. Specific interactions PA and the C-terminal tail of PLC-β1 will promote its activation [83].…”

The Molecular Basis of Differential Subcellular Localization of C2 Domains of Protein Kinase C-α and Group IVa Cytosolic Phospholipase A2

“…Most Ca 2ϩ -dependent membrane binding C2 domains have higher affinity for anionic membranes than for zwitterionic ones. In particular, the C2 domains of PKC-␣ (PKC-␣-C2) and phospholipase C-␦1 show selectivity for phosphatidylserine (PS) (15)(16)(17). In contrast, the C2 domains of group IVa cytosolic phospholipase A 2 (cPLA 2 ) (18) and 5-lipoxygenase (19) strongly favor zwitterionic phosphatidylcholine (PC).…”