Phospholipase Cβ3 Membrane Adsorption and Activation Are Regulated by Its C-Terminal Domains and Phosphatidylinositol 4,5-Bisphosphate

Abstract

Phospholipase Cβ (PLCβ) enzymes hydrolyze phosphatidylinositol 4,5-bisphosphate to produce second messengers that regulate intracellular Ca, cell proliferation, and survival. Their activity is dependent upon interfacial activation that occurs upon localization to cell membranes. However, the molecular basis for how these enzymes productively interact with the membrane is poorly understood. Herein, atomic force microscopy demonstrates that the ∼300-residue C-terminal domain promotes adsorption to monolayers and is required for spatial organization of the protein on the monolayer surface. PLCβ variants lacking this C-terminal domain display differences in their distribution on the surface. In addition, a previously identified autoinhibitory helix that binds to the PLCβ catalytic core negatively impacts membrane binding, providing an additional level of regulation for membrane adsorption. Lastly, defects in phosphatidylinositol 4,5-bisphosphate hydrolysis also alter monolayer adsorption, reflecting a role for the active site in this process. Together, these findings support a model in which multiple elements of PLCβ modulate adsorption, distribution, and catalysis at the cell membrane.

Phospholipase C families: Common themes and versatility in physiology and pathology

“…the distal C-terminal domain is a coiled-coil domain with highly conserved clusters of lysine and arginine residues arrayed along one face that function as a major membrane binding determinant. Indeed, the membrane acts as an allosteric activator of PLCs [198,199]. Thus conceptually, the model that PLCβ3 only hydrolyzes ORP4L-extracted lipid is at odds with our general understanding of how PLCβ enzymes work in general.…”

Direct observation of conformational dynamics of the PH domain in phospholipases Cɛ and β may contribute to subfamily-specific roles in regulation

“…PLC␤3-⌬892 and reduced PLC␤3-⌬892 E60C/V164C were purified as described previously (54). Oxidized PLC␤3-⌬892 E60C/V164C was purified similarly, but reducing agents were omitted in each step.…”

“…DSF assays were carried out as described previously (40,54), with minor modifications. Purified PLC⑀ (0.2-0.6 mg/ml) and PLC␤ variants (0.5 mg/ml) were incubated with 5ϫ SYPRO orange dye in the presence of 5 mM CaCl 2 .…”

“…PLC⑀ and PLC␤ activity was measured as described previously (24,38,54). The PLC⑀ PH-COOH variant was assayed at a final concentration of 0.075 ng/l, PH-C2 at 0.1-1 ng/l, and EF-COOH at 0.5 ng/l.…”

Characterization of the membrane interactions of phospholipase Cγ reveals key features of the active enzyme

“…Comprehensive analyses of some of the PLC enzymes also identified specific features involved in regulation of a particular family and suggested that membrane interactions could be quite complex, with multiple roles. In this respect, members of the PLC family have been studied most extensively; in addition to structural studies (13)(14)(15)(16)(17), regulation of these enzymes has been analyzed in the presence of different model membranes in vitro (21)(22)(23)(24)(25). In its basal state, PLC is autoinhibited by the XY-linker from the catalytic TIM barrel and by elements from the C-terminal domain, representing a unique feature of PLC enzymes.…”