Rac2 Regulation of Phospholipase C-β2 Activity and Mode of Membrane Interactions in Intact Cells

Abstract

Phospholipase C-␤ (PLC␤) isozymes play important roles in transmembrane signaling. Their activity is regulated by heterotrimeric G proteins. The PLC␤ 2 isozyme is unique in being stimulated also by Rho GTPases (Rac and Cdc42). However, the mechanism(s) of this stimulation are still unclear. Here, we employed fluorescence recovery after photobleaching to investigate the interaction of green fluorescent protein (GFP)-PLC␤ 2 with the plasma membrane. For either GFP-PLC␤ 2 or GFP-PLC␤ 2 ⌬, a C-terminal deletion mutant lacking the region required for stimulation by G␣ q , these interactions were characterized by a mixture of exchange with a cytoplasmic pool and lateral diffusion. Constitutively active Rac2(12V) stimulated the activity of both GFP-PLC␤ 2 and GFP-PLC␤ 2 ⌬ in live cells, and enhanced their membrane association as evidenced by the marked reduction in their fluorescence recovery rates. Both effects required the putative N-terminal pleckstrin homology (PH) domain of PLC␤ 2 . Importantly, Rac2(12V) dramatically increased the contribution of exchange to the fluorescence recovery of GFP-PLC␤ 2 , but had the opposite effect on GFP-PLC␤ 2 ⌬, where lateral diffusion became dominant. Our results demonstrate for the first time the regulation of membrane association of a PLC␤ isozyme by a GTP-binding protein and assign a novel function to the PLC␤ 2 C-terminal region, regulating its exchange between membrane-bound and cytosolic states.The activity of phospholipase C-␤ (PLC␤) 1 enzymes that hydrolyze phosphatidylinositol 4,5-bisphosphate (PtdInsP 2 ) is stimulated with different orders of efficacy by G protein ␣ q subunits and by G protein ␤␥ dimers (1-3). In addition, the PLC␤ 2 isozyme is specifically activated in vitro by the Rho GTPases Rac and Cdc42, but not by RhoA (4 -6). As for all PLC␤ isozymes, activation by ␣ q requires the C-terminal region of PLC␤ 2 , and mutants carrying deletions in this region, such as the mutant PLC␤ 2 ⌬ that lacks the Phe 819 -Glu 1166 segment, are resistant to stimulation by ␣ q but are susceptible to activation by Rho GTPases and G protein ␤␥ subunits (3, 4, 7). Recent studies (4 -6) show that ␤␥ dimers and Rho GTPases activate PLC␤ 2 by interacting with different regions of the effector enzyme. Thus, the PLC␤ 2 catalytic subdomains X and Y are sufficient for efficient stimulation by ␤␥, whereas the putative pleckstrin homology (PH) domain of PLC␤ 2 is absolutely required for stimulation by Rho GTPases (6). Among the Rho GTPases, Rac1 and Rac2 are more potent stimulators than Cdc42 (6). Evidence for a tight connection between PLC␤ 2 and Rho GTPases in cells is provided by the chemoattractant receptor system, whose activation stimulates PLC␤ 2 and Rac1, Rac2 and Cdc42 (8 -11). Moreover, in accord with our in vitro studies on PLC␤ 2 activation by Rho GTPases (4, 6), a recent study conducted on myeloid-differentiated HL-60 cells demonstrated that dominant-negative Cdc42 disrupted the stimulation of inositol 1,4,5-trisphosphate formation mediated via the chemoattractant receptors whi...

Differential Regulation of Phospholipase C-β2 Activity and Membrane Interaction by Gαq, Gβ1γ2, and Rac2

“…PLC␦ 1 and the Dictyostelium discoideum CRAC protein) were found to undergo dynamic membrane-cytoplasm exchange along with lateral diffusion over short distances (35,36). Interestingly, we found a similar behavior for PLC␤ 2 (7), although this protein is likely to interact with the membrane through several distinct sites; these include the catalytic triosephosphate isomerase barrel, the C-terminal region, and possibly the EF hands motif and the C2 domain, as well as its PH domain, which, unlike that of PLC␦, appears to be unable to bind phosphoinositides (16,18).…”

“…Cell Culture and Transfection-Cells were grown in Dulbecco's modified Eagle's medium with 10% fetal calf serum (7). For FRAP experiments, COS-7 cells grown on glass coverslips in 35-mm dishes for 24 h were transfected using DEAE-dextran (46) with 150 ng of plasmid DNA encoding one of the PLC␤ 2 -GFP derivatives together with 850 ng of empty vector or expression vectors encoding the various activating proteins: human ␤ 1 along with bovine ␥ 2 (wt) (␤ 1 ␥ 2 ), ␤ 1 plus the isoprenylationdefective ␥ 2 (C68S) (␤ 1 ␥ 2 (C68S)), murine ␣ q (wt), constitutively active ␣ q (R183C), and human Rac2(wt) or Rac2(G12V).…”

“…-Glu 1166 segment) are resistant to stimulation by ␣ q , but undergo activation by ␤␥ and Rac/Cdc42 (7,(13)(14)(15). Recent results show that ␤␥ and Rac/Cdc42 activate PLC␤ 2 by interacting, at least in part, with different regions of the effector enzyme.…”

“…We have formerly studied the interactions of green fluorescent protein (GFP)-tagged PLC␤ 2 (PLC␤ 2 -GFP) and PLC␤ 2 ⌬-GFP with the plasma membrane in live cells (7). Using FRAP beam-size analysis (37), which discriminates between recovery by lateral diffusion and exchange, we demonstrated that activation by Rac2 enhances the association of PLC␤ 2 and PLC␤ 2 ⌬ with the plasma membrane via binding to slow diffusing membrane proteins.…”

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

“…Rac1, 2, 3, and to a lesser extent Cdc42Hs, were shown to activate PLC-β2 and β3 [60,61] by binding to their PH domains [11,61]. This binding was thought to activate the PLC-βs by recruiting them to the membrane surface [62]. In contrast, heterotrimeric G proteins activate PLC-βs without promoting its binding to membranes [37,39] and also without affecting the calcium dependence of its activity [10,37,38].…”