Claudia Walliser

Isozyme-specific Stimulation of Phospholipase C-γ2 by Rac GTPases

The regulation of the two isoforms of phospholipase C-γ, PLCγ1 and PLCγ2, by cell surface receptors involves protein tyrosine phosphorylation as well as interaction with adapter proteins and phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3) generated by inositol phospholipid 3-kinases (PI3Ks). All three processes may lead to recruitment of the PLCγ isozymes to the plasma membrane and/or stimulation of their catalytic activity. Recent evidence suggests that PLCγ may also be regulated by Rho GTPases. In this study, PLCγ1 and PLCγ2 were reconstituted in intact cells and in a cell-free system with Rho GTPases to examine their influence on PLCγ activity. PLCγ2, but not PLCγ1, was markedly activated in intact cells by constitutively active Rac1G12V, Rac2G12V, and Rac3G12V but not by Cdc42G12V and RhoAG14V. The mechanism of PLCγ2 activation was apparently independent of phosphorylation of tyrosine residues known to be modified by PLCγ2-activating protein-tyrosine kinases. Activation of PLCγ2 by Rac2G12V in intact cells coincided with a translocation of PLCγ2 from the soluble to the particulate fraction. PLCγ isozyme-specific activation of PLCγ2 by Rac GTPases (Rac1 ≈ Rac2 > Rac3), but not by Cdc42 or RhoA, was also observed in a cell-free system. Herein, activation of wild-type Rac GTPases with guanosine 5′-(3-O-thio)triphosphate caused a marked stimulation of PLCγ2 but had no effect on the activity of PLCγ1. PLCγ1 and PLCγ2 have previously been shown to be indiscriminately activated by PtdInsP3 in vitro. Thus, the results suggest a novel mechanism of PLCγ2 activation by Rac GTPases involving neither protein tyrosine phosphorylation nor PI3K-mediated generation of PtdInsP3.

Specificity and Structural Requirements of Phospholipase C-β Stimulation by Rho GTPases Versus G Protein βγ Dimers

Phospholipase C-β2(PLCβ2) is activated both by heterotrimeric G protein α- and βγ- subunits and by Rho GTPases. In this study, activated Rho GTPases are shown to stimulate PLCβ isozymes with the rank order of PLCβ2 > PLCβ3 ≥ PLCβ1. The sensitivity of PLCβ isozymes to Rho GTPases was clearly different from that observed for G protein βγ dimers, which decreased in the following order: PLCβ3 > PLCβ2 > PLCβ1 for β1γ1/2 and PLCβ2 > PLCβ1 >>> PLCβ3 for β5γ2. Rac1 and Rac2 were found to be more potent and efficacious activators of PLCβ2 than was Cdc42Hs. The stimulation of PLCβ2 by Rho GTPases and G protein βγ dimers was additive, suggesting that PLCβ2 activation can be augmented by independent regulation of the enzyme by the two stimuli. Using chimeric PLCβ1-PLCβ2 enzymes, βγ dimers, and Rho GTPases are shown to require different regions of PLCβ2 to mediate efficient stimulation of the enzyme. Although the catalytic subdomains X and Y of PLCβ2 were sufficient for efficient stimulation by βγ, the presence of the putative pleckstrin homology domain of PLCβ2 was absolutely required for the stimulation of the enzyme by Rho GTPases. Taken together, these results identify Rho GTPases as novel PLCβ regulators, which mediate PLCβ isozyme-specific stimulation and are potentially involved in coordinating the activation of PLCβ2 by extracellular mediators in intact cells.

Structural insights into formation of an active signaling complex between Rac and phospholipase C gamma 2.

Rho family GTPases are important cellular switches and control a number of physiological functions. Understanding the molecular basis of interaction of these GTPases with their effectors is crucial in understanding their functions in the cell. Here we present the crystal structure of the complex of Rac2 bound to the split pleckstrin homology (spPH) domain of phospholipase C-gamma(2) (PLCgamma(2)). Based on this structure, we illustrate distinct requirements for PLCgamma(2) activation by Rac and EGF and generate Rac effector mutants that specifically block activation of PLCgamma(2), but not the related PLCbeta(2) isoform. Furthermore, in addition to the complex, we report the crystal structures of free spPH and Rac2 bound to GDP and GTPgammaS. These structures illustrate a mechanism of conformational switches that accompany formation of signaling active complexes and highlight the role of effector binding as a common feature of Rac and Cdc42 interactions with a variety of effectors.

Rac Regulates Its Effector Phospholipase Cγ2 through Interaction with a Split Pleckstrin Homology Domain

Several isoforms of phospholipase C (PLC) are regulated through interactions with Ras superfamily GTPases, including Rac proteins. Interestingly, of two closely related PLCγ isoforms, only PLCγ2 has previously been shown to be activated by Rac. Here, we explore the molecular basis of this interaction as well as the structural properties of PLCγ2 required for activation. Based on reconstitution experiments with isolated PLCγ variants and Rac2, we show that an unusual pleckstrin homology (PH) domain, designated as the split PH domain (spPH), is both necessary and sufficient to effect activation of PLCγ2 by Rac2. We also demonstrate that Rac2 directly binds to PLCγ2 as well as to the isolated spPH of this isoform. Furthermore, through the use of NMR spectroscopy and mutational analysis, we determine the structure of spPH, define the structural features of spPH required for Rac interaction, and identify critical amino acid residues at the interaction interface. We further discuss parallels and differences between PLCγ1 and PLCγ2 and the implications of our findings for their respective signaling roles.

Differential regulation of phospholipase C-β2 activity and membrane interaction by Gαq, Gβ1γ2, and Rac2

We combined fluorescence recovery after photobleaching (FRAP) beam-size analysis with biochemical assays to investigate the mechanisms of membrane recruitment and activation of phospholipase C-β2 (PLCβ2) by G protein αq and βγ dimers. We show that activation by αq and βγ differ from activation by Rac2 and from each other. Stimulation by αq enhanced the plasma membrane association of PLCβ2, but not of PLCβ2Δ, which lacks the αq-interacting region. Although αq resembled Rac2 in increasing the contribution of exchange to the FRAP of PLCβ2 and in enhancing its membrane association, the latter effect was weaker than with Rac2. Moreover, the membrane recruitment of PLCβ2 by αq occurred by enhancing PLCβ2 association with fast-diffusing (lipid-like) membrane components, whereas stimulation by Rac2 led to interactions with slow diffusing membrane sites. On the other hand, activation by βγ shifted the FRAP of PLCβ2 and PLCβ2Δ to pure lateral diffusion 3- to 5-fold faster than lipids, suggesting surfing-like diffusion along the membrane. We propose that these different modes of PLCβ2 membrane recruitment may accommodate contrasting functional needs to hydrolyze phosphatidylinositol 4,5-bisphosphate (PtdInsP2) in localized versus dispersed populations. PLCβ2 activation by Rac2, which leads to slow lateral diffusion and much faster exchange, recruits PLCβ2 to act locally on PtdInsP2 at specific domains. Activation by αq leads to lipid-like diffusion of PLCβ2 accompanied by exchange, enabling the sampling of larger, yet limited, areas prior to dissociation. Finally, activation by βγ recruits PLCβ2 to the membrane by transient interactions, leading to fast “surfing” diffusion along the membrane, sampling large regions for dispersed PtdInsP2 populations.

Membrane Environment Exerts an Important Influence on Rac-Mediated Activation of Phospholipase Cγ2

ABSTRACT We performed analyses of the molecular mechanisms involved in the regulation of phospholipase Cγ2 (PLCγ2). We identified several regions in the PLCγ-specific array, γSA, that contribute to autoinhibition in the basal state by occlusion of the catalytic domain. While the activation of PLCγ2 by Rac2 requires stable translocation to the membrane, the removal of the domains required for membrane translocation in the context of an enzyme with impaired autoinhibition generated constitutive, highly active PLC in cells. We further tested the possibility that the interaction of PLCγ2 with its activator protein Rac2 was sufficient for activation through the release of autoinhibition. However, we found that Rac2 binding in the absence of lipid surfaces was not able to activate PLCγ2. Together with other observations, these data suggest that an important consequence of Rac2 binding and translocation to the membrane is that membrane proximity, on its own or together with Rac2, has a role in the release of autoinhibition, resulting in interfacial activation.

The Phospholipase Cγ2 Mutants R665W and L845F Identified in Ibrutinib-resistant Chronic Lymphocytic Leukemia Patients Are Hypersensitive to the Rho GTPase Rac2 Protein.

Mutations in the gene encoding phospholipase C-γ2 (PLCγ2) have been shown to be associated with resistance to targeted therapy of chronic lymphocytic leukemia (CLL) with the Brutons tyrosine kinase inhibitor ibrutinib. The fact that two of these mutations, R665W and L845F, imparted upon PLCγ2 an ∼2–3-fold ibrutinib-insensitive increase in the concentration of cytosolic Ca2+ following ligation of the B cell antigen receptor (BCR) led to the assumption that the two mutants exhibit constitutively enhanced intrinsic activity. Here, we show that the two PLCγ2 mutants are strikingly hypersensitive to activation by Rac2 such that even wild-type Rac2 suffices to activate the mutant enzymes upon its introduction into intact cells. Enhanced “basal” activity of PLCγ2 in intact cells is shown using the pharmacologic Rac inhibitor EHT 1864 and the PLCγ2F897Q mutation mediating Rac resistance to be caused by Rac-stimulated rather than by constitutively enhanced PLCγ2 activity. We suggest that R665W and L845F be referred to as allomorphic rather than hypermorphic mutations of PLCG2. Rerouting of the transmembrane signals emanating from BCR and converging on PLCγ2 through Rac in ibrutinib-resistant CLL cells may provide novel drug treatment strategies to overcome ibrutinib resistance mediated by PLCG2 mutations or to prevent its development in ibrutinib-treated CLL patients.

Rac-mediated Stimulation of Phospholipase Cγ2 Amplifies B Cell Receptor-induced Calcium Signaling

Background: Phospholipase Cγ2 (PLCγ2) is stimulated by Rac GTPases through direct protein-protein interaction. Results: The Rac-PLCγ2 interaction markedly enhances B cell-receptor-mediated Ca2+ mobilization and nuclear translocation of the Ca2+-regulated transcription factor NFAT in B cells. Conclusion: Rac-mediated stimulation of PLCγ2 activity amplifies B cell receptor-induced Ca2+ signaling. Significance: A specific Rac-resistant PLCγ2 variant is used to determine the physiological cell signaling relevance of a functional Rac-PLCγ2 interaction in an appropriate cellular context. The Rho GTPase Rac is crucially involved in controlling multiple B cell functions, including those regulated by the B cell receptor (BCR) through increased cytosolic Ca2+. The underlying molecular mechanisms and their relevance to the functions of intact B cells have thus far remained unknown. We have previously shown that the activity of phospholipase Cγ2 (PLCγ2), a key constituent of the BCR signalosome, is stimulated by activated Rac through direct protein-protein interaction. Here, we use a Rac-resistant mutant of PLCγ2 to functionally reconstitute cultured PLCγ2-deficient DT40 B cells and to examine the effects of the Rac-PLCγ2 interaction on BCR-mediated changes of intracellular Ca2+ and regulation of Ca2+-regulated and nuclear-factor-of-activated-T-cell-regulated gene transcription at the level of single, intact B cells. The results show that the functional Rac-PLCγ2 interaction causes marked increases in the following: (i) sensitivity of B cells to BCR ligation; (ii) BCR-mediated Ca2+ release from intracellular stores; (iii) Ca2+ entry from the extracellular compartment; and (iv) nuclear translocation of the Ca2+-regulated nuclear factor of activated T cells. Hence, Rac-mediated stimulation of PLCγ2 activity serves to amplify B cell receptor-induced Ca2+ signaling.

Cool-temperature-mediated activation of phospholipase C-γ2 in the human hereditary disease PLAID

Deletions in the gene encoding signal-transducing inositol phospholipid-specific phospholipase C-γ2 (PLCγ2) are associated with the novel human hereditary disease PLAID (PLCγ2-associated antibody deficiency and immune dysregulation). PLAID is characterized by a rather puzzling concurrence of augmented and diminished functions of the immune system, such as cold urticaria triggered by only minimal decreases in temperature, autoimmunity, and immunodeficiency. Understanding of the functional effects of the genomic alterations at the level of the affected enzyme, PLCγ2, is currently lacking. PLCγ2 is critically involved in coupling various cell surface receptors to regulation of important functions of immune cells such as mast cells, B cells, monocytes/macrophages, and neutrophils. PLCγ2 is unique by carrying three Src (SH) and one split pleckstrin homology domain (spPH) between the two catalytic subdomains (spPHn-SH2n-SH2c-SH3-spPHc). Prevailing evidence suggests that activation of PLCγ2 is primarily due to loss of SH-region-mediated autoinhibition and/or enhanced plasma membrane translocation. Here, we show that the two PLAID PLCγ2 mutants lacking portions of the SH region are strongly (>100-fold), rapidly, and reversibly activated by cooling by only a few degrees. We found that the mechanism(s) underlying PLCγ2 PLAID mutant activation by cool temperatures is distinct from a mere loss of SH-region-mediated autoinhibition and dependent on both the integrity and the pliability of the spPH domain. The results suggest a new mechanism of PLCγ activation with unique thermodynamic features and assign a novel regulatory role to its spPH domain. Involvement of this mechanism in other human disease states associated with cooling such as exertional asthma and certain acute coronary events appears an intriguing possibility.

Activated PLCγ Breaking Loose

In this issue of Structure, Bunney and colleagues use a combination of NMR, SAXS, crystallography, ITC, and biochemical methods to elucidate, in molecular detail, the sequence of events causing receptor-mediated activation of phospholipase C-γ(1) by protein tyrosine phosphorylation.