Evaluation of vecabrutinib as a model for noncovalent BTK/ITK inhibition for treatment of chronic lymphocytic leukemia.

Covalent Bruton tyrosine kinase (BTK) inhibitors, such as ibrutinib, have proven to be highly beneficial in the treatment of chronic lymphocytic leukemia (CLL). Interestingly, the off-target inhibition of IL-2-inducible T-cell kinase (ITK) by ibrutinib may also play a role in modulating the tumor microenvironment, potentially enhancing the treatment benefit. However, resistance to covalently binding BTK inhibitors can develop as the result of a mutation in cysteine 481 of BTK (C481S), which prevents irreversible binding of the drugs. In the present study we performed preclinical characterization of vecabrutinib, a next-generation noncovalent BTK inhibitor that has ITK-inhibitory properties similar to those of ibrutinib. Unlike ibrutinib and other covalent BTK inhibitors, vecabrutinib showed retention of the inhibitory effect on C481S BTK mutants in vitro, similar to that of wild-type BTK. In the murine Eµ-TCL1 adoptive transfer model, vecabrutinib reduced tumor burden and significantly improved survival. Vecabrutinib treatment led to a decrease in CD8+ effector and memory T-cell populations, whereas the naive populations were increased. Of importance, vecabrutinib treatment significantly reduced the frequency of regulatory CD4+ T cells in vivo. Unlike ibrutinib, vecabrutinib treatment showed minimal adverse impact on the activation and proliferation of isolated T cells. Lastly, combination treatment with vecabrutinib and venetoclax augmented treatment efficacy, significantly improved survival, and led to favorable reprogramming of the microenvironment in the murine Eµ-TCL1 model. Thus, noncovalent BTK/ITK inhibitors, such as vecabrutinib, may be efficacious in C481S BTK mutant CLL while preserving the T-cell immunomodulatory function of ibrutinib.

Noncatalytic Bruton's tyrosine kinase activates PLCγ2 variants mediating ibrutinib resistance in human chronic lymphocytic leukemia cells.

Treatment of patients with chronic lymphocytic leukemia (CLL) with inhibitors of Bruton's tyrosine kinase (BTK), such as ibrutinib, is limited by primary or secondary resistance to this drug. Examinations of CLL patients with late relapses while on ibrutinib, which inhibits BTK's catalytic activity, revealed several mutations in BTK, most frequently resulting in the C481S substitution, and disclosed many mutations in PLCG2, encoding phospholipase C-γ2 (PLCγ2). The PLCγ2 variants typically do not exhibit constitutive activity in cell-free systems, leading to the suggestion that in intact cells they are hypersensitive to Rac family small GTPases or to the upstream kinases spleen-associated tyrosine kinase (SYK) and Lck/Yes-related novel tyrosine kinase (LYN). The sensitivity of the PLCγ2 variants to BTK itself has remained unknown. Here, using genetically-modified DT40 B lymphocytes, along with various biochemical assays, including analysis of PLCγ2-mediated inositol phosphate formation, inositol phospholipid assessments, fluorescence recovery after photobleaching (FRAP) static laser microscopy, and determination of intracellular calcium ([Ca2+] i ), we show that various CLL-specific PLCγ2 variants such as PLCγ2S707Y are hyper-responsive to activated BTK, even in the absence of BTK's catalytic activity and independently of enhanced PLCγ2 phospholipid substrate supply. At high levels of B-cell receptor (BCR) activation, which may occur in individual CLL patients, catalytically-inactive BTK restored the ability of the BCR to mediate increases in [Ca2+] i Because catalytically-inactive BTK is insensitive to active-site BTK inhibitors, the mechanism involving the noncatalytic BTK uncovered here may contribute to preexisting reduced sensitivity or even primary resistance of CLL to these drugs.

cAMP guided his way: a life for G protein-mediated signal transduction and molecular pharmacology-tribute to Karl H. Jakobs.

Karl H. Jakobs, former editor-in-chief of Naunyn-Schmiedeberg's Archives of Pharmacology and renowned molecular pharmacologist, passed away in April 2018. In this article, his scientific achievements regarding G protein-mediated signal transduction and regulation of canonical pathways are summarized. Particularly, the discovery of inhibitory G proteins for adenylyl cyclase, methods for the analysis of receptor-G protein interactions, GTP supply by nucleoside diphosphate kinases, mechanisms in phospholipase C and phospholipase D activity regulation, as well as the development of the concept of sphingosine-1-phosphate as extra- and intracellular messenger will presented. His seminal scientific and methodological contributions are put in a general and timely perspective to display and honor his outstanding input to the current knowledge in molecular pharmacology.

Functional and Phenotypic Characteristics of Human Leptin Receptor Mutations.

Several case series of extreme early-onset obesity due to mutations in the human leptin receptor (LEPR) gene have been reported. In this review we summarize published functional and phenotypic data on mutations in the human LEPR gene causing severe early-onset obesity. Additionally, we included data on six new cases from our obesity center. Literature research was performed using PubMed and OMIM. Functional relevance of mutations was estimated based on reported functional analysis, mutation size, and location, as well as phenotypic characteristics of affected patients. We identified 57 cases with 38 distinct LEPR mutations. We found severe early-onset obesity, hyperphagia, and hypogonadotropic hypogonadism as cardinal features of a complete loss of LEPR function. Other features, for example, metabolic disorders and recurring infections, were variable in manifestation. Obesity degree or other manifestations did not aggregate by genotype. Few patients underwent bariatric surgery with variable success. Most mutations occurred in the fibronectin III and cytokine receptor homology II domains, whereas none was found in cytoplasmic domain. In silico data were available for 25 mutations and in vitro data were available for four mutations, revealing residual activity in one case. By assessing provided information on the clinical phenotype, functional analysis, and character of the 38 mutations, we assume residual LEPR activity for five additional mutations. Functional in vitro analysis is necessary to confirm this assumption.

Functional characterization of phospholipase C-γ2 mutant protein causing both somatic ibrutinib resistance and a germline monogenic autoinflammatory disorder.

Depending on its occurrence in the germline or somatic context, a single point mutation, S707Y, of phospholipase C-γ2 (PLCγ2) gives rise to two distinct human disease states: acquired resistance of chronic lymphocytic leukemia cells (CLL) to inhibitors of Brutons´s tyrosine kinase (Btk) and dominantly inherited autoinflammation and PLCγ2-associated antibody deficiency and immune dysregulation, APLAID, respectively. The functional relationships of the PLCγ2S707Y mutation to other PLCG2 mutations causing (i) Btk inhibitor resistance of CLL cells and (ii) the APLAID-related human disease PLCγ2-associated antibody deficiency and immune dysregulation, PLAID, revealing different clinical characteristics including cold-induced urticaria, respectively, are currently incompletely understood. Here, we show that PLCγ2S707 point mutants displayed much higher activities at 37° C than the CLL Btk inhibitor resistance mutants R665W and L845F and the two PLAID mutants, PLCγ2Δ19 and PLCγ2Δ20-22. Combinations of CLL Btk inhibitor resistance mutations synergized to enhance PLCγ2 activity, with distinct functional consequences for different temporal orders of the individual mutations. Enhanced activity of PLCγ2S707Y was not observed in a cell-free system, suggesting that PLCγ2 activation in intact cells is dependent on regulatory rather than mutant-enzyme-inherent influences. Unlike the two PLAID mutants, PLCγ2S707Y was insensitive to activation by cooling and retained marked hyperresponsiveness to activated Rac upon cooling. In contrast to the PLAID mutants, which are insensitive to activation by endogenously expressed EGF receptors, the S707Y mutation markedly enhanced the stimulatory effect of EGF, explaining some of the pathophysiological discrepancies between immune cells of PLAID and APLAID patients in response to receptor-tyrosine-kinase activation.

Estimated prevalence of potentially damaging variants in the leptin gene.

BACKGROUND: Mutations in the leptin gene (LEP) can alter the secretion or interaction of leptin with its receptor, leading to extreme early-onset obesity. The purpose of this work was to estimate the prevalence of heterozygous and homozygous mutations in the leptin gene with the help of the Exome Aggregation Consortium (ExAC) database ( http://exac.broadinstitute.org/about ). RESULTS: The ExAC database encompasses exome sequencing data from 60,706 individuals. We searched for listed leptin variants and identified 36 missense, 1 in-frame deletion, and 3 loss-of-function variants. The functional relevance of these variants was assessed by the in silico prediction tools PolyPhen-2, Sorting Intolerant from Tolerant (SIFT), and Loss-Of-Function Transcript Effect Estimator (LOFTEE). PolyPhen-2 predicted 7 of the missense variants to be probably damaging and 10 to be possibly damaging. SIFT predicted 7 of the missense variants to be deleterious. Three loss-of-function variants were predicted by LOFTEE. Excluding double counts, we can summarize 21 variants as potentially damaging. Considering the allele count, we identified 31 heterozygous but no homozygous subjects with at least probably damaging variants. In the ExAC population, the estimated prevalence of heterozygous carriers of these potentially damaging variants was 1:2000. The probability of homozygosity was 1:15,000,000. We furthermore tried to assess the functionality of ExAC-listed leptin variants by applying a knowledge-driven approach. By this approach, additional 6 of the ExAC-listed variants were considered potentially damaging, increasing the number of heterozygous subjects to 58, the prevalence of heterozygosity to 1:1050, and the probability of homozygosity to 1:4,400,000. CONCLUSION: Using exome sequencing data from ExAC, in silico prediction tools and by applying a knowledge-driven approach, we identified 27 probably damaging variants in the leptin gene of 58 heterozygous subjects. With this information, we estimate the prevalence for heterozygosity at 1:1050 corresponding to an incidence of homozygosity of 1:4,400,000 in this large pluriethnic cohort.

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 Bruton's 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.

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.

Severe Early-Onset Obesity Due to Bioinactive Leptin Caused by a p.N103K Mutation in the Leptin Gene.

CONTEXT: Congenital leptin deficiency is a very rare cause of severe early-onset obesity. We recently characterized a mutation in the leptin gene (p.D100Y), which was associated with detectable leptin levels and bioinactivity of the hormone. CASE DESCRIPTION: We now describe two siblings, a 9-year-old girl and a 6-year-old boy with severe early-onset obesity and hyperphagia, both homozygous for a c.309C>A substitution in the leptin gene leading to a p.N103K amino acid exchange in the protein and detectable circulating levels of leptin. In vitro experiments in a heterologous cell system demonstrated that the mutated protein was biologically inactive. Treatment with sc recombinant human leptin led to rapid improvement of eating behavior and weight loss. CONCLUSIONS: Sequencing of the leptin gene may need to be considered in hyperphagic, severely obese children with detectable levels of circulating leptin.

Discovery and characterization of an endogenous CXCR4 antagonist.

CXCL12-CXCR4 signaling controls multiple physiological processes and its dysregulation is associated with cancers and inflammatory diseases. To discover as-yet-unknown endogenous ligands of CXCR4, we screened a blood-derived peptide library for inhibitors of CXCR4-tropic HIV-1 strains. This approach identified a 16 amino acid fragment of serum albumin as an effective and highly specific CXCR4 antagonist. The endogenous peptide, termed EPI-X4, is evolutionarily conserved and generated from the highly abundant albumin precursor by pH-regulated proteases. EPI-X4 forms an unusual lasso-like structure and antagonizes CXCL12-induced tumor cell migration, mobilizes stem cells, and suppresses inflammatory responses in mice. Furthermore, the peptide is abundant in the urine of patients with inflammatory kidney diseases and may serve as a biomarker. Our results identify EPI-X4 as a key regulator of CXCR4 signaling and introduce proteolysis of an abundant precursor protein as an alternative concept for chemokine receptor regulation.

Phenotypic analysis of chemokine-driven actin reorganization in primary human neutrophils.

The chemokine-driven activation of CXC-type chemokine receptors 1/2 (CXCR1/2) and the subsequent reorganization of the neutrophilic actin are early key events in the induction of neutrophil migration toward centers of inflammation. In this study, an image analysis algorithm was developed to detect subtle chemokine-induced changes in the actin cytoskeleton of primary human neutrophils. By this means, a discrete early step of neutrophil activation was dissected that could be initiated by concentrations of growth-related oncogen α (Gro-α) or interleukin-8 (IL-8) just above their resting-state plasma levels. The associated half-maximal effective concentration (EC50) values for Gro-α and IL-8 of 8 and 22 pM, respectively, are between two and three orders of magnitude below the so-far reported EC50 values of these chemokines for the induction of neutrophilic calcium release, integrin expression, degranulation, and receptor internalization. Sch527123, a known inhibitor of CXCR2 (KD=49 pM) and with a lower potency/affinity also of CXCR1 (KD=3.9 nM), antagonized actin remodeling with half-maximal inhibitory concentration (IC50) values of 400 pM for the CXCR2-specific agonist Gro-α and of 36 nM for the CXCR1/2-promiscuous agonist IL-8. This observation indicates that the here-described early step of chemokine-driven actin reorganization is modulated by both CXCR1 and CXCR2. Thus, the imaging-based assay format, as developed in this work, may be employed in a phenotypic screening campaign to identify inhibitors of an early step in CXCR1/2-induced neutrophilic chemotaxis.

C2-streptavidin mediates the delivery of biotin-conjugated tumor suppressor protein p53 into tumor cells.

We have previously generated a recombinant C2-streptavidin fusion protein for the delivery of biotin-labeled molecules of low molecular weight into the cytosol of mammalian cells. A nontoxic moiety of Clostridium botulinum C2 toxin mediates the cellular uptake, whereas the streptavidin unit serves as a binding platform for biotin-labeled cargo molecules. In the present study, we used the C2-streptavidin transporter to introduce biotin-conjugated p53 protein into various mammalian cell lines. The p53 tumor suppressor protein is inactivated in many human cancers by multiple mechanisms and therefore the restoration of its activity in tumor cells is of great therapeutic interest. Recombinant p53 was expressed in insect cells and biotin-labeled. Biotin-p53 retained its specific high-affinity DNA-binding as revealed by gel-shift analysis. Successful conjugation of biotin-p53 to the C2-streptavidin transporter was monitored by an overlay blot technique and confirmed by real-time surface plasmon resonance, providing a KD-value in the low nM range. C2-streptavidin significantly enhanced the uptake of biotin-p53 into African Green Monkey (Vero) epithelial cells as shown by flow cytometry. Using cell fractionation, the cytosolic translocation of biotin-p53 was detected in Vero cells as well as in HeLa cervix carcinoma cells. In line with this finding, confocal microscopy displayed cytoplasmic staining of biotin-p53 in HeLa and HL60 leukemia cells. Internalized biotin-p53 partially colocalized with early endosomes, as confirmed by confocal microscopy. In conclusion, our results demonstrate the successful conjugation of biotin-p53 to C2-streptavidin and its subsequent receptor-mediated endocytosis into different human tumor cell lines.

LARG links histamine-H1-receptor-activated Gq to Rho-GTPase-dependent signaling pathways.

Activation of heterotrimeric G proteins, such as G(12/13) and G(q), by cell surface receptors is coupled to the regulation of numerous cellular functions controlled by activated Rho GTPases. Previous studies have implicated the Rho guanine nucleotide exchange factor (RhoGEF) leukemia-associated RhoGEF (LARG) as a regulatory protein receiving stimulatory inputs from activated Gα(12/13) and Gα(q). However, the molecular mechanisms of the Gα(q)-mediated LARG activation are not fully understood and the structural elements of LARG involved in this process have remained unclear. In the present work, the specific coupling of the histamine H1 receptor (HRH1) exogenously expressed in COS-7 cells to G(q), but not to G(12/13), was used to conduct a detailed analysis of receptor- and Gα(q)-mediated LARG activation and to define its structural requirements. The results show that HRH1-mediated activation of the strictly Rho-dependent transcriptional activity of serum response factor requires the PDZ domain of LARG and can be mimicked by activated Gα(q)(Q209L). The functional interaction between activated Gα(q) and LARG requires no more than the catalytic DH-PH tandem of LARG, and is independent of PLCß activation and distinct from the mechanisms of Gα(q)-mediated p63RhoGEF and PLCß(3) activation. Activated Gα(q) physically interacts with the relevant portions of LARG in COS-7 cells and histamine causes activation of LARG in native HeLa cells endogenously expressing HRH1, G(q), and LARG. This work is the first positive demonstration of a stimulatory effect of LARG on the ability of a strictly G(q)-coupled receptor to cause activation of a Rho-GTPase-dependent signaling pathway.

Functional analysis of Rho GTPase activation and inhibition in a bead-based miniaturized format.

Extensive knowledge about protein-protein interactions is fundamental to fully understand signaling pathways and for the development of new drugs. Rho GTPases are key molecules in cellular signaling processes and their deregulation is implicated in the development of a variety of diseases such as neurofibromatosis type 2 and cancer. Here, we describe a bead-based protein-protein interaction assay for overexpressed HA-tagged Rho GTPases to study the GTPγS-dependent interaction with the regulatory protein RhoGDIα. This assay provides a useful tool for the analysis of both macromolecular and small molecule activators and inhibitors of the protein-protein interactions of Rho GTPases with their regulatory proteins in a multiplexed miniaturized format.

High-content analysis of CCR2 antagonists on human primary monocytes.

The monocyte chemoattractant protein 1 (MCP-1)-driven activation of CC-type chemokine receptor 2 (CCR2) is one of the early key events to induce monocyte migration toward centers of inflammation. In this work, the authors analyzed MCP-1 internalization into primary human monocytes using partially automated liquid handling, automated fluorescence microscopic imaging, and a specific image analysis algorithm. A fluorophore-conjugated form of MCP-1 was rapidly endocytosed and retained by the monocytes. The CCR2 dependency of the MCP-1 internalization was demonstrated by the use of BMS CCR2 22, a CCR2-specific antagonist. The apparent inhibitory potencies of a series of small-molecule CCR2 antagonists were determined and compared in five assay formats, including the high-content analysis assay described in this work. Interestingly, some but not all antagonists showed markedly different inhibitory behaviors in the five readout systems, with an up to more than 100-fold difference between the highest and the lowest apparent inhibitory potencies. These findings raise the distinct possibility that some CCR2 antagonists are capable of discriminating between different functional states of the CCR2 receptor(s) and suggest strategies for the identification of functionally selective CCR2 antagonists with increased therapeutic advantage over nonselective antagonists.

Bead-based protein-protein interaction assays for the analysis of Rho GTPase signaling.

Bead-based interaction assays are excellently suited to study protein-protein interactions, as they require only minimal amounts of sample material. Miniaturized protein-protein interaction assays were designed to analyze Rho GTPase activation based on its interaction with Rho GDI or p21-activated kinase (PAK).Rho GDI plays a key role in the regulation of a variety of cellular functions through its interaction with Rho GTPases. Rho GDI is frequently overexpressed in many human cancers. Therefore, there is a growing and as yet unfulfilled demand for screening assays to identify biologically active compounds that may inhibit the Rho GTPase-Rho GDI interaction. Bead-based interaction assays provide an interesting alternative that facilitate such assays to be performed faster with only small amounts of material compared to routinely used co-immunoprecipitation followed by Western Blot analysis.Bead-based protein interaction assays for overexpressed HA-tagged Rho GTPases were established to study the GTPγS-dependent interaction of five different Rho GTPases with the regulatory protein Rho GDIα and the downstream effector PAK1. In addition, it was demonstrated that the ability of Rho GTPases to interact with Rho GDI in this experimental system was markedly, but differentially sensitive to post-translational modification of their carboxyl terminus. Importantly, this modification also notably affected the ability of Rac1 and Rac2, but not of Cdc42, to interact with PAK1.

CXCR2 inverse agonism detected by arrestin redistribution.

To study CXCR2 modulated arrestin redistribution, the authors employed arrestin as a fusion protein containing either the Aequorea victoria-derived enhanced green fluorescent protein (EGFP) or a recently developed mutant of eqFP611, a red fluorescent protein derived from Entacmaea quadricolor. This mutant, referred to as RFP611, had earlier been found to assume a dimeric quarternary structure. It was therefore employed in this work as a "tandem" (td) construct for pseudo-monomeric fusion protein labeling. Both arrestin fusion proteins, containing either td-RFP611 (Arr-td-RFP611) or enhanced green fluorescent protein (EGFP; Arr-EGFP), were found to colocalize with internalized fluorescently labeled Gro-alpha a few minutes after Gro-alpha addition. Intriguingly, however, Arr-td-RFP611 and Arr-EGFP displayed distinct cellular distribution patterns in the absence of any CXCR2-activating ligand. Under these conditions, Arr-td-RFP611 showed a largely homogeneous cytosolic distribution, whereas Arr-EGFP segregated, to a large degree, into granular spots. These observations indicate a higher sensitivity of Arr EGFP to the constitutive activity of CXCR2 and, accordingly, an increased arrestin redistribution to coated pits and endocytic vesicles. In support of this interpretation, the authors found the known CXCR2 antagonist Sch527123 to act as an inverse agonist with respect to Arr-EGFP redistribution. The inverse agonistic properties of Sch527123 were confirmed in vitro in a guanine nucleotide binding assay, revealing an IC(50) value similar to that observed for Arr-EGFP redistribution. Thus, the redistribution assay, when based on Arr-EGFP, enables the profiling of antagonistic test compounds with respect to inverse agonism. When based on Arr-td-RFP611, the assay may be employed to study CXCR2 agonism or neutral antagonism.

In vitro reconstitution of activation of PLCepsilon by Ras and Rho GTPases.

Phosphatidylinositol-specific phospholipase C (PLC) enzymes catalyze the hydrolysis of phophatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] to diacylglycerol (DAG) and inositol 1,4,5-triphosphate [Ins(1,4,5)P3]. PLCepsilon is a recently discovered isoform that has been shown to be activated by members of the Ras and Rho families of guanosine trisphosphatases (GTPases) as well as subunits of heterotrimeric G-proteins. We describe a method for expressing a truncated PLCepsilon variant as an MBP fusion protein in E. coli. Subsequently, we describe the methodology necessary to reconstitute this protein with K-Ras-4B and RhoA GTPases and measure its activation.

rac regulates its effector phospholipase Cgamma2 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 PLCgamma isoforms, only PLCgamma(2) 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 PLCgamma(2) required for activation. Based on reconstitution experiments with isolated PLCgamma 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 PLCgamma(2) by Rac2. We also demonstrate that Rac2 directly binds to PLCgamma(2) 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 PLCgamma(1) and PLCgamma(2) and the implications of our findings for their respective signaling roles.

Structural and mechanistic insights into ras association domains of phospholipase C epsilon.

Ras proteins signal to a number of distinct pathways by interacting with diverse effectors. Studies of ras/effector interactions have focused on three classes, Raf kinases, ral guanylnucleotide-exchange factors, and phosphatidylinositol-3-kinases. Here we describe ras interactions with another effector, the recently identified phospholipase C epsilon (PLCepsilon). We solved structures of PLCepsilon RA domains (RA1 and RA2) by NMR and the structure of the RA2/ras complex by X-ray crystallography. Although the similarity between ubiquitin-like folds of RA1 and RA2 proves that they are homologs, only RA2 can bind ras. Some of the features of the RA2/ras interface are unique to PLCepsilon, while the ability to make contacts with both switch I and II regions of ras is shared only with phosphatidylinositol-3-kinase. Studies of PLCepsilon regulation suggest that, in a cellular context, the RA2 domain, in a mode specific to PLCepsilon, has a role in membrane targeting with further regulatory impact on PLC activity.