Treatment of Relapsing HPV Diseases by Restored Function of Natural Killer Cells.

Human papillomavirus (HPV) infections underlie a wide spectrum of both benign and malignant epithelial diseases. In this report, we describe the case of a young man who had encephalitis caused by herpes simplex virus during adolescence and currently presented with multiple recurrent skin and mucosal lesions caused by HPV. The patient was found to have a pathogenic germline mutation in the X-linked interleukin-2 receptor subunit gamma gene (IL2RG), which was somatically reverted in T cells but not in natural killer (NK) cells. Allogeneic hematopoietic-cell transplantation led to restoration of NK cytotoxicity, with normalization of the skin microbiome and persistent remission of all HPV-related diseases. NK cytotoxicity appears to play a role in containing HPV colonization and the ensuing HPV-related hyperplastic or dysplastic lesions. (Funded by the National Institutes of Health and the Herbert Irving Comprehensive Cancer Center Flow Cytometry Shared Resources.).

A post-transcriptional regulon controlled by TtpA, the single tristetraprolin family member expressed in Dictyostelium discoideum.

Post-transcriptional processes mediated by mRNA binding proteins represent important control points in gene expression. In eukaryotes, mRNAs containing specific AU-rich motifs are regulated by binding of tristetraprolin (TTP) family tandem zinc finger proteins, which promote mRNA deadenylation and decay, partly through interaction of a conserved C-terminal CNOT1 binding (CNB) domain with CCR4-NOT protein complexes. The social ameba Dictyostelium discoideum shared a common ancestor with humans more than a billion years ago, and expresses only one TTP family protein, TtpA, in contrast to three members expressed in humans. Evaluation of ttpA null-mutants identified six transcripts that were consistently upregulated compared to WT during growth and early development. The 3'-untranslated regions (3'-UTRs) of all six 'TtpA-target' mRNAs contained multiple TTP binding motifs (UUAUUUAUU), and one 3'-UTR conferred TtpA post-transcriptional stability regulation to a heterologous mRNA that was abrogated by mutations in the core TTP-binding motifs. All six target transcripts were upregulated to similar extents in a C-terminal truncation mutant, in contrast to less severe effects of analogous mutants in mice. All six target transcripts encoded probable membrane proteins. In Dictyostelium, TtpA may control an 'RNA regulon', where a single RNA binding protein, TtpA, post-transcriptionally co-regulates expression of several functionally related proteins.

Prospective Study of a Novel, Radiation-Free, Reduced-Intensity Bone Marrow Transplantation Platform for Primary Immunodeficiency Diseases.

Allogeneic blood or marrow transplantation (BMT) is a potentially curative therapy for patients with primary immunodeficiency (PID). Safe and effective reduced-intensity conditioning (RIC) approaches that are associated with low toxicity, use alternative donors, and afford good immune reconstitution are needed to advance the field. Twenty PID patients, ranging in age from 4 to 58 years, were treated on a prospective clinical trial of a novel, radiation-free and serotherapy-free RIC, T-cell-replete BMT approach using pentostatin, low-dose cyclophosphamide, and busulfan for conditioning with post-transplantation cyclophosphamide-based graft-versus-host-disease (GVHD) prophylaxis. This was a high-risk cohort with a median hematopoietic cell transplantation comorbidity index of 3. With median follow-up of survivors of 1.9 years, 1-year overall survival was 90% and grade III to IV acute GVHD-free, graft-failure-free survival was 80% at day +180. Graft failure incidence was 10%. Split chimerism was frequently observed at early post-BMT timepoints, with a lower percentage of donor T cells, which gradually increased by day +60. The cumulative incidences of grade II to IV and grade III to IV acute GVHD (aGVHD) were 15% and 5%, respectively. All aGVHD was steroid responsive. No patients developed chronic GVHD. Few significant organ toxicities were observed. Evidence of phenotype reversal was observed for all engrafted patients, even those with significantly mixed chimerism (n = 2) or with unknown underlying genetic defect (n = 3). All 6 patients with pre-BMT malignancies or lymphoproliferative disorders remain in remission. Most patients have discontinued immunoglobulin replacement. All survivors are off immunosuppression for GVHD prophylaxis or treatment. This novel RIC BMT approach for patients with PID has yielded promising results, even for high-risk patients.

Five Residues in the Apical Loop of the Respiratory Syncytial Virus Fusion Protein F2 Subunit Are Critical for Its Fusion Activity.

The respiratory syncytial virus (RSV) fusion (F) protein is a trimeric, membrane-anchored glycoprotein capable of mediating both virus-target cell membrane fusion to initiate infection and cell-cell fusion, even in the absence of the attachment glycoprotein. The F protein is initially expressed in a precursor form, whose functional capabilities are activated by proteolysis at two sites between the F1 and F2 subunits. This cleavage results in expression of the metastable and high-energy prefusion conformation. To mediate fusion, the F protein is triggered by an unknown stimulus, causing the F1 subunit to refold dramatically while F2 changes minimally. Hypothesizing that the most likely site for interaction with a target cell component would be the top, or apex, of the protein, we determined the importance of the residues in the apical loop of F2 by alanine scanning mutagenesis analysis. Five residues were not important, two were of intermediate importance, and all four lysines and one isoleucine were essential. Alanine replacement did not result in the loss of the pre-F conformation for any of these mutants. Each of the four lysines required its specific charge for fusion function. Alanine replacement of the three essential lysines on the ascent to the apex hindered fusion following a forced fusion event, suggesting that these residues are involved in refolding. Alanine mutations at Ile64, also on the ascent to the apex, and Lys75 did not prevent fusion following forced triggering, suggesting that these residues are not involved in refolding and may instead be involved in the natural triggering of the F protein.IMPORTANCE RSV infects virtually every child by the age of 3 years, causing nearly 33 million acute lower respiratory tract infections (ALRI) worldwide each year in children younger than 5 years of age (H. Nair et al., Lancet 375:1545-1555, 2010). RSV is also the second leading cause of respiratory system-related death in the elderly (A. R. Falsey and E. E. Walsh, Drugs Aging 22:577-587, 2005; A. R. Falsey, P. A. Hennessey, M. A. Formica, C. Cox, and E. E. Walsh, N Engl J Med 352:1749-1759, 2005). The monoclonal antibody palivizumab is approved for prophylactic use in some at-risk infants, but healthy infants remain unprotected. Furthermore, its expense limits its use primarily to developed countries. No vaccine or effective small-molecule drug is approved for preventing disease or treating infection (H. M. Costello, W. Ray, S. Chaiwatpongsakorn, and M. E. Peeples, Infect Disord Drug Targets, 12:110-128, 2012). The essential residues identified in the apical domain of F2 are adjacent to the apical portion of F1, which, upon triggering, refolds into a long heptad repeat A (HRA) structure with the fusion peptide at its N terminus. These essential residues in F2 are likely involved in triggering and/or refolding of the F protein and, as such, may be ideal targets for antiviral drug development.

Functional equivalence of an evolutionarily conserved RNA binding module.

Members of the tristetraprolin (TTP) family of proteins participate in the regulation of mRNA turnover after initially binding to AU-rich elements in target mRNAs. Related proteins from most groups of eukaryotes contain a conserved tandem zinc finger (TZF) domain consisting of two closely spaced, similar CCCH zinc fingers that form the primary RNA binding domain. There is considerable sequence variation within the TZF domains from different family members within a single organism and from different organisms, raising questions about sequence-specific effects on RNA binding and decay promotion. We hypothesized that TZF domains from evolutionarily distant species are functionally interchangeable. The single family member expressed in the fission yeast Schizosaccharomyces pombe, Zfs1, promotes the turnover of several dozen transcripts, some of which are involved in cell-cell interactions. Using knockin techniques, we replaced the TZF domain of S. pombe Zfs1 with the equivalent domains from human TTP and the single family member proteins expressed in the silkworm Bombyx mori, the pathogenic yeast Candida guilliermondii, and the plant Chromolaena odorata. We found that the TZF domains from these widely disparate species could completely substitute for the native S. pombe TZF domain, as determined by measurement of target transcript levels and the flocculation phenotype characteristic of Zfs1 deletion. Recombinant TZF domain peptides from several of these species bound to an AU-rich RNA oligonucleotide with comparably high affinity. We conclude that the TZF domains from TTP family members in these evolutionarily widely divergent species are functionally interchangeable in mRNA binding and decay.

Post-transcriptional regulation of transcript abundance by a conserved member of the tristetraprolin family in Candida albicans.

Members of the tristetraprolin (TTP) family of CCCH tandem zinc finger proteins bind to AU-rich regions in target mRNAs, leading to their deadenylation and decay. Family members in Saccharomyces cerevisiae influence iron metabolism, whereas the single protein expressed in Schizosaccharomyces pombe, Zfs1, regulates cell-cell interactions. In the human pathogen Candida albicans, deep sequencing of mutants lacking the orthologous protein, Zfs1, revealed significant increases (> 1.5-fold) in 156 transcripts. Of these, 113 (72%) contained at least one predicted TTP family member binding site in their 3'UTR, compared with only 3 of 56 (5%) down-regulated transcripts. The zfs1Δ/Δ mutant was resistant to 3-amino-1,2,4-triazole, perhaps because of increased expression of the potential target transcript encoded by HIS3. Sequences of the proteins encoded by the putative Zfs1 targets were highly conserved among other species within the fungal CTG clade, while the predicted Zfs1 binding sites in these mRNAs often 'disappeared' with increasing evolutionary distance from the parental species. C. albicans Zfs1 bound to the ideal mammalian TTP binding site with high affinity, and Zfs1 was associated with target transcripts after co-immunoprecipitation. Thus, the biochemical activities of these proteins in fungi are highly conserved, but Zfs1-like proteins may target different transcripts in each species.

General and versatile autoinhibition of PLC isozymes.

Phospholipase C (PLC) isozymes are directly activated by heterotrimeric G proteins and Ras-like GTPases to hydrolyze phosphatidylinositol 4,5-bisphosphate into the second messengers diacylglycerol and inositol 1,4,5-trisphosphate. Although PLCs play central roles in myriad signaling cascades, the molecular details of their activation remain poorly understood. As described here, the crystal structure of PLC-beta2 illustrates occlusion of the active site by a loop separating the two halves of the catalytic TIM barrel. Removal of this insertion constitutively activates PLC-beta2 without ablating its capacity to be further stimulated by classical G protein modulators. Similar regulation occurs in other PLC members, and a general mechanism of interfacial activation at membranes is presented that provides a unifying framework for PLC activation by diverse stimuli.

Mutational and structural analysis of the tandem zinc finger domain of tristetraprolin.

Tristetraprolin (TTP), the best known member of a class of tandem (R/K)YKTELCX8CX5CX3H zinc finger proteins, can destabilize target mRNAs by first binding to AU-rich elements (AREs) in their 3'-untranslated regions (UTRs) and subsequently promoting deadenylation and ultimate destruction of those mRNAs. This study sought to determine the roles of selected amino acids in the RNA binding domain, known as the tandem zinc finger (TZF) domain, in the ability of the full-length protein to bind to AREs within the tumor necrosis factor α (TNF) mRNA 3'-UTR. Within the CX8C region of the TZF domain, mutation of some of the residues specific to TTP, not found in other members of the TTP protein family, resulted in decreased binding to RNA as well as inhibited mRNA deadenylation and decay. Evaluation of simulation solution models revealed a distinct structure in the second zinc finger of TTP that was induced by the presence of these TTP-specific residues. In addition, mutations within the lead-in sequences preceding the first C of highly conserved residues within the CX5C or CX3H regions or within the linker region between the two fingers also perturbed both RNA binding and the simulation model of the TZF domain in complex with RNA. We conclude that, although the majority of conserved residues within the TZF domain of TTP are required for productive binding, not all residues at sequence-equivalent positions in the two zinc fingers of the TZF domain of TTP are functionally equivalent.

RNA-Binding Protein-Mediated mRNA Deadenylation in Mammalian Cell Extracts.

Removal of the poly(A) tail, or deadenylation, is a crucial step in destabilizing mRNAs in eukaryotes. In this chapter, we describe a cell-free deadenylation assay that uses cytoplasmic cell extracts from human HEK293 cells transiently transfected with DNA encoding RNA-binding proteins (RBP), and in vitro-transcribed, radiolabeled, RNA probes. We include methods to evaluate the effects of RBPs or deadenylases on various in vitro-transcribed probes, with or without poly(A) tails. Finally, we also demonstrate the adaptability of these assays to test purified protein components in our cell-free deadenylation assay. In our experience, these methods are well suited for the initial assessment of the effects of RBPs on the deadenylation of mRNAs.

Acireductone dioxygenase 1 (ARD1) is an effector of the heterotrimeric G protein beta subunit in Arabidopsis.

Heterotrimeric G protein complexes are conserved from plants to mammals, but the complexity of each system varies. Arabidopsis thaliana contains one Gα, one Gß (AGB1), and at least three Gγ subunits, allowing it to form three versions of the heterotrimer. This plant model is ideal for genetic studies because mammalian systems contain hundreds of unique heterotrimers. The activation of these complexes promotes interactions between both the Gα subunit and the Gßγ dimer with enzymes and scaffolds to propagate signaling to the cytoplasm. However, although effectors of Gα and Gß are known in mammals, no Gß effectors were previously known in plants. Toward identifying AGB1 effectors, we genetically screened for dominant mutations that suppress Gß-null mutant (agb1-2) phenotypes. We found that overexpression of acireductone dioxygenase 1 (ARD1) suppresses the 2-day-old etiolated phenotype of agb1-2. ARD1 is homologous to prokaryotic and eukaryotic ARD proteins; one function of ARDs is to operate in the methionine salvage pathway. We show here that ARD1 is an active metalloenzyme, and AGB1 and ARD1 both control embryonic hypocotyl length by modulating cell division; they also may contribute to the production of ethylene, a product of the methionine salvage pathway. ARD1 physically interacts with AGB1, and ARD enzymatic activity is stimulated by AGB1 in vitro. The binding interface on AGB1 was deduced using a comparative evolutionary approach and tested using recombinant AGB1 mutants. A possible mechanism for AGB1 activation of ARD1 activity was tested using directed mutations in a loop near the substrate-binding site.

Direct activation of human phospholipase C by its well known inhibitor u73122.

Phospholipase C (PLC) enzymes are an important family of regulatory proteins involved in numerous cellular functions, primarily through hydrolysis of the polar head group from inositol-containing membrane phospholipids. U73122 (1-(6-((17ß-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione), one of only a few small molecules reported to inhibit the activity of these enzymes, has been broadly applied as a pharmacological tool to implicate PLCs in diverse experimental phenotypes. The purpose of this study was to develop a better understanding of molecular interactions between U73122 and PLCs. Hence, the effects of U73122 on human PLCß3 (hPLCß3) were evaluated in a cell-free micellar system. Surprisingly, U73122 increased the activity of hPLCß3 in a concentration- and time-dependent manner; up to an 8-fold increase in enzyme activity was observed with an EC50=13.6±5 µm. Activation of hPLCß3 by U73122 required covalent modification of cysteines as evidenced by the observation that enzyme activation was attenuated by thiol-containing nucleophiles, l-cysteine and glutathione. Mass spectrometric analysis confirmed covalent reaction with U73122 at eight cysteines, although maximum activation was achieved without complete alkylation; the modified residues were identified by LC/MS/MS peptide sequencing. Interestingly, U73122 (10 µm) also activated hPLCγ1 (>10-fold) and hPLCß2 (∼2-fold); PLCδ1 was neither activated nor inhibited. Therefore, in contrast to its reported inhibitory potential, U73122 failed to inhibit several purified PLCs. Most of these PLCs were directly activated by U73122, and a simple mechanism for the activation is proposed. These results strongly suggest a need to re-evaluate the use of U73122 as a general inhibitor of PLC isozymes.

Backbone and sidechain 1H, 15N and 13C resonance assignments of the free and RNA-bound tandem zinc finger domain of the tristetraprolin family member from Selaginella moellendorffii.

Members of the tristetraprolin (TTP) family of RNA binding proteins (RBPs) regulate the metabolism of a variety of mRNA targets. In mammals, these proteins modulate many physiological processes, including immune cell activation, hematopoiesis, and embryonic development. Regulation of mRNA stability by these proteins requires that the tandem zinc finger (TZF) domain binds initially and directly to target mRNAs, ultimately leading to their deadenylation and decay. Proteins of this type throughout eukarya possess a highly conserved TZF domain, suggesting that they are all capable of high-affinity RNA binding. However, the mechanism of TTP-mediated mRNA decay is largely undefined. Given the vital role that these TTP family proteins play in maintaining RNA homeostasis throughout eukaryotes, we focused here on the first, key step in this process: recognition and binding of the TZF domain to target RNA. For these studies, we chose a primitive plant, the spikemoss Selaginella moellendorffii, which last shared a common ancestor with humans more than a billion years ago. Here we report the near complete backbone and side chain resonance assignments of the spikemoss TZF domain, including: (1) the assignment of the RNA-TZF domain complex, representing one of only two data sets currently available for the entire TTP family of proteins; and (2) the first NMR resonance assignments of the entire TZF domain, in the RNA-free form. This work will serve as the basis for further NMR structural investigations aimed at gaining insights into the process of RNA recognition and the mechanisms of TTP-mediated mRNA decay.

Mechanism of phosphorylation-induced activation of phospholipase C-gamma isozymes.

The lipase activity of most phospholipases C (PLCs) is basally repressed by a highly degenerate and mostly disordered X/Y linker inserted within the catalytic domain. Release of this auto-inhibition is driven by electrostatic repulsion between the plasma membrane and the electronegative X/Y linker. In contrast, PLC-γ isozymes (PLC-γ1 and -γ2) are structurally distinct from other PLCs because multiple domains are present in their X/Y linker. Moreover, although many tyrosine kinases directly phosphorylate PLC-γ isozymes to enhance their lipase activity, the underlying molecular mechanism of this activation remains unclear. Here we define the mechanism for the unique regulation of PLC-γ isozymes by their X/Y linker. Specifically, we identify the C-terminal SH2 domain within the X/Y linker as the critical determinant for auto-inhibition. Tyrosine phosphorylation of the X/Y linker mediates high affinity intramolecular interaction with the C-terminal SH2 domain that is coupled to a large conformational rearrangement and release of auto-inhibition. Consequently, PLC-γ isozymes link phosphorylation to phospholipase activation by elaborating upon primordial regulatory mechanisms found in other PLCs.

Importance of the Conserved Carboxyl-Terminal CNOT1 Binding Domain to Tristetraprolin Activity In Vivo.

Tristetraprolin (TTP) is an anti-inflammatory protein that modulates the stability of certain cytokine/chemokine mRNAs. After initial high-affinity binding to AU-rich elements in 3' untranslated regions of target mRNAs, mediated through its tandem zinc finger (TZF) domain, TTP promotes the deadenylation and ultimate decay of target transcripts. These transcripts and their encoded proteins accumulate abnormally in TTP knockout (KO) mice, leading to a severe inflammatory syndrome. To assess the importance of the highly conserved C-terminal CNOT1 binding domain (CNBD) of TTP to the TTP deficiency phenotype in mice, we created a mouse model in which TTP lacked its CNBD. CNBD deletion mice exhibited a less severe phenotype than the complete TTP KO mice. In macrophages, the stabilization of target transcripts seen in KO mice was partially normalized in the CNBD deletion mice. In cell-free experiments, recombinant TTP lacking its CNBD could still activate target mRNA deadenylation by purified recombinant Schizosaccharomyces pombe CCR4/NOT complexes, although to a lesser extent than full-length TTP. Thus, TTP lacking its CNBD can still act to promote target mRNA instability in vitro and in vivo These data have implications for TTP family members throughout the eukarya, since species from all four kingdoms contain proteins with linked TZF and CNOT1 binding domains.

A fluorogenic, small molecule reporter for mammalian phospholipase C isozymes.

Phospholipase C isozymes (PLCs) catalyze the conversion of the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP(2)) into two second messengers, inositol 1,4,5-trisphosphate and diacylglycerol. This family of enzymes are key signaling proteins that regulate the physiological responses of many extracellular stimuli such as hormones, neurotransmitters, and growth factors. Aberrant regulation of PLCs has been implicated in various diseases including cancer and Alzheimer's disease. How, when, and where PLCs are activated under different cellular contexts are still largely unknown. We have developed a fluorogenic PLC reporter, WH-15, that can be cleaved in a cascade reaction to generate fluorescent 6-aminoquinoline. When applied in enzymatic assays with either pure PLCs or cell lysates, this reporter displays more than a 20-fold fluorescence enhancement in response to PLC activity. Under assay conditions, WH-15 has comparable K(m) and V(max) with the endogenous PIP(2). This novel reporter will likely find broad applications that vary from imaging PLC activity in live cells to high-throughput screening of PLC inhibitors.

Kinetic scaffolding mediated by a phospholipase C-beta and Gq signaling complex.

Transmembrane signals initiated by a broad range of extracellular stimuli converge on nodes that regulate phospholipase C (PLC)-dependent inositol lipid hydrolysis for signal propagation. We describe how heterotrimeric guanine nucleotide-binding proteins (G proteins) activate PLC-ßs and in turn are deactivated by these downstream effectors. The 2.7-angstrom structure of PLC-ß3 bound to activated Gα(q) reveals a conserved module found within PLC-ßs and other effectors optimized for rapid engagement of activated G proteins. The active site of PLC-ß3 in the complex is occluded by an intramolecular plug that is likely removed upon G protein-dependent anchoring and orientation of the lipase at membrane surfaces. A second domain of PLC-ß3 subsequently accelerates guanosine triphosphate hydrolysis by Gα(q), causing the complex to dissociate and terminate signal propagation. Mutations within this domain dramatically delay signal termination in vitro and in vivo. Consequently, this work suggests a dynamic catch-and-release mechanism used to sharpen spatiotemporal signals mediated by diverse sensory inputs.

Phospholipase C isozymes as effectors of Ras superfamily GTPases.

The physiological effects of many extracellular stimuli are initiated through receptor-promoted activation of phospholipase C and inositol lipid signaling pathways. The historical view that phospholipase C-promoted signaling primarily occurs through activation of heterotrimeric G proteins or tyrosine kinases has expanded in recent years with the realization that at least three different mammalian phospholipase C isozymes are directly activated by members of the Ras superfamily of GTPases. Thus, Ras, Rap, Rac, and Rho GTPases all specifically regulate certain phospholipase C isozymes, and insight into the physiological significance of these signaling responses is beginning to accrue. High resolution three-dimensional structures of phospholipase C isozymes also are beginning to shed light on their mechanism of activation.

Prediction of protein-protein interfaces on G-protein beta subunits reveals a novel phospholipase C beta2 binding domain.

Gbeta subunits from heterotrimeric G-proteins (guanine nucleotide-binding proteins) directly bind diverse proteins, including effectors and regulators, to modulate a wide array of signaling cascades. These numerous interactions constrained the evolution of the molecular surface of Gbeta. Although mammals contain five Gbeta genes comprising two classes (Gbeta1-like and Gbeta5-like), plants and fungi have a single ortholog, and organisms such as Caenorhabditis elegans and Drosophila melanogaster contain one copy from each class. A limited number of crystal structures of complexes containing Gbeta subunits and complementary biochemical data highlight specific sites within Gbetas needed for protein interactions. It is difficult to determine from these interaction sites what, if any, additional regions of the Gbeta molecular surface comprise interaction interfaces essential to Gbeta's role as a nexus in numerous signaling cascades. We used a comparative evolutionary approach to identify five known and eight previously unknown putative interfaces on the surface of Gbeta. We show that one such novel interface occurs between Gbeta and phospholipase C beta2 (PLC-beta2), a mammalian Gbeta interacting protein. Substitutions of residues within this Gbeta-PLC-beta2 interface reduce the activation of PLC-beta2 by Gbeta1, confirming that our de novo comparative evolutionary approach predicts previously unknown Gbeta-protein interfaces. Similarly, we hypothesize that the seven remaining untested novel regions contribute to putative interfaces for other Gbeta interacting proteins. Finally, this comparative evolutionary approach is suitable for application to any protein involved in a significant number of protein-protein interactions.

Dual activation of phospholipase C-epsilon by Rho and Ras GTPases.

Phospholipase C-epsilon (PLC-epsilon) is a highly elaborated PLC required for a diverse set of signaling pathways. Here we use a combination of cellular assays and studies with purified proteins to show that activated RhoA and Ras isoforms directly engage distinct regions of PLC-epsilon to stimulate its phospholipase activity. Purified PLC-epsilon was activated in a guanine nucleotide- and concentration-dependent fashion by purified lipidated K-Ras reconstituted in PtdIns(4,5)P(2)-containing phospholipid vesicles. Whereas mutation of two critical lysine residues within the second Ras-association domain of PLC-epsilon prevented K-Ras-dependent activation of the purified enzyme, guanine nucleotide-dependent activation by RhoA was retained. Deletion of a loop unique to PLC-epsilon eliminated its activation by RhoA but not H-Ras. In contrast, removal of the autoinhibitory X/Y-linker region of the catalytic core of PLC-epsilon markedly activates the enzyme (Hicks, S. N., Jezyk, M. R., Gershburg, S., Seifert, J. P., Harden, T. K., and Sondek, J. (2008) Mol. Cell, 31, 383-394), but PLC-epsilon lacking this regulatory region retained activation by both Rho and Ras GTPases. Additive activation of PLC-epsilon by RhoA and K- or H-Ras was observed in intact cell studies, and this additivity was recapitulated in experiments in which activation of purified PLC-epsilon was quantified with PtdIns(4,5)P(2)-containing phospholipid vesicles reconstituted with purified, isoprenylated GTPases. A maximally effective concentration of activated RhoA also increased the sensitivity of purified PLC-epsilon to activation by K-Ras. These results indicate that PLC-epsilon can be directly and concomitantly activated by both RhoA and individual Ras GTPases resulting in diverse upstream control of signaling cascades downstream of PLC-epsilon.