Intrinsic GTPase Activity Research Articles

RGS Redundancy and Implications in GPCR-GIRK Signaling.

Regulators of G protein signaling (RGS proteins) are key components of GPCR complexes, interacting directly with G protein α-subunits to enhance their intrinsic GTPase activity. The functional consequence is an accelerated termination of G protein effectors including certain ion channels. RGS proteins have a profound impact on the membrane-delimited gating behavior of G-protein-activated inwardly rectifying K(+) (GIRK) channels as demonstrated in reconstitution assays and recent RGS knockout mice studies. Akin to GPCRs and G protein αβγ subunits, multiple RGS isoforms are expressed within single GIRK-expressing neurons, suggesting functional redundancy and/or specificity in GPCR-GIRK channel signaling. The extent and impact of RGS redundancy in neuronal GPCR-GIRK channel signaling is currently not fully appreciated; however, recent studies from RGS knockout mice are providing important new clues on the impact of individual endogenous RGS proteins and the extent of RGS functional redundancy. Incorporating "tools" such as engineered RGS-resistant Gαi/o subunits provide an important assessment method for determining the impact of all endogenous RGS proteins on a given GPCR response and an accounting benchmark to assess the impact of individual RGS knockouts on overall RGS redundancy within a given neuron. Elucidating the degree of regulation attributable to specific RGS proteins in GIRK channel function will aid in the assessment of individual RGS proteins as viable therapeutic targets in epilepsy, ataxia's, memory disorders, and a growing list of neurological disorders.

Introduction: G Protein-coupled Receptors and RGS Proteins.

Here, we provide an overview of the role of regulator of G protein-signaling (RGS) proteins in signaling by G protein-coupled receptors (GPCRs), the latter of which represent the largest class of cell surface receptors in humans responsible for transducing diverse extracellular signals into the intracellular environment. Given that GPCRs regulate virtually every known physiological process, it is unsurprising that their dysregulation plays a causative role in many human diseases and they are targets of 40-50% of currently marketed pharmaceuticals. Activated GPCRs function as GTPase exchange factors for Gα subunits of heterotrimeric G proteins, promoting the formation of Gα-GTP and dissociated Gβγ subunits that regulate diverse effectors including enzymes, ion channels, and protein kinases. Termination of signaling is mediated by the intrinsic GTPase activity of Gα subunits leading to reformation of the inactive Gαβγ heterotrimer. RGS proteins determine the magnitude and duration of cellular responses initiated by many GPCRs by functioning as GTPase-accelerating proteins (GAPs) for specific Gα subunits. Twenty canonical mammalian RGS proteins, divided into four subfamilies, act as functional GAPs while almost 20 additional proteins contain nonfunctional RGS homology domains that often mediate interaction with GPCRs or Gα subunits. RGS protein biochemistry has been well elucidated in vitro, but the physiological functions of each RGS family member remain largely unexplored. This book summarizes recent advances employing modified model organisms that reveal RGS protein functions in vivo, providing evidence that RGS protein modulation of G protein signaling and GPCRs can be as important as initiation of signaling by GPCRs.

Structural perspective of ARHI mediated inhibition of STAT3 signaling: An insight into the inactive to active transition of ARHI and its interaction with STAT3 and importinβ

ARHI, a putative tumor suppressor protein with unique 32 amino acid extension in the N-terminal region, differs from oncogenes Ras and Rap, negatively regulates STAT3 signaling and inhibits the migration of ovarian cancer cells. ARHI associates directly with STAT3, also forms complex with importinβ, and prevents formation of RanGTPase–importinβ complex, which is essential for transporting STAT3 into the nucleus. Hence, the structural aspects pertaining to ARHI mediated inhibition of STAT3 translocation can provide hints on the regulation of STAT3 signaling mechanism. Accordingly, in the present study, the structure of ARHI was predicted and its transition from inactive to active state studied using MD simulations and free energy landscape analysis. The transition of ARHI is marked by the movement of switch I region towards γ-phosphate of GTP, in addition, the hydrophobic interaction between N-terminal helix and switch II region of ARHI accounts for its low intrinsic GTPase activity. Further, the protein–protein interaction studies reveal that the residues of N-terminal helix, effector domain, P-loop and G box motif of ARHI actively form polar and non-polar interaction with NTD of STAT3 and make them compact thereby rendering STAT3 inaccessible for Ran–importinβ mediated translocation. On the other hand, ARHI competes with RanGTPase and interacts with importinβ via basic–acidic patch interaction, which leads to inhibition of STAT3 translocation. The interacting residues involved for this structural mechanism would be instrumental in designing inhibitors for STAT3, which mimics ARHI thereby leading to the suppression of cancer cell growth.

Biochemical and Functional Analysis of Novel KRAS Insertions in MPN and Other Cancers

The somatic KRAS mutations found in ~25% of human cancers commonly encode amino acid substitutions at codons 12, 13, and 61. Each of these mutations lead to elevated levels of active Ras-GTP by reducing intrinsic Ras GTPase activity and conferring resistance to GTPase activating proteins (GAPs). Amino acids 12, 13, and 61 play key roles in binding the γ phosphate of GTP and facilitating GAP-mediated catalysis. The germline KRAS point mutations found in Noonan syndrome encode different amino acid substitutions that have less severe biochemical consequences. Together, these observations and the structural conservation of the Ras/GAP switch through evolution suggest that a limited spectrum of mutations can constitutively activate Ras and cause disease. Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm characterized by driver Ras pathway mutations in 85% of cases, including common somatic KRAS and NRAS substitutions. We unexpectedly identified a novel somatic KRAS insertion, 66GQEEYSA67, in the bone marrow of a patient who meets most, but not all diagnostic criteria, for JMML and lacks any other Ras pathway mutation. This seven amino acid insertion is located in the conserved Switch II domain of Ras, which binds to and activates PI3 kinase and other Ras effectors. We characterized the functional and biochemical consequences of this mutation, and of a similar five amino acid insertion identified in two lung cancers and one colon cancer archived in the COSMIC database (66EEYSA67). We find that unlike WT K-Ras, the expression of both insertion mutants supports cytokine independent growth of IL-3 dependent pro-B Ba/F3 cells similar to oncogenic K-RasG12D. Ba/F3 cells expressing K-Ras66GQEEYSA67 or K-Ras66EEYSA67 mutant proteins have markedly elevated levels of Ras-GTP that are insensitive to IL-3 stimulation, and result in constitutive ERK and Akt phosphorylation. Confirming that these Switch II insertion mutants have oncogenic potential in the hematopoietic system, fetal liver cells transduced with KRas66GQEEYSA67, KRas66EEYSA67, or KRasG12D formed cytokine independent colony forming unit granulocyte macrophage (CFU-GM) colonies and also exhibited GM-CSF hypersensitivity, which is a cellular hallmark of JMML. Biochemical characterization of recombinant K-Ras66GQEEYSA67 and K-Ras66EEYSA67 proteins showed them to have reduced intrinsic GTP hydrolysis, and to accumulate in the GTP-bound conformation when compared to oncogenic K-RasG12D. Furthermore, whereas recombinant p120 GAP markedly accelerated the rate of GTP hydrolysis by WT K-Ras, K-Ras66GQEEYSA67 and K-Ras66EEYSA67 were completely resistant. The most likely structural explanation is that these insertions perturb critical contacts between glutamine 61 in the Switch II domain of Ras and GAPs. We conclude that KRAS66GQEEYSA67 and KRAS66EEYSA67 are oncogenic driver mutations in JMML and other cancers. These data illuminate a new class of oncogenic RAS mutations and are of immediate diagnostic relevance. Furthermore, these data reveal unanticipated structural flexibility in a domain of Ras that plays a critical role in effector binding, which has implications for potential mechanism of resistance to emerging therapeutic approaches. DisclosuresBollag:Plexxikon: Employment. Zhang:Plexxikon: Employment.

Application of a multi-channel microfluidic chip on the simultaneous detection of DNAs by using microbead-quantum dots.

Several researches have shown that cancer is caused by genetic mutations especially in genes involved in cell growth and regulation. Ras family members are frequently found in their mutated, oncogenic forms in human tumors. Mutant RAS proteins are constitutively active, owing to reduce intrinsic GTPase activity and insensitivity to GTPase-activating protein (GAPs). In total, activating mutations in the RAS genes occur in approximately 20% of all human cancers, mainly in codon 12, 13 or 61. Activating mutations in the NRAS gene not only result in the reduction of intrinsic GTPase activity but also in the induction of resistance against molecules inducing such activity. In this paper, we reported a rapid, simple and portable method for detecting the mutant types of NRAS genes codon 12 and 61 simultaneously by using bead-quantum dots (QDs) based multi-channel microfluidic chip. Probe DNAs are conjugated to bead-QDs and packed in the pillars of channels in the microfluidic chip. After injection of target DNAs and intercalating dyes, the fluorescence quenching of QDs by intercalating dye was observed due to FRET phenomena. The platform can be effortlessly applied in other biological and clinical areas.

Mutation-specific RAS oncogenicity explains NRAS codon 61 selection in melanoma.

NRAS mutation at codons 12, 13, or 61 is associated with transformation; yet, in melanoma, such alterations are nearly exclusive to codon 61. Here, we compared the melanoma susceptibility of an NrasQ61R knock-in allele to similarly designed KrasG12D and NrasG12D alleles. With concomitant p16INK4a inactivation, KrasG12D or NrasQ61R expression efficiently promoted melanoma in vivo, whereas NrasG12D did not. In addition, NrasQ61R mutation potently cooperated with Lkb1/Stk11 loss to induce highly metastatic disease. Functional comparisons of NrasQ61R and NrasG12D revealed little difference in the ability of these proteins to engage PI3K or RAF. Instead, NrasQ61R showed enhanced nucleotide binding, decreased intrinsic GTPase activity, and increased stability when compared with NrasG12D. This work identifies a faithful model of human NRAS-mutant melanoma, and suggests that the increased melanomagenecity of NrasQ61R over NrasG12D is due to heightened abundance of the active, GTP-bound form rather than differences in the engagement of downstream effector pathways. This work explains the curious predominance in human melanoma of mutations of codon 61 of NRAS over other oncogenic NRAS mutations. Using conditional "knock-in" mouse models, we show that physiologic expression of NRASQ61R, but not NRASG12D, drives melanoma formation.

Abstract A17: Differential role of Ras isoforms in regulating PI3KC2β: Novel role for nucleotide-free H-Ras in cell signaling

Abstract Ras proteins, like all GTPases, cycle through an active GTP-bound state and an inactive GDP-bound state that arises through the intrinsic GTPase activity of the protein. Activation of Ras requires the activity of GEFs which promote the release of GDP leading to the subsequent binding of GTP due to the relatively higher levels of GTP in cells. Given the high affinity of nucleotide-free Ras (nf-Ras) for nucleotides in vitro, it has been proposed that nf-Ras is a transient intermediate in vivo. However, we have discovered a potentially new role for nf-H-Ras in the regulation of PI3KC2β (1). Previously, we discovered that the scaffold protein intersectin 1 (ITSN1) interacts with and activates H-Ras on endocytic vesicles (2). In addition, ITSN1 binds and activates PI3KC2β on a similar population of vesicles (3). PI3KC2β, like Class I PI3Ks, contains a Ras binding domain (RBD) although PI3KC2β does not interact with RasGTP or RasGDP (4). Our recent studies, however, indicate that nf-H-Ras binds the PI3KC2β RBD and inhibits PI3KC2β activity in vitro (1). Furthermore, the complex of nf-H-Ras with PI3KC2β RBD was not disrupted by addition of 1mM GTP or GDP suggesting that this complex may exist in the cellular milieu of high guanine nucleotides (1). Consistent with this in vitro data, PI3KC2β preferentially interacts with H-Ras dominant-negative (Ras17N)>H-Ras WT>activated H-Ras (Ras61L or 12V) in cells (1). To determine whether these results can be extended to other GTPases, we have examined the interaction of PI3KC2β with K-Ras and various Ras-related GTPases (see poster by RS Smith et. al.). Interestingly, PI3KC2β preferentially interacts with K-Ras12V>WT> dominant-negative (K-Ras15A). Furthermore, K-Ras localizes PI3KC2β to the plasma membrane whereas H-Ras localizes PI3KC2β to the perinuclear region. Thus, different Ras GTPases interact with PI3KC2β in distinct manners. We will present data on the contrasting role of K-Ras and H-Ras on regulation of PI3KC2β. Our findings suggest that PI3KC2β stabilizes nf-H-Ras and that interaction of H-Ras and PI3KC2β mutually inhibit one another. However, the role of K-Ras in regulation of PI3KC2β appears distinct. These findings have important implications for Ras-dependent tumorigenesis.

Abstract 2482A: ARHI (DIRAS3) downregulates Ras-MAPK signaling and inhibits transformation of NIH3T3 cells through direct interaction with GTP-bound Ras

Abstract Somatic mutation of Ras was the first genetic alteration documented to drive human oncogenesis. Activating mutations of H-Ras, N-Ras, or K-Ras are associated with 30% of all human cancers. Characterization of wild type Ras proteins has identified their essential role in intra-cellular signaling networks controlling cell proliferation, differentiation and survival. Despite intensive efforts, effective targeted therapy directed against mutated Ras has remained elusive. Regulation of Ras function has been studied in depth, but relatively little attention has been paid to physiological inhibitors of Ras. Our group has previously reported that the maternally imprinted Ras-related tumor suppressor gene ARHI (DIRAS3) is downregulated in more than 60% of ovarian cancers and several other tumor types. Re-expression of ARHI at physiologic levels can inhibit ovarian cancer cell proliferation, block migration, induce autophagy, mediate tumor dormancy and block both PI3K and Ras-MAPK signaling. ARHI shares 50-60% homology with the H- and K-Ras genes, differing primarily in a 34-amino acid N-terminal extension that is required for most of ARHI's functions. The mechanism by which ARHI regulates Ras-MAPK signaling is not fully understood. Here, we show that re-expression of ARHI in NIH3T3 murine fibroblasts inhibits cell proliferation, suppresses clonogenic growth and blocks cell transformation by constitutively active mutant H-Ras. Addition of the N-terminal extension of ARHI to mutant H-Ras also prevents malignant transformation. ARHI interacts directly with mutated Ras on co-immunoprecipitation and can inhibit GTP-bound Ras activity following overexpression of the constitutively active construct. We hypothesize that inhibition of the Ras-MAPK signaling pathways following re-expression of ARHI (DIRAS3) may be, in part, the effect of a direct interaction between GTP-bound Ras and ARHI, resulting in the restoration of intrinsic Ras GTPase activity. Our results suggest that ARHI (DIRAS3) may serve as a novel inhibitor of activated Ras, and re-expression of ARHI in tumors exhibiting an oncogenic mutation of Ras might permit more specifically targeted therapy. Citation Format: Margie N. Sutton. ARHI (DIRAS3) downregulates Ras-MAPK signaling and inhibits transformation of NIH3T3 cells through direct interaction with GTP-bound Ras. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2482A. doi:10.1158/1538-7445.AM2014-2482A

Abstract 4443: The tumor suppressive small GTPase DiRas1 binds the RhoGEF SmgGDS and antagonizes RhoA activation

Abstract A number of malignancies are promoted by the activation of Rho GTPases. Regulators of RhoA activity, including the RhoGEF, SmgGDS, can also promote cancer development. Both RhoA and SmgGDS have been shown to enhance NF-ĸB activation in a number of tumors. Utilizing mass spectrometry, we identified the tumor suppressive small GTPase DiRas1 as a novel binding partner for SmgGDS. DiRas1 is a tumor suppressor in malignant gliomas and esophageal squamous cell carcinomas. Examining DiRas gene expression using the ONCOMINE mRNA microarray database (www.oncomine.org) revealed that DIRAS1 and DIRAS2 are markedly decreased in central nervous system tumors, including anaplastic astrocytomas and GBMs, when compared to normal tissue. The role of DiRas1 in other cancers is largely undetermined, and the ways in which DiRas1 may antagonize pro-oncogenic small GTPase signaling is largely unknown. To define how DiRas1 expression may affect SmgGDS functions, we tested the association of DiRas family proteins with SmgGDS. SmgGDS robustly bound wildtype DiRas1, and its closely-related family member DiRas2, but only weakly bound DiRas3 in vitro and in cell lines. In addition, DiRas1, DiRas2, or DiRas3 protein expression decreased proliferation of HEK293T cells. DiRas1 expression could promote less activated RhoA, as a low level of DiRas1 expression caused markedly decreased RhoA-SmgGDS binding in a number of cell lines. DiRas1 expression also abrogated SmgGDS- and RhoA-mediated NF-ĸB activation in a concentration-dependent manner. SmgGDS may exhibit GEF activity toward DiRas1, as a dominant negative DiRas1 (S21N) binds to SmgGDS more than the wildtype protein. However, DiRas1 is predominantly GTP-bound in cells, likely due to a high rate of guanine nucleotide dissociation and low intrinsic GTPase activity. Thus, DiRas1 expression may in part promote tumor suppression by non-productively binding SmgGDS, resulting in less RhoA activity. Taken together, these results suggest that the tumor suppressive small GTPase DiRas1 can antagonize RhoA signaling by competing for binding of the RhoGEF SmgGDS. Further characterizing these actions may lead to novel therapeutic targeting of RhoA activation in cancer malignancies. Citation Format: Andrew D. Hauser, Kristen M. Barr, Anne C. Frei, Patrick Gonyo, Ellen L. Lorimer, Carol L. Williams, Carmen Bergom. The tumor suppressive small GTPase DiRas1 binds the RhoGEF SmgGDS and antagonizes RhoA activation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4443. doi:10.1158/1538-7445.AM2014-4443

Rice serine/threonine kinase 1 is required for the stimulation of OsNug2 GTPase activity

Several GTPases are required for ribosome biogenesis and assembly. We recently identified rice (Oryza sativa) nuclear/nucleolar GTPase 2 (OsNug2), a YlqF/YawG family GTPase, as having a role in pre-60S ribosomal subunit maturation. To investigate the potential factors involved in regulating OsNug2 function, yeast two-hybrid screens were performed using OsNug2 as bait. Rice serine/threonine kinase 1 (OsSTK1) was identified as a candidate interacting protein. OsSTK1 appeared to interact with OsNug2 both in vitro and in vivo. OsSTK1 was found to have no effect on the GTP-binding activity of OsNug2; however, the presence of recombinant OsSTK1 in OsNug2 assay reaction mixtures increased OsNug2 GTPase activity. A kinase assay showed that OsSTK1 had weak autophosphorylation activity and strongly phosphorylated serine 209 of OsNug2. Using yeast complementation testing, we identified a GAL::OsNug2(S209N) mutation-harboring yeast strain that exhibited a growth-defective phenotype on galactose medium at 39°C, which was divergent from that of a yeast strain harboring GAL::OsNug2. The intrinsic GTPase activity of OsNug2(S209N), which was found to be similar to that of OsNug2, was not fully enhanced upon weak binding of OsSTK1. Our findings indicate that OsSTK1 functions as a positive regulator of OsNug2 by enhancing OsNug2 GTPase activity. In addition, phosphorylation of OsNug2 serine 209 is essential for its complete function in biological functional pathway.

A Dynamin Mutant Defines a Superconstricted Prefission State

Dynamin is a 100kDa GTPase that organizes into helical assemblies at the base of nascent clathrin-coated vesicles. Formation of these oligomers stimulates the intrinsic GTPase activity of dynamin, whichis necessary for efficient membrane fission during endocytosis. Recent evidence suggests that the transition state of dynamin's GTP hydrolysis reaction serves as a key determinant of productive fission. Here, we present the structure of a transition-state-defective dynamin mutant K44A trapped in a prefission state at 12.5Å resolution. This structure constricts to 3.7nm, reaching the theoretical limit required for spontaneous membrane fission. Computational docking indicates that the ground-state conformation of the dynamin polymer is sufficient to achieve this superconstricted prefission state and reveals how a two-start helical symmetry promotes the most efficient packing of dynamin tetramers around the membrane neck. These data suggest a model for the assembly and regulation of the minimal dynamin fission machine.

Braking bad: stopping translation in hard times.

Braking bad: stopping translation in hard times.

Structural and functional analyses confirm the presence of functional P‐loops and intrinsic GTPase activity in Evc protein (1013.9)

Ellis van Creveld (EvC) syndrome is a rare multi‐symptom human genetic disorder clinically characterized by mild short stature, postaxial polydactyly, dysplastic nail and teeth. Evc is one of the novel proteins that have been implicated in the pathogenesis of EvC syndrome and its milder form, Weyers acrofacial dysostosis (WAD). Mutations in EVC and EVC2 genes that cause these syndromes introduce premature termination codons and frameshifts, resulting in production of defective Evc and Evc2 proteins respectively. Genetic and biochemical experiments have identified Evc and Evc2 as novel proteins that are essential for positive regulation of both Indian and sonic hedgehog (Hh) signaling pathways during bone and heart development, and pathogenesis of EvC syndrome. Two separate research reports demonstrated that Evc‐Evc2 complex interacts with smoothened (smo) at the base of the cilia to transduce Hh‐induced signal to downstream proteins. We present spectroscopic and structural evidence to show that the putative P‐loop motif in Evc is functional and that it confers GTPase activity on the protein. The implications of our findings on the mechanism of transduction and regulation of Hh‐induced signals will be discussed.

Characterization of Recombinant ELMOD (Cell Engulfment and Motility Domain) Proteins as GTPase-activating Proteins (GAPs) for ARF Family GTPases

The ARF family of regulatory GTPases, within the RAS superfamily, is composed of ~30 members in mammals, including up to six ARF and at least 18 ARF-like (ARL) proteins. They exhibit significant structural and biochemical conservation and regulate a variety of essential cellular processes, including membrane traffic, cell division, and energy metabolism; each with links to human diseases. We previously identified members of the ELMOD family as GTPase-activating proteins (GAPs) for ARL2 that displayed crossover activity for ARFs as well. To further characterize the GAP activities of the three human ELMODs as GAPs we developed new preparations of each after overexpression in human embryonic kidney (HEK293T) cells. This allowed much higher specific activities and enhanced stability and solubility of the purified proteins. The specificities of ELMOD1-3 as GAPs for six different members of the ARF family were determined and found to display wide variations, which we believe will reveal differences in cellular functions of family members. The non-opioid sigma-1 receptor (S1R) was identified as a novel effector of GAP activity of ELMOD1-3 proteins as its direct binding to either ELMOD1 or ELMOD2 resulted in loss of GAP activity. These findings are critical to understand the roles of ELMOD proteins in cell signaling in general and in the inner ear specifically, and open the door to exploration of the regulation of their GAP activities via agonists or antagonists of the S1R.

The Human Papillomavirus E7 Proteins Associate with p190RhoGAP and Alter Its Function

Using mass spectrometry, we identified p190RhoGAP (p190) as a binding partner of human papillomavirus 16 (HPV16) E7. p190 belongs to the GTPase activating protein (GAP) family and is one of the primary GAPs for RhoA. GAPs stimulate the intrinsic GTPase activity of the Rho proteins, leading to Rho inactivation and influencing numerous biological processes. RhoA is one of the best-characterized Rho proteins and is specifically involved in formation of focal adhesions and stress fibers, thereby regulating cell migration and cell spreading. Since this is the first report that E7 associates with p190, we carried out detailed interaction studies. We show that E7 proteins from other HPV types also bind p190. Furthermore, we found that conserved region 3 (CR3) of E7 and the middle domain of p190 are important for this interaction. More specifically, we identified two residues in CR3 of E7 that are necessary for p190 binding and used mutants of E7 with mutations of these residues to determine the biological consequences of the E7-p190 interaction. Our data suggest that the interaction of E7 with p190 dysregulates this GAP and alters the actin cytoskeleton. We also found that this interaction negatively regulates cell spreading on a fibronectin substrate and therefore likely contributes to important aspects of the HPV life cycle or HPV-induced tumorigenesis. This study identifies p190RhoGAP as a novel cellular binding partner for the human papillomavirus (HPV) E7 protein. Our study shows that a large number of different HPV E7 proteins bind p190RhoGAP, and it identifies regions in both E7 and p190RhoGAP which are important for the interaction to occur. This study also highlights the likelihood that the E7-p190RhoGAP interaction may have important biological consequences related to actin organization in the infected cell. These changes could be an important contributor to the viral life cycle and during progression to cancer in HPV-infected cells. Importantly, this work also emphasizes the need for further study in a field which has largely been unexplored as it relates to the HPV life cycle and HPV-induced transformation.

Modification and Functional Inhibition of Regulator of G-Protein Signaling 4 (RGS4) by 4-Hydroxy-2-nonenal

Oxidative stress has been implicated as a component of various pathologies including ischemia/reperfusion injury (IRI) and neurodegenerative diseases such as Parkinson's disease (PD) and schizophrenia. Similarly, regulator of G-protein signaling 4 (RGS4) has been implicated as an important player in each of these pathologies. RGS4, like other RGS proteins, is responsible for temporally regulating G-protein coupled receptor signaling by increasing the intrinsic GTPase activity of Gα subunit of the heterotrimeric signaling complex. In this study we evaluated whether modification by 4-hydroxy-2-nonenal (4HNE), a common lipid peroxidation product, inhibits RGS4. Using immunoprecipitation, we first determined RGS4 modification was occurring in cells at concentrations of 4HNE within reported physiological conditions. Following this determination, we evaluated modification of RGS4 by 4HNE by both Western blot and mass spectrometry (MS). Once it was established that covalent modification occurred only on cysteine containing constructs, tryptic digest followed by mass spectrometry analysis revealed modification occurs at cysteine residues 71, 148, and 183. In order to determine the effect 4HNE had on RGS4 activity, a steady-state colorimetric assay was used to analyze the GAP activity of Δ51-RGS4 as well as the cysteine null mutant. From the data, we determined that RGS4 activity can be modulated by 4HNE through modification at cysteine residues similar to previously reported small molecule inhibition of RGS4.

RhoGTPases control specific cytoskeleton‐dependent functions of hematopoietic stem cells

The Rho family of guanosine triphosphatases (GTPases) is composed of members of the Ras superfamily of proteins. They are GTP-bound molecules with a modest intrinsic GTPase activity that can be accelerated upon activation/localization of specialized guanine nucleotide exchange factors. Members of this family act as molecular switches and are required for coordinated cytoskeletal rearrangements that are crucial in a set of specialized functions of mammalian stem cells. These functions include self-renewal, adhesion, and migration. Mouse gene-targeting studies have provided convincing evidence of the indispensable and dispensable roles of individual members of the Rho GTPase family and the putative upstream and downstream mediators in stem cell-specific functions. The role of Rho GTPases and related signaling pathways previously seen in other cell types and organisms have been confirmed in mammalian hematopoietic stem cells (HSCs), and new signaling pathways and unexpected functions unique to HSCs have been identified and dissected. This review summarizes our current understanding of the role of Rho family of GTPases on HSC and progenitor activity through cytoskeleton-mediated signaling pathways, providing insight about relevant signaling pathways that regulate mammalian stem cell self-renewal, adhesion, and migration.

Autoinhibition and Signaling by the Switch II Motif in the G-protein Chaperone of a Radical B12 Enzyme

MeaB is an accessory GTPase protein involved in the assembly, protection, and reactivation of 5'-deoxyadenosyl cobalamin-dependent methylmalonyl-CoA mutase (MCM). Mutations in the human ortholog of MeaB result in methylmalonic aciduria, an inborn error of metabolism. G-proteins typically utilize conserved switch I and II motifs for signaling to effector proteins via conformational changes elicited by nucleotide binding and hydrolysis. Our recent discovery that MeaB utilizes an unusual switch III region for bidirectional signaling with MCM raised questions about the roles of the switch I and II motifs in MeaB. In this study, we addressed the functions of conserved switch II residues by performing alanine-scanning mutagenesis. Our results demonstrate that the GTPase activity of MeaB is autoinhibited by switch II and that this loop is important for coupling nucleotide-sensitive conformational changes in switch III to elicit the multiple chaperone functions of MeaB. Furthermore, we report the structure of MeaB·GDP crystallized in the presence of AlFx(-) to form the putative transition state analog, GDP·AlF4(-). The resulting crystal structure and its comparison with related G-proteins support the conclusion that the catalytic site of MeaB is incomplete in the absence of the GTPase-activating protein MCM and therefore unable to stabilize the transition state analog. Favoring an inactive conformation in the absence of the client MCM protein might represent a strategy for suppressing the intrinsic GTPase activity of MeaB in which the switch II loop plays an important role.

The Human Minor Histocompatibility Antigen1 Is a RhoGAP

The human minor Histocompatibility Antigen HMHA-1 is a major target of immune responses after allogeneic stem cell transplantation applied for the treatment of leukemia and solid tumors. The restriction of its expression to hematopoietic cells and many solid tumors raised questions regarding its cellular functions. Sequence analysis of the HMHA-1 encoding HMHA1 protein revealed the presence of a possible C-terminal RhoGTPase Activating Protein (GAP) domain and an N-terminal BAR domain. Rho-family GTPases, including Rac1, Cdc42, and RhoA are key regulators of the actin cytoskeleton and control cell spreading and migration. RhoGTPase activity is under tight control as aberrant signaling can lead to pathology, including inflammation and cancer. Whereas Guanine nucleotide Exchange Factors (GEFs) mediate the exchange of GDP for GTP resulting in RhoGTPase activation, GAPs catalyze the low intrinsic GTPase activity of active RhoGTPases, resulting in inactivation. Here we identify the HMHA1 protein as a novel RhoGAP. We show that HMHA1 constructs, lacking the N-terminal region, negatively regulate the actin cytoskeleton as well as cell spreading. Furthermore, we show that HMHA1 regulates RhoGTPase activity in vitro and in vivo. Finally, we demonstrate that the HMHA1 N-terminal BAR domain is auto-inhibitory as HMHA1 mutants lacking this region, but not full-length HMHA1, showed GAP activity towards RhoGTPases. In conclusion, this study shows that HMHA1 acts as a RhoGAP to regulate GTPase activity, cytoskeletal remodeling and cell spreading, which are crucial functions in normal hematopoietic and cancer cells.

A Physiologically Required G Protein-coupled Receptor (GPCR)-Regulator of G Protein Signaling (RGS) Interaction That Compartmentalizes RGS Activity

G protein-coupled receptors (GPCRs) can interact with regulator of G protein signaling (RGS) proteins. However, the effects of such interactions on signal transduction and their physiological relevance have been largely undetermined. Ligand-bound GPCRs initiate by promoting exchange of GDP for GTP on the Gα subunit of heterotrimeric G proteins. Signaling is terminated by hydrolysis of GTP to GDP through intrinsic GTPase activity of the Gα subunit, a reaction catalyzed by RGS proteins. Using yeast as a tool to study GPCR signaling in isolation, we define an interaction between the cognate GPCR (Mam2) and RGS (Rgs1), mapping the interaction domains. This reaction tethers Rgs1 at the plasma membrane and is essential for physiological signaling response. In vivo quantitative data inform the development of a kinetic model of the GTPase cycle, which extends previous attempts by including GPCR-RGS interactions. In vivo and in silico data confirm that GPCR-RGS interactions can impose an additional layer of regulation through mediating RGS subcellular localization to compartmentalize RGS activity within a cell, thus highlighting their importance as potential targets to modulate GPCR signaling pathways.