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1
Cantor, S. B., T. Urano, and L. A. Feig. "Identification and characterization of Ral-binding protein 1, a potential downstream target of Ral GTPases." Molecular and Cellular Biology 15, no. 8 (August 1995): 4578–84. http://dx.doi.org/10.1128/mcb.15.8.4578.
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Ral proteins constitute a distinct family of Ras-related GTPases. Although similar to Ras in amino acid sequence, Ral proteins are activated by a unique nucleotide exchange factor and inactivated by a distinct GTPase-activating protein. Unlike Ras, they fail to promote transformed foci when activated versions are expressed in cells. To identify downstream targets that might mediate a Ral-specific function, we used a Saccharomyces cerevisiae-based interaction assay to clone a novel cDNA that encodes a Ral-binding protein (RalBP1). RalBP1 binds specifically to the active GTP-bound form of RalA and not to a mutant Ral with a point mutation in its putative effector domain. In addition to a Ral-binding domain, RalBP1 also contains a Rho-GTPase-activating protein domain that interacts preferentially with Rho family member CDC42. Since CDC42 has been implicated in bud site selection in S. cerevisiae and filopodium formation in mammalian cells, Ral may function to modulate the actin cytoskeleton through its interactions with RalBP1.
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Gupta, A., B. Bastani, P. Chardin, and K. A. Hruska. "Localization of ral, a small Mr GTP-binding protein, to collecting duct cells in bovine and rat kidney." American Journal of Physiology-Renal Physiology 261, no. 6 (December 1, 1991): F1063—F1070. http://dx.doi.org/10.1152/ajprenal.1991.261.6.f1063.
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Plasma membranes from bovine kidney cortex were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Blotting with [alpha-32P]GTP and [35S]GTP gamma S demonstrated specific binding to three and six distinct protein bands, respectively, in the 20,000- to 29,000-Mr range. This indicated the presence of small Mr GTP binding proteins (smg) in bovine kidney cortex. Only one smg with 28,000 Mr was labeled with hydrolysis-resistant GTP photoaffinity probe p3-(4-azidoanilido)-p1-5GTP (AAGTP). The major smg in platelet membranes that binds GTP on nitrocellulose blots has been identified as ral-Mr 29,000. With the use of an antiserum against the ral A gene product, one of the smg with Mr of 29,000 present in bovine renal cortical plasma membranes was identified as ral. Ral was absent from glomerular homogenate, suggesting that it is localized to the tubular segments of the nephron. Ral was detected only in the particulate fraction and not the cytosol. Further subcellular localization of ral was investigated by immunohistochemical staining. Anti-ral antibody immunostained the apical and basolateral membranes of cells in the cortical and medullary collecting ducts in a speckled pattern in the bovine kidney. In the rat kidney, however, uniform linear staining of cortical and medullary collecting ducts predominantly localized to the apical membrane was observed. To date, no function has been assigned to ral. Localization of the ral gene product to the collecting duct suggests a specific functional role for this GTP-binding protein.
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Bhullar, Rajinder P., Richard R. Clough, Juddy Kanungo, Sherif M. Elsaraj, and Ognjen Grujic. "Ral-GTPase interacts with the β1 subunit of Na+/K+-ATPase and is activated upon inhibition of the Na+/K+ pumpThis paper is one of a selection of papers published in this Special Issue, entitled The Cellular and Molecular Basis of Cardiovascular Dysfunction, Dhalla 70th Birthday Tribute." Canadian Journal of Physiology and Pharmacology 85, no. 3-4 (March 2007): 444–54. http://dx.doi.org/10.1139/y07-027.
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Na+/K+-ATPase functions as both an ion pump and a signal transducer. Cardiac glycosides partially inhibit Na+/K+-ATPase, causing activation of multiple interrelated growth pathways via the Na+/K+-ATPase/c-Src/epidermal growth factor receptor complex. Such pathways include Ras/MEK/ERK and Ral/RalGDS cascades, which can lead to cardiac hypertrophy. In search of novel Ral-GTPase binding proteins, we used RalB as the bait to screen a human testes cDNA expression library using the yeast 2-hybrid system. The results demonstrated that 1 of the RalB interacting clones represented the C-terminal region of the β1 subunit of Na+/K+-ATPase. Further analysis using the yeast 2-hybrid system and full-length β1 subunit of Na+/K+-ATPase confirmed the interaction with RalA and RalB. In vitro binding and pull-down assays demonstrated that the β1 subunit of Na+/K+-ATPase interacts directly with RalA and RalB. Ral-GTP pull-down assays demonstrated that short-term ouabain treatment of A7r5 cells, a rat aorta smooth muscle cell line, caused activation of Ral GTPase. Maximal activation was observed 10 min after ouabain treatment. Ouabain-mediated Ral activation was inhibited upon the stimulation of Na+/K+-ATPase activity by Ang II. We propose that Ral GTPase is involved in the signal transducing function of Na+/K+-ATPase and provides a possible molecular mechanism connecting Ral to cardiac hypertrophy during diseased conditions.
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Bauer, Bettina, Gladys Mirey, Ingrid R. Vetter, Juan A. Garcı́a-Ranea, Alfonso Valencia, Alfred Wittinghofer, Jacques H. Camonis, and Robbert H. Cool. "Effector Recognition by the Small GTP-binding Proteins Ras and Ral." Journal of Biological Chemistry 274, no. 25 (June 18, 1999): 17763–70. http://dx.doi.org/10.1074/jbc.274.25.17763.
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Wildey, G. M., M. Viggeswarapu, S. Rim, and J. K. Denker. "Isolation of cDNA Clones and Tissue Expression of Rat Ral A and Ral B GTP-Binding Proteins." Biochemical and Biophysical Research Communications 194, no. 1 (July 1993): 552–59. http://dx.doi.org/10.1006/bbrc.1993.1855.
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Karunanithi, Sheelarani, Tingting Xiong, Maeran Uhm, Dara Leto, Jingxia Sun, Xiao-Wei Chen, and Alan R. Saltiel. "A Rab10:RalA G protein cascade regulates insulin-stimulated glucose uptake in adipocytes." Molecular Biology of the Cell 25, no. 19 (October 2014): 3059–69. http://dx.doi.org/10.1091/mbc.e14-06-1060.
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Insulin-stimulated glucose uptake in fat and muscle is mediated by the major facilitative glucose transporter Glut4. Insulin controls the trafficking of Glut4 to the plasma membrane via regulation of a series of small G proteins, including RalA and Rab10. We demonstrate here that Rab10 is a bona fide target of the GTPase-activating protein AS160, which is inhibited after phosphorylation by the protein kinase Akt. Once activated, Rab10 can increase the GTP binding of RalA by recruiting the Ral guanyl nucleotide exchange factor, Rlf/Rgl2. Rab10 and RalA reside in the same pool of Glut4-storage vesicles in untreated cells, and, together with Rlf, they ensure maximal glucose transport. Overexpression of membrane-tethered Rlf compensates for the loss of Rab10 in Glut4 translocation, suggesting that Rab10 recruits Rlf to membrane compartments for RalA activation and that RalA is downstream of Rab10. Together these studies identify a new G protein cascade in the regulation of insulin-stimulated Glut4 trafficking and glucose uptake.
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Kikuchi, A., S. D. Demo, Z. H. Ye, Y. W. Chen, and L. T. Williams. "ralGDS family members interact with the effector loop of ras p21." Molecular and Cellular Biology 14, no. 11 (November 1994): 7483–91. http://dx.doi.org/10.1128/mcb.14.11.7483.
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Using a yeast two-hybrid system, we identified a novel protein which interacts with ras p21. This protein shares 69% amino acid homology with ral guanine nucleotide dissociation stimulator (ralGDS), a GDP/GTP exchange protein for ral p24. We designated this protein RGL, for ralGDS-like. Using the yeast two-hybrid system, we found that an effector loop mutant of ras p21 was defective in interacting with the ras p21-interacting domain of RGL, suggesting that this domain binds to ras p21 through the effector loop of ras p21. Since ralGDS contained a region highly homologous with the ras p21-interacting domain of RGL, we examined whether ralGDS could interact with ras p21. In the yeast two-hybrid system, ralGDS failed to interact with an effector loop mutant of ras p21. In insect cells, ralGDS made a complex with v-ras p21 but not with a dominant negative mutant of ras p21. ralGDS interacted with the GTP-bound form of ras p21 but not with the GDP-bound form in vitro. ralGDS inhibited both the GTPase-activating activity of the neurofibromatosis gene product (NF1) for ras p21 and the interaction of Raf with ras p21 in vitro. These results demonstrate that ralGDS specifically interacts with the active form of ras p21 and that ralGDS can compete with NF1 and Raf for binding to the effector loop of ras p21. Therefore, ralGDS family members may be effector proteins of ras p21 or may inhibit interactions between ras p21 and its effectors.
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Kikuchi, A., S. D. Demo, Z. H. Ye, Y. W. Chen, and L. T. Williams. "ralGDS family members interact with the effector loop of ras p21." Molecular and Cellular Biology 14, no. 11 (November 1994): 7483–91. http://dx.doi.org/10.1128/mcb.14.11.7483-7491.1994.
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Using a yeast two-hybrid system, we identified a novel protein which interacts with ras p21. This protein shares 69% amino acid homology with ral guanine nucleotide dissociation stimulator (ralGDS), a GDP/GTP exchange protein for ral p24. We designated this protein RGL, for ralGDS-like. Using the yeast two-hybrid system, we found that an effector loop mutant of ras p21 was defective in interacting with the ras p21-interacting domain of RGL, suggesting that this domain binds to ras p21 through the effector loop of ras p21. Since ralGDS contained a region highly homologous with the ras p21-interacting domain of RGL, we examined whether ralGDS could interact with ras p21. In the yeast two-hybrid system, ralGDS failed to interact with an effector loop mutant of ras p21. In insect cells, ralGDS made a complex with v-ras p21 but not with a dominant negative mutant of ras p21. ralGDS interacted with the GTP-bound form of ras p21 but not with the GDP-bound form in vitro. ralGDS inhibited both the GTPase-activating activity of the neurofibromatosis gene product (NF1) for ras p21 and the interaction of Raf with ras p21 in vitro. These results demonstrate that ralGDS specifically interacts with the active form of ras p21 and that ralGDS can compete with NF1 and Raf for binding to the effector loop of ras p21. Therefore, ralGDS family members may be effector proteins of ras p21 or may inhibit interactions between ras p21 and its effectors.
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Polakis, P. G., R. F. Weber, B. Nevins, J. R. Didsbury, T. Evans, and R. Snyderman. "Identification of the ral and rac1 Gene Products, Low Molecular Mass GTP-binding Proteins from Human Platelets." Journal of Biological Chemistry 264, no. 28 (October 1989): 16383–89. http://dx.doi.org/10.1016/s0021-9258(19)84717-8.
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Rosário, Marta, Hugh F. Paterson, and Christopher J. Marshall. "Activation of the Ral and Phosphatidylinositol 3′ Kinase Signaling Pathways by the Ras-Related Protein TC21." Molecular and Cellular Biology 21, no. 11 (June 1, 2001): 3750–62. http://dx.doi.org/10.1128/mcb.21.11.3750-3762.2001.
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ABSTRACT TC21 is a member of the Ras superfamily of small GTP-binding proteins that, like Ras, has been implicated in the regulation of growth-stimulating pathways. We have previously identified the Raf/mitogen-activated protein kinase pathway as a direct TC21 effector pathway required for TC21-induced transformation (M. Rosário, H. F. Paterson, and C. J. Marshall, EMBO J. 18:1270–1279, 1999). In this study we have identified two further effector pathways for TC21, which contribute to TC21-stimulated transformation: the phosphatidylinositol 3′ kinase (PI-3K) and Ral signaling pathways. Expression of constitutively active TC21 leads to the activation of Ral A and the PI-3K-dependent activation of Akt/protein kinase B. Strong activation of the PI-3K/Akt pathway is seen even with very low levels of TC21 expression, suggesting that TC21 may be a key small GTPase-regulator of PI-3K. TC21-induced alterations in cellular morphology in NIH 3T3 and PC12 cells are also PI-3K dependent. On the other hand, activation of the Ral pathway by TC21 is required for TC21-stimulated DNA synthesis but not transformed morphology. We show that inhibition of Ral signaling blocks DNA synthesis in human tumor cell lines containing activating mutations in TC21, demonstrating for the first time that this pathway is required for the proliferation of human tumor cells. Finally, we provide mechanisms for the activation of these pathways, namely, the direct in vivo interaction of TC21 with guanine nucleotide exchange factors for Ral, resulting in their translocation to the plasma membrane, and the direct interaction of TC21 with PI-3K. In both cases, the effector domain region of TC21 is required since point mutations in this region can interfere with activation of downstream signaling.
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HENRIKSSON, Maria L., Charlotta SUNDIN, Anna L. JANSSON, Åke FORSBERG, Ruth H. PALMER, and Bengt HALLBERG. "Exoenzyme S shows selective ADP-ribosylation and GTPase-activating protein (GAP) activities towards small GTPases in vivo." Biochemical Journal 367, no. 3 (November 1, 2002): 617–28. http://dx.doi.org/10.1042/bj20020714.
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Intracellular targeting of the Pseudomonas aeruginosa toxins exoenzyme S (ExoS) and exoenzyme T (ExoT) initially results in disruption of the actin microfilament structure of eukaryotic cells. ExoS and ExoT are bifunctional cytotoxins, with N-terminal GTPase-activating protein (GAP) and C-terminal ADP-ribosyltransferase activities. We show that ExoS can modify multiple GTPases of the Ras superfamily in vivo. In contrast, ExoT shows no ADP-ribosylation activity towards any of the GTPases tested in vivo. We further examined ExoS targets in vivo and observed that ExoS modulates the activity of several of these small GTP-binding proteins, such as Ras, Rap1, Rap2, Ral, Rac1, RhoA and Cdc42. We suggest that ExoS is the major ADP-ribosyltransferase protein modulating small GTPase function encoded by P. aeruginosa. Furthermore, we show that the GAP activity of ExoS abrogates the activation of RhoA, Cdc42 and Rap1.
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Voorberg, Jan, Mariska G. Rondaij, Karina A. Gijzen, Ruben Bierings, Erica Sellink, Mar Fernandez-Borja, and Jan A. van Mourik. "The Guanine Exchange Factor RalGDS Is Involved in Regulated Exocytosis of Weibel-Palade Bodies from Endothelial Cells." Blood 106, no. 11 (November 16, 2005): 3688. http://dx.doi.org/10.1182/blood.v106.11.3688.3688.
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Abstract Endothelial cells contribute to vascular homeostasis and mediate pathophysiological responses to hypoxia-induced injury and inflammatory events. To ensure rapid responses to vascular perturbation endothelial cells contain intracellular storage pools for inflammatory mediators and pro-thrombotic compounds. One of the best characterized storage granules within endothelial cells are the Weibel-Palade bodies (WPB), rod-shaped organelles that contains P-selectin, Von Willebrand Factor (VWF), interleukin-8 (IL-8) and a number of other proteins with diverse biological activities. Agonist-induced triggering of heterotrimeric G protein coupled receptors (GPCR) present on endothelial cells promote exocytosis of WPB. We have previously shown that the small GTP binding protein RalA is involved in thrombin induced exocytosis of Weibel-Palade bodies. Exocytosis of Weibel-Palade bodies was found to coincide with the activation of RalA in response to thrombin. More recently, we have shown that cAMP-raising stimuli such as epinephrine also coincide with the activation of RalA. Consistent with these findings constitutively active RalG23V was capable of inducing release of WPB from endothelial cells. Small GTPases are activated by guanine exchange factors (GEFs) that induce GDP release thereby enhancing GTP binding to the small GTPase. RalGDS is a widely expressed GEF for Ral that has recently been implicated in cytoskeletal rearrangements that result from activation of GPCRs. Here, we investigated whether RalGDS is involved in thrombin- and/or epinephrine-induced exocytosis of WPB from endothelial cells. First, we showed by RT-PCR that human endothelial cells express RalGDS. Overexpression of a GFP-tagged variant of RalGDS in endothelial cells reduces the number of WPB in endothelial cells suggesting that RalGDS can promote exocytosis of these subcellular organelles. To investigate whether endogenously synthesized RalGDS plays a role in exocytosis of WPBs we designed short hairpin RNAs that acts as small interfering RNA (siRNA). Co-expression of siRalGDS and GFP-RalGDS in heterologously transfected 293 cells markedly reduced expression levels of GFP-RalGDS. Subsequently, we addressed the effect of siRalGDS on exocytosis of WPB in endothelial cells. Knockdown of endogenous RalGDS using siRNA prevented thrombin-induced release of WPBs. Expression of siRalGDS also interfered with WPB release in response to epinephrine. These results show that knock down of RalGDS interferes with exocytosis of WPB in endothelial cells. A dominant negative RalGDS variant, RalGDSΔGEF, lacking its catalytic exchange domain, was subsequently introduced in endothelial cells. As expected, no release of WPB was observed in endothelial cells expressing RalGDSΔGEF. Remarkably, both thrombin- and epinephrine-induced exocytosis were impaired in cells expressing RalGDSΔGEF. Together, these findings indicate that the Ral-specific guanine exchange factor RalGDS is involved in exocytosis of WPB from endothelial cells.
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Hertzog, Maud, and Philippe Chavrier. "Cell polarity during motile processes: keeping on track with the exocyst complex." Biochemical Journal 433, no. 3 (January 14, 2011): 403–9. http://dx.doi.org/10.1042/bj20101214.
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Motile processes are critical for several physiological and pathological situations such as embryonic development, tumour dissemination and metastasis. Migrating cells, or developing neurons, need to establish front–rear polarity consisting of actin-driven extension of the leading edge and traffic of components that are essential for membrane extension and cell adhesion at the front. Previously, several studies have suggested that the exocyst complex is critical for the establishment and maintenance of cell polarity. This octameric complex controls the docking and insertion of exocytic vesicles to growing areas of the plasma membrane. The aim of the present review is to detail recent advances concerning the molecular and structural organization of the exocyst complex that help to elucidate its role in cell polarity. We will also review the function of the exocyst complex and some of its key interacting partners [including the small GTP-binding protein Ral, aPKCs (atypical protein kinase Cs) and proteins involved in actin assembly] in the formation of plasma extensions at the leading edge, growth cone formation during axonal extension and generation of cell movement.
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Herrmann, Christian, Gudrun Horn, Marcel Spaargaren, and Alfred Wittinghofer. "Differential Interaction of the Ras Family GTP-binding Proteins H-Ras, Rap1A, and R-Ras with the Putative Effector Molecules Raf Kinase and Ral-Guanine Nucleotide Exchange Factor." Journal of Biological Chemistry 271, no. 12 (March 22, 1996): 6794–800. http://dx.doi.org/10.1074/jbc.271.12.6794.
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Van Meter, Margaret E. M., Ernesto Diaz-Flores, Joehleen A. Archard, Nikesh Kotecha, Amit J. Sabnis, Jonathan M. Irish, Garry P. Nolan, Kevin M. Shannon, and Benjamin S. Braun. "Multiparameter Flow Cytometric Analysis Reveals Aberrant Phosphorylation of a Network of Effector Molecules in Defined Populations of Kras Mutant Bone Marrow." Blood 106, no. 11 (November 16, 2005): 239. http://dx.doi.org/10.1182/blood.v106.11.239.239.
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Abstract Ras proteins regulate cell fates by cycling between an inactive, GDP-bound and an active, GTP-bound state. Ras activation is controlled by the opposing effects of guanine nucleotide exchange factors, which promote GTP binding, and GTPase activating proteins (GAPs), which markedly accelerate the slow intrinsic Ras GTPase. The mutant RAS alleles found in leukemia and other cancers encode proteins that accumulate in the GTP-bound conformation due to defective intrinsic GTPase activity and resistance to GAPs. These mutant Ras proteins, in turn, aberrantly activate effector pathways such as Raf/MEK/ERK, PI3 kinase/Akt, and Ral-GDS. To investigate the mechanisms by which hyperactive Ras deregulates myeloid growth, we previously generated Mx1-Cre, LSL-KrasG12D mice, which harbor a conditional oncogenic KrasG12D allele and uniformly develop a fatal myeloproliferative disease (MPD) upon induction of KrasG12D expression (Braun et al., PNAS10, 597, 2004). This robust in vivo phenotype is associated with hypersensitivity of myeloid progenitors to cytokines in methylcellulose assays and elevated levels of Ras-GTP. However, we surprisingly found no increase in basal or growth factor-induced levels of phosphorylated MEK, ERK, and Akt in bone marrow cells from KrasG12D mice with MPD. To investigate this apparent paradox, we analyzed the activation states of MEK, ERK, Akt, and STAT5 under various conditions. By increasing the stringency of serum starvation prior to stimulation with GM-CSF, serum, or both, we uncovered reproducible hyperphosphorylation of MEK, Akt, and STAT5 in KrasG12D marrow. We observed similar phosphorylation patterns in marrow from mice one week after activation of oncogenic KrasG12D expression and from mice with advanced MPD, suggesting that myeloid lineage cells do not adapt to oncogenic Kras by modulating activation of downstream effectors on this time scale. Finally, we used multiparameter flow cytometric analysis of intracellular phosphoproteins to directly assay Ras effector pathways in defined populations of primary cells. We demonstrated that the c-kit-positive, lineage-negative (c-kit+/lin−) fraction contains the colony-forming unit granulocyte-macrophage (CFU-GM) progenitors in control and KrasG12D marrow and that the GM-CSF hypersensitivity characteristic of KrasG12D marrow is retained in this fraction. This population comprises 0.2–0.9% of nucleated marrow cells, making analysis by traditional methods difficult. Cells in this population exhibited distinct patterns of ERK and STAT5 phosphorylation in comparison with whole bone marrow. Importantly, c-kit+/lin− cells from KrasG12D mice demonstrated marked hyperphosphorylation of multiple signal transduction molecules under both starved and stimulated conditions. We conclude that KrasG12D expression from the endogenous Kras locus deregulates a network of signaling molecules in hematopoietic cells. This is most evident in a defined subset of progenitors that exhibits aberrant proliferation and differentiation in vitro. Multiparameter flow cytometry is a robust methodology for analyzing the activation status of signaling networks in primary cells that can be used to monitor responses to targeted therapeutics in disease-relevant populations of cells.
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Bhattacharya, M., A. V. Babwah, and S. S. G. Ferguson. "Small GTP-binding protein-coupled receptors." Biochemical Society Transactions 32, no. 6 (October 26, 2004): 1040–44. http://dx.doi.org/10.1042/bst0321040.
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Heterotrimeric GPCRs (G-protein-coupled receptors) form the largest group of integral membrane receptor proteins and mediate diverse physiological processes. In addition to signalling via heterotrimeric G-proteins, GPCRs can also signal by interacting with various small G-proteins to regulate downstream effector pathways. The small G-protein superfamily is structurally classified into at least five families: the Ras, Rho/Rac/cdc42, Rab, Sar1/Arf and Ran families. They are monomeric G-proteins with molecular masses over the range 20–30 kDa, which function as molecular switches to control many eukaryotic cell functions. Several studies have provided evidence of crosstalk between GPCRs and small G-proteins. It is well documented that GPCR signalling through heterotrimeric G-proteins can lead to the activation of Ras and Rho GTPases. In addition, RhoA, Rabs, ARFs and ARF GEFs (guanine nucleotide-exchange factors) can associate directly with GPCRs, and GPCRs may also function as GEFs for small GTPases. In this review, we summarize the recent progress made in understanding the interaction between GPCRs and small GTPases, focusing on understanding how the association of small G-proteins with GPCRs and GPCR-regulatory proteins may influence GPCR signalling and intracellular trafficking.
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Takai, Yoshimi, Takuya Sasaki, and Takashi Matozaki. "Small GTP-Binding Proteins." Physiological Reviews 81, no. 1 (January 1, 2001): 153–208. http://dx.doi.org/10.1152/physrev.2001.81.1.153.
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Small GTP-binding proteins (G proteins) exist in eukaryotes from yeast to human and constitute a superfamily consisting of more than 100 members. This superfamily is structurally classified into at least five families: the Ras, Rho, Rab, Sar1/Arf, and Ran families. They regulate a wide variety of cell functions as biological timers (biotimers) that initiate and terminate specific cell functions and determine the periods of time for the continuation of the specific cell functions. They furthermore play key roles in not only temporal but also spatial determination of specific cell functions. The Ras family regulates gene expression, the Rho family regulates cytoskeletal reorganization and gene expression, the Rab and Sar1/Arf families regulate vesicle trafficking, and the Ran family regulates nucleocytoplasmic transport and microtubule organization. Many upstream regulators and downstream effectors of small G proteins have been isolated, and their modes of activation and action have gradually been elucidated. Cascades and cross-talks of small G proteins have also been clarified. In this review, functions of small G proteins and their modes of activation and action are described.
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Wang, Li, Gang Li, and Shuzo Sugita. "RalA-Exocyst Interaction Mediates GTP-dependent Exocytosis." Journal of Biological Chemistry 279, no. 19 (February 20, 2004): 19875–81. http://dx.doi.org/10.1074/jbc.m400522200.
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Many secretory cells utilize a GTP-dependent pathway, in addition to the well characterized Ca2+-dependent pathway, to trigger exocytotic secretion. However, little is currently known about the mechanism by which this may occur. Here we show the key signaling pathway that mediates GTP-dependent exocytosis. Incubation of permeabilized PC12 cells with soluble RalA GTPase, but not RhoA or Rab3A GTPases, strongly inhibited GTP-dependent exocytosis. A Ral-binding fragment from Sec5, a component of the exocyst complex, showed a similar inhibition. Point mutations in both RalA (RalAE38R) and the Sec5 (Sec5T11A) fragment, which abolish RalA-Sec5 interaction also abolished the inhibition of GTP-dependent exocytosis. Moreover, transfection with wild-type RalA, but not RalAE38R, enhanced GTP-dependent exocytosis. In contrast the RalA and the Sec5 fragment showed no inhibition of Ca2+-dependent exocytosis, but cleavage of a SNARE (solubleN-ethylmaleimide-sensitive factor attachment protein receptor) protein byBotulinumneurotoxin blocked both GTP- and Ca2+-dependent exocytosis. Our results indicate that the interaction between RalA and the exocyst complex (containing Sec5) is essential for GTP-dependent exocytosis. Furthermore, GTP- and Ca2+-dependent exocytosis use different sensors and effectors for triggering exocytosis whereas their final fusion steps are both SNARE-dependent.
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Zohn, Irene E., Marc Symons, Magdalena Chrzanowska-Wodnicka, John K. Westwick, and Channing J. Der. "Mas Oncogene Signaling and Transformation Require the Small GTP-Binding Protein Rac." Molecular and Cellular Biology 18, no. 3 (March 1, 1998): 1225–35. http://dx.doi.org/10.1128/mcb.18.3.1225.
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ABSTRACT The Mas oncogene encodes a novel G-protein-coupled receptor that was identified originally as a transforming protein when overexpressed in NIH 3T3 cells. The mechanism and signaling pathways that mediate Mas transformation have not been determined. We observed that the foci of transformed NIH 3T3 cells caused by Mas were similar to those caused by activated Rho and Rac proteins. Therefore, we determined if Mas signaling and transformation are mediated through activation of a specific Rho family protein. First, we observed that, like activated Rac1, Mas cooperated with activated Raf and caused synergistic transformation of NIH 3T3 cells. Second, both Mas- and Rac1-transformed NIH 3T3 cells retained actin stress fibers and showed enhanced membrane ruffling. Third, like Rac, Mas induced lamellipodium formation in porcine aortic endothelial cells. Fourth, Mas and Rac1 strongly activated the JNK and p38, but not ERK, mitogen-activated protein kinases. Fifth, Mas and Rac1 stimulated transcription from common DNA promoter elements: NF-κB, serum response factor (SRF), Jun/ATF-2, and the cyclin D1 promoter. Finally, Mas transformation and some of Mas signaling (SRF and cyclin D1 but not NF-κB activation) were blocked by dominant negative Rac1. Taken together, these observations suggest that Mas transformation is mediated in part by activation of Rac-dependent signaling pathways. Thus, Rho family proteins are common mediators of transformation by a diverse variety of oncogene proteins that include Ras, Dbl family, and G-protein-coupled oncogene proteins.
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Shieh, Angell C., and Kevin M. Shannon. "Role of Downstream Effectors in Kras-Driven Myeloproliferative Disease." Blood 110, no. 11 (November 16, 2007): 1617. http://dx.doi.org/10.1182/blood.v110.11.1617.1617.
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Abstract Mutations that deregulate Ras signaling are highly prevalent in myeloid malignancies. Previous work has shown that expressing oncogenic KrasG12D in hematopoietic cells from the endogenous Kras promoter leads to growth factor-independent and hypersensitive myeloid progenitor colony formation, and causes a fatal myeloproliferative disorder (MPD) (Braun et al PNAS101:597, 2004; Chan et al JCI113:528, 2004). These characteristics mirror the human juvenile and chronic myelomonocytic leukemias (JMML and CMML) - diseases that are associated with Ras pathway mutations. Oncogenic Ras accumulates in its active GTP-bound form and constitutively activates a number of downstream effectors. However, it is unclear which effectors are necessary for the maintenance of disease as well as how they contribute to specific phenotypes such as enhanced proliferation and defective apoptosis. We constructed second site mutants - secondary mutations on a backbone of KrasG12D(G12D) that prevent K-RasG12D from binding to a subset of effectors - to begin to elucidate the individual contributions of downstream effector pathways to hematologic disease. Murine fetal liver cells engineered to express KrasG12D,E37G(E37G) and KrasG12D,Y64G(Y64G) second site mutants maintain a hypersensitive pattern of colony-forming unit granulocyte-macrophage (CFU-GM) colony growth in response to granulocyte-macrophage colony-stimulating factor (GM-CSF), but are no longer able to grow in the absence of GM-CSF. Interestingly, both mutant proteins that display hypersensitivity also hyperactivate two major Ras effector pathways, as opposed to other second site mutants tested which only hyperactivate one major Ras effector pathway and do not show growth factor hypersensitivity. Whereas E37G activates both PI3 kinase and Ral-GDS but not Raf, Y64G stimulates Raf/MEK/ERK and PI3 kinase but not Ral-GDS. Consistent with the idea that activation of at least two effector cascades is required for aberrant in vitro growth, expression of activated effectors BrafV600E, p110a-CAAX, and RalGDS-CAAX did not confer GM-CSF hypersensitivity on CFU-GM colonies. We transplanted bone marrow cells transduced with MSCV-E37G-IRES-GFP or MSCV-Y64G-IRES-GFP retroviruses into lethally irradiated Balb/c mice. Seven of 8 mice that received Y64G cells died of T lineage acute lymphoid leukemia/lymphoma (T-ALL/L) with a median survival of 112 days. Diseased animals showed very high levels of GFP in the thymus, spleen, peripheral blood and bone marrow. Four of 9 mice injected with E37G-expressing cells mice also died of T-ALL/L with a median survival of 106 days and three of 9 died of anemia. None of the MIG mice (n=8) have died of leukemia/lymphoma. The Y64G and E37G T ALL/Ls studied to date are transplantable into sublethally irradiated recipients. We conclude that second site mutations in the KrasG12D oncogene that are defective for activation of either PI3 kinase or Raf/MEK/ERK are able to deregulate the growth of primary hematopoietic cells in vitro and in vivo. These data argue that targeting a single effector pathway downstream of oncogenic Ras may not be effective in many hematologic malignancies. We are interrogating Ras signaling networks in T-ALL/L cells and cloning MSCV integration sites to further characterize the effects of these second site mutant alleles.
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Sanford, J. C., Y. Pan, and M. Wessling-Resnick. "Properties of Rab5 N-terminal domain dictate prenylation of C-terminal cysteines." Molecular Biology of the Cell 6, no. 1 (January 1995): 71–85. http://dx.doi.org/10.1091/mbc.6.1.71.
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Rab5 is a Ras-related GTP-binding protein that is post-translationally modified by prenylation. We report here that an N-terminal domain contained within the first 22 amino acids of Rab5 is critical for efficient geranylgeranylation of the protein's C-terminal cysteines. This domain is immediately upstream from the "phosphate binding loop" common to all GTP-binding proteins and contains a highly conserved sequence recognized among members of the Rab family, referred to here as the YXYLFK motif. A truncation mutant that lacks this domain (Rab5(23-215) fails to become prenylated. However, a chimeric peptide with the conserved motif replacing cognate Rab5 sequence (MAYDYLFKRab5(23-215) does become post-translationally modified, demonstrating that the presence of this simple six amino acid N-terminal element enables prenylation at Rab5's C-terminus. H-Ras/Rab5 chimeras that include the conserved YXYLFK motif at the N-terminus do not become prenylated, indicating that, while this element may be necessary for prenylation of Rab proteins, it alone is not sufficient to confer properties to a heterologous protein to enable substrate recognition by the Rab geranylgeranyl transferase. Deletion analysis and studies of point mutants further reveal that the lysine residue of the YXYLFK motif is an absolute requirement to enable geranylgeranylation of Rab proteins. Functional studies support the idea that this domain is not required for guanine nucleotide binding since prenylation-defective mutants still bind GDP and are protected from protease digestion in the presence of GTP gamma S. We conclude that the mechanism of Rab geranylgeranylation involves key elements of the protein's tertiary structure including a conserved N-terminal amino acid motif (YXYLFK) that incorporates a critical lysine residue.
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Heyworth, P. G., U. G. Knaus, J. Settleman, J. T. Curnutte, and G. M. Bokoch. "Regulation of NADPH oxidase activity by Rac GTPase activating protein(s)." Molecular Biology of the Cell 4, no. 11 (November 1993): 1217–23. http://dx.doi.org/10.1091/mbc.4.11.1217.
Full textAbstract:
Activation of the NADPH oxidase of phagocytic cells requires the action of Rac2 or Rac1, members of the Ras superfamily of GTP-binding proteins. Rac proteins are active when in the GTP-bound form and can be regulated by a variety of proteins that modulate the exchange of GDP for GTP and/or GTP hydrolysis. The p190 Rac GTPase Activating Protein (GAP) inhibits human neutrophil NADPH oxidase activity in a cell-free assay system with a K1 of approximately 100 nM. Inhibition by p190 was prevented by GTP gamma S, a nonhydrolyzable analogue of GTP. Similar inhibition was seen with a second protein exhibiting Rac GAP activity, CDC42Hs GAP. The effect of p190 on superoxide (O2-) formation was reversed by the addition of a constitutively GTP-bound Rac2 mutant or Rac1-GTP gamma S but not by RhoA-GTP gamma S. Addition of p190 to an activated oxidase produced no inhibitory effect, suggesting either that p190 no longer has access to Rac in the assembled oxidase or that Rac-GTP is not required for activity once O2- generation has been initiated. These data confirm the role of Rac in NADPH oxidase regulation and support the view that it is the GTP form of Rac that is necessary for oxidase activation. Finally, they raise the possibility that NADPH oxidase may be regulated by the action of GAPs for Rac proteins.
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KITAMURA, Yukari, Tadahiro KITAMURA, Hiroshi SAKAUE, Tetsuo MAEDA, Hikaru UENO, Shoko NISHIO, Shigeo OHNO, et al. "Interaction of Nck-associated protein 1 with activated GTP-binding protein Rac." Biochemical Journal 322, no. 3 (March 15, 1997): 873–78. http://dx.doi.org/10.1042/bj3220873.
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Bacterially expressed glutathione S-transferase fusion proteins containing Rac1 were used to identify binding proteins of this Rho family GTPase present in a bovine brain extract. Five proteins of 85, 110, 125, 140 and 170 kDa were detected, all of which were associated exclusively with guanosine 5´-[γ-thio]triphosphate-bound Rac1, not with GDP-bound Rac1. The 85 and 110 kDa proteins were identified as the regulatory and catalytic subunits respectively of phosphatidylinositol 3-kinase. Several lines of evidence suggested that the 125 kDa protein is identical with Nck-associated protein 1 (Nap1). The mobilities of the 125 kDa protein and Nap1 on SDS/PAGE were indistinguishable, and the 125 kDa protein was depleted from brain extract by preincubation with the Src homology 3 domain of Nck to which Nap1 binds. Furthermore, antibodies to Nap1 reacted with the 125 kDa protein. Nap1 was co-immunoprecipitated with a constitutively active form of Rac expressed in Chinese hamster ovary cells. The observation that complex formation between activated Rac and PAK, but not that between Rac and Nap1, could be reproduced in vitro with recombinant proteins indicates that the interaction of Nap1 with Rac is indirect. The 140 kDa Rac-binding protein is a potential candidate for a link that connects Nap1 to Rac. The multimolecular complex comprising Rac, Nap1 and probably the 140 kDa protein might mediate some of the biological effects transmitted by the multipotent GTPase.
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de Leeuw, Hubert, Pauline Wijers-Koster, Jan van Mourik, and Jan Voorberg. "Small GTP-binding Protein RalA Associates with Weibel-Palade Bodies in Endothelial Cells." Thrombosis and Haemostasis 82, no. 09 (1999): 1177–81. http://dx.doi.org/10.1055/s-0037-1614349.
Full textAbstract:
SummaryIn endothelial cells von Willebrand factor (vWF) and P-selectin are stored in dense granules, so-called Weibel-Palade bodies. Upon stimulation of endothelial cells with a variety of agents including thrombin, these organelles fuse with the plasma membrane and release their content. Small GTP-binding proteins have been shown to control release from intracellular storage pools in a number of cells. In this study we have investigated whether small GTP-binding proteins are associated with Weibel-Palade bodies. We isolated Weibel-Palade bodies by centrifugation on two consecutive density gradients of Percoll. The dense fraction in which these subcellular organelles were highly enriched, was analysed by SDS-PAGE followed by GTP overlay. A distinct band with an apparent molecular weight of 28,000 was observed. Two-dimensional gel electrophoresis followed by GTP overlay revealed the presence of a single small GTP-binding protein with an isoelectric point of 7.1. A monoclonal antibody directed against RalA showed reactivity with the small GTP-binding protein present in subcellular fractions that contain Weibel-Palade bodies. The small GTPase RalA was previously identified on dense granules of platelets and on synaptic vesicles in nerve terminals. Our observations suggest that RalA serves a role in regulated exocytosis of Weibel-Palade bodies in endothelial cells.
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BOKOCH, Gary M., Chris J. VLAHOS, Yan WANG, Ulla G. KNAUS, and Alexis E. TRAYNOR-KAPLAN. "Rac GTPase interacts specifically with phosphatidylinositol 3-kinase." Biochemical Journal 315, no. 3 (May 1, 1996): 775–79. http://dx.doi.org/10.1042/bj3150775.
Full textAbstract:
The Rac GTP-binding proteins are members of the Rho family and regulate growth factor-stimulated actin assembly in a variety of cells. The formation of phosphorylated inositol lipids has been implicated in control of the processes initiating and regulating such actin polymerization. Associations of Rho family GTP-binding proteins with enzymes involved in lipid metabolism have been described. Here we demonstrate a direct and specific interaction of Rac proteins with phosphatidylinositol (PI) 3-kinase. This interaction is dependent upon Rac being in a GTP-bound state and requires an intact Rac effector domain. In contrast, direct binding of RhoA to PI 3-kinase could not be detected. Rac–GTP also bound to PI 3-kinase in Swiss 3T3 fibroblast and human neutrophil lysates, and increased PI 3-kinase activity became associated with Rac–GTP in platelet-derived growth factor-stimulated cells. Interaction of Rac–GTP with PI 3-kinase in vitro stimulated the activity of the enzyme by 2–9-fold. A specific interaction of active Rac with PI 3-kinase might be important in regulation of the actin cytoskeleton.
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Iida, H., S. Tanaka, and Y. Shibata. "Small GTP-binding protein, Rab6, is associated with secretory granules in atrial myocytes." American Journal of Physiology-Cell Physiology 272, no. 5 (May 1, 1997): C1594—C1601. http://dx.doi.org/10.1152/ajpcell.1997.272.5.c1594.
Full textAbstract:
Rab proteins, a subfamily of small GTP-binding proteins, have been shown to play key roles in regulation of vesicular traffic in eukaryotic cells. In this study, we have intended to identify, the atrial granule-associated Rab proteins that seem to be required for formation or intracellular transport of the granules. Atrial granules contained at least four small GTP-binding proteins, and we have demonstrated by biochemical analysis that one of the small GTP-binding proteins associated with the atrial granules is a Rab6 protein (Rab6p). Rab6p was also detected in highly purified zymogen granules of pancreatic exocrine gland. Immunogold electron microscopy performed on ultrathin cryosections of rat auricle revealed that Rab6p was associated with the atrial granule membranes. Association of Rab6p with the atrial granule membranes was also confirmed by immunodiffusion electron microscopy in agarose-embedded atrial granules. These data indicate that Rab6p is associated with the atrial granules and that it might function in the intracellular traffic of the secretory granules in the atrial myocytes.
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Prekeris, Rytis. "Rabs, Rips, FIPs, and Endocytic Membrane Traffic." Scientific World JOURNAL 3 (2003): 870–80. http://dx.doi.org/10.1100/tsw.2003.69.
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Rab GTPases, proteins belonging to the Ras-like small GTP-binding protein superfamily, have emerged as master regulators of cellular membrane transport. Rab11 GTPase, a member of the Rab protein family, plays a role in regulating various cellular functions, including plasma membrane recycling, phagocytosis, and cytokinesis. Rab11 acts by forming mutually exclusive complexes with Rab11-family binding proteins, known as FIPs. Rab11-FIP complexes serve a role of �targeting complexes� by recruiting various membrane traffic factors to cellular membranes. Recent studies have identified several Rab11-FIP complex-binding proteins that regulate distinct membrane traffic pathways.
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Schwaninger, R., H. Plutner, G. M. Bokoch, and W. E. Balch. "Multiple GTP-binding proteins regulate vesicular transport from the ER to Golgi membranes." Journal of Cell Biology 119, no. 5 (December 1, 1992): 1077–96. http://dx.doi.org/10.1083/jcb.119.5.1077.
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Using indirect immunofluorescence we have examined the effects of reagents which inhibit the function of ras-related rab small GTP-binding proteins and heterotrimeric G alpha beta gamma proteins in ER to Golgi transport. Export from the ER was inhibited by an antibody towards rab1B and an NH2-terminal peptide which inhibits ARF function (Balch, W. E., R. A. Kahn, and R. Schwaninger. 1992. J. Biol. Chem. 267:13053-13061), suggesting that both of these small GTP-binding proteins are essential for the transport vesicle formation. Export from the ER was also potently inhibited by mastoparan, a peptide which mimics G protein binding regions of seven transmembrane spanning receptors activating and uncoupling heterotrimeric G proteins from their cognate receptors. Consistent with this result, purified beta gamma subunits inhibited the export of VSV-G from the ER suggesting an initial event in transport vesicle assembly was regulated by a heterotrimeric G protein. In contrast, incubation in the presence of GTP gamma S or AIF(3-5) resulted in the accumulation of transported protein in different populations of punctate pre-Golgi intermediates distributed throughout the cytoplasm of the cell. Finally, a peptide which is believed to antagonize the interaction of rab proteins with putative downstream effector molecules inhibited transport at a later step preceding delivery to the cis Golgi compartment, similar to the site of accumulation of transported protein in the absence of NSF or calcium (Plutner, H., H. W. Davidson, J. Saraste, and W. E. Balch. 1992. J. Cell Biol. 119:1097-1116). These results are consistent with the hypothesis that multiple GTP-binding proteins including a heterotrimeric G protein(s), ARF and rab1 differentially regulate steps in the transport of protein between early compartments of the secretory pathway. The concept that G protein-coupled receptors gate the export of protein from the ER is discussed.
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Campbell, Sharon, Helen Mott, Sheng Zhong, Jonelle Drugan, and John Carpenter. "New insights into the Ras onco-protein and its interactions with the Raf-1-1 kinase." Proceedings, annual meeting, Electron Microscopy Society of America 54 (August 11, 1996): 878–79. http://dx.doi.org/10.1017/s0424820100166853.
Full textAbstract:
The Ras proteins are members of a large superfamily of nucleotide-binding proteins that cycle between active GTP- and inactive GDP-bound states. They are positively regulated by guanine nucleotide exchange factors (GEFs) that promote formation of the active GTP-bound state and negatively regulated by GTPase activating proteins (GAPs) that stimulate formation of the inactive GDP-complexed protein. Structural mutations that activate Ras oncogenic potential either impair GAP-stimulated GTPase activity or promote enhanced intrinsic nucleotide exchange. The net result of either biochemical effect is to favor elevated levels of Ras-GTP in vivo.We are investigating selected Ras variants containing substitutions at conserved sites thought to be important for guanine nucleotide binding, GTP hydrolysis, GTP/GDP interconversion and protein recognition, to better understand the role of these conserved amino acids in Ras-mediated signal transduction. Recent studies conducted at the U. of North Carolina will be presented, where we have characterized a novel activating mutation in Ras using multi-dimensional NMR spectroscopy and established a region involved in direct interaction with guanine nucleotide exchange factors.
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Le, Nguyen Quoc Khanh, Quang-Thai Ho, and Yu-Yen Ou. "Using two-dimensional convolutional neural networks for identifying GTP binding sites in Rab proteins." Journal of Bioinformatics and Computational Biology 17, no. 01 (February 2019): 1950005. http://dx.doi.org/10.1142/s0219720019500057.
Full textAbstract:
Deep learning has been increasingly and widely used to solve numerous problems in various fields with state-of-the-art performance. It can also be applied in bioinformatics to reduce the requirement for feature extraction and reach high performance. This study attempts to use deep learning to predict GTP binding sites in Rab proteins, which is one of the most vital molecular functions in life science. A functional loss of GTP binding sites in Rab proteins has been implicated in a variety of human diseases (choroideremia, intellectual disability, cancer, Parkinson’s disease). Therefore, creating a precise model to identify their functions is a crucial problem for understanding these diseases and designing the drug targets. Our deep learning model with two-dimensional convolutional neural network and position-specific scoring matrix profiles could identify GTP binding residues with achieved sensitivity of 92.3%, specificity of 99.8%, accuracy of 99.5%, and MCC of 0.92 for independent dataset. Compared with other published works, this approach achieved a significant improvement. Throughout the proposed study, we provide an effective model for predicting GTP binding sites in Rab proteins and a basis for further research that can apply deep learning in bioinformatics, especially in nucleotide binding site prediction.
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Memon, Abdul Razaque, Christiane Katja Schwager, and Karsten Niehaus. "Expression of small GTPases in the roots and nodules of Medicago truncatula cv. Jemalong." Acta Botanica Croatica 78, no. 1 (April 1, 2019): 1–8. http://dx.doi.org/10.2478/botcro-2019-0008.
Full textAbstract:
Abstract In this study we used Medicago truncatula, to identify and analyze the expression of small GTP-binding proteins (Arf1, Arl1, Sar1, Rabs, Rop/Rac) and their interacting partners in the infection process in the roots and nodules. A real-time polymerase chain reaction analysis was carried out and our results showed that Arf1 (AtArfB1c-like), MtSar1, AtRabA1e-like, AtRabC1-like, MsRab11-like and AtRop7-like genes were highly expressed in the nodules of rhizobium inoculated plants compared to the non-inoculated ones. On the contrary, AtRabA3 like, AtRab5c and MsRac1-like genes were highly expressed in non-infected nitrogen supplied roots of M. truncatula. Other Rab genes (AtRabA4a, AtRabA4c and AtRabG3a-like genes) were nearly equally expressed in both treatments. Interestingly, RbohB (a respiratory burst NADPH oxidase homologue) was more highly expressed in rhizobium infected than in non-infected roots and nodules. Our data show a differential expression pattern of small GTP-binding proteins in roots and nodules of the plants. This study demonstrates an important role of small GTP-binding proteins in symbiosome biogenesis and root nodule development in legumes.
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Ngsee, J. K., A. M. Fleming, and R. H. Scheller. "A rab protein regulates the localization of secretory granules in AtT-20 cells." Molecular Biology of the Cell 4, no. 7 (July 1993): 747–56. http://dx.doi.org/10.1091/mbc.4.7.747.
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Low molecular weight (LMW) GTP-binding proteins are hypothesized to play a role in the vectorial transport of intracellular vesicles. Mutational studies in yeast and subcellular localization in mammalian cells suggest that a family of LMW GTP-binding proteins, termed rab, target intracellular vesicles to their appropriate acceptor compartment. In this report, we demonstrate that an elasmobranch homologue of rab3A, o-rab3, plays a significant role in the sequestration of regulated secretory vesicles. When transfected into the murine endocrine cell line AtT-20, the wild-type o-rab3 protein is localized exclusively to the tips of the processes, a region of the cell known to accumulate proteins associated with regulated secretory vesicles. Two mutations, Gln81 to Leu (Q81L) and Asn135 Ile (N135I), which alter GTP binding or rate of hydrolysis, blocked the localization of the o-rab3 protein to the tips of cell processes. These mutations also hindered the sequestration of ACTH-containing secretory vesicles to the process tips but did not affect the basal or stimulated release of ACTH. Moreover, the sequestration of the protein VAMP to the process tip was also hindered by the mutation. The results demonstrate a role for the rab3 proteins in localization, sequestration, and storage of secretory vesicles near their release site.
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Zick, Michael, and William Wickner. "Phosphorylation of the effector complex HOPS by the vacuolar kinase Yck3p confers Rab nucleotide specificity for vacuole docking and fusion." Molecular Biology of the Cell 23, no. 17 (September 2012): 3429–37. http://dx.doi.org/10.1091/mbc.e12-04-0279.
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The homotypic fusion of yeast vacuoles requires the Rab-family GTPase Ypt7p and its effector complex, homotypic fusion and vacuole protein sorting complex (HOPS). Although the vacuolar kinase Yck3p is required for the sensitivity of vacuole fusion to proteins that regulate the Rab GTPase cycle—Gdi1p (GDP-dissociation inhibitor [GDI]) or Gyp1p/Gyp7p (GTPase-activating protein)—this kinase phosphorylates HOPS rather than Ypt7p. We addressed this puzzle in reconstituted proteoliposome fusion reactions with all-purified components. In the presence of HOPS and Sec17p/Sec18p, there is comparable fusion of 4-SNARE (soluble N-ethylmaleimide–sensitive factor attachment protein receptor) proteoliposomes when they have Ypt7p bearing either GDP or GTP, a striking exception to the rule that only GTP-bound forms of Ras-superfamily GTPases have active conformations. However, the phosphorylation of HOPS by recombinant Yck3p confers a strict requirement for GTP-bound Ypt7p for binding phosphorylated HOPS, for optimal membrane tethering, and for proteoliposome fusion. Added GTPase-activating protein promotes GTP hydrolysis by Ypt7p, and added GDI captures Ypt7p in its GDP-bound state during nucleotide cycling. In either case, the net conversion of Ypt7:GTP to Ypt7:GDP has no effect on HOPS binding or activity but blocks fusion mediated by phosphorylated HOPS. Thus guanine nucleotide specificity of the vacuolar fusion Rab Ypt7p is conferred through downstream posttranslational modification of its effector complex.
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Nobes, C. D., P. Hawkins, L. Stephens, and A. Hall. "Activation of the small GTP-binding proteins rho and rac by growth factor receptors." Journal of Cell Science 108, no. 1 (January 1, 1995): 225–33. http://dx.doi.org/10.1242/jcs.108.1.225.
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The small GTP-binding proteins, rho and rac, control signal transduction pathways that link growth factor receptors to the activation of actin polymerization. In Swiss 3T3 cells, rho proteins mediate the lysophosphatidic acid and bombesin-induced formation of focal adhesions and actin stress fibres, whilst rac proteins are required for the platelet-derived growth factor-, insulin-, bombesin- and phorbol ester (phorbol 12-myristate 13-acetate)-stimulated actin polymerization at the plasma membrane that results in membrane ruffling. To investigate the role of p85/p110 phosphatidylinositol 3-kinase in the rho and rac signalling pathways, we have used a potent inhibitor of this activity, wortmannin. Wortmannin has no effect on focal adhesion or actin stress fibre formation induced by lysophosphatidic acid, bombesin or microinjected recombinant rho protein. In contrast, it totally inhibits plasma membrane edge-ruffling induced by platelet-derived growth factor and insulin though not by bombesin, phorbol ester or microinjected recombinant rac protein. We conclude that phosphatidylinositol 3,4,5 trisphosphate mediates activation of rac by the platelet-derived growth factor and insulin receptors. The effects of lysophosphatidic acid on the Swiss 3T3 actin cytoskeleton can be blocked by the tyrosine kinase inhibitor, tyrphostin. Since tyrphostin does not inhibit the effects of microinjected rho protein, we conclude that lysophosphatidic acid activation of rho is mediated by a tyrosine kinase.
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Gorynia, Sabine, Todd C. Lorenz, Giancarlo Costaguta, Lydia Daboussi, Duilio Cascio, and Gregory S. Payne. "Yeast Irc6p is a novel type of conserved clathrin coat accessory factor related to small G proteins." Molecular Biology of the Cell 23, no. 22 (November 15, 2012): 4416–29. http://dx.doi.org/10.1091/mbc.e12-07-0507.
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Clathrin coat accessory proteins play key roles in transport mediated by clathrin-coated vesicles. Yeast Irc6p and the related mammalian p34 are putative clathrin accessory proteins that interact with clathrin adaptor complexes. We present evidence that Irc6p functions in clathrin-mediated traffic between the trans-Golgi network and endosomes, linking clathrin adaptor complex AP-1 and the Rab GTPase Ypt31p. The crystal structure of the Irc6p N-terminal domain revealed a G-protein fold most related to small G proteins of the Rab and Arf families. However, Irc6p lacks G-protein signature motifs and high-affinity GTP binding. Also, mutant Irc6p lacking candidate GTP-binding residues retained function. Mammalian p34 rescued growth defects in irc6∆ cells, indicating functional conservation, and modeling predicted a similar N-terminal fold in p34. Irc6p and p34 also contain functionally conserved C-terminal regions. Irc6p/p34-related proteins with the same two-part architecture are encoded in genomes of species as diverse as plants and humans. Together these results define Irc6p/p34 as a novel type of conserved clathrin accessory protein and founding members of a new G protein–like family.
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Uphues, I., T. Kolter, B. Goud, and J. Eckel. "Insulin-induced translocation of the glucose transporter GLUT4 in cardiac muscle: studies on the role of small-molecular-mass GTP-binding proteins." Biochemical Journal 301, no. 1 (July 1, 1994): 177–82. http://dx.doi.org/10.1042/bj3010177.
Full textAbstract:
Subcellular fractions obtained from rat cardiac ventricular tissue were used to elucidate a possible functional relationship between small-molecular-mass G-proteins and the insulin-responsive glucose transporter GLUT4. Proteins were separated by SDS/PAGE and transferred to nitrocellulose membranes. Incubation with [alpha-32P]GTP revealed the presence of two major distinct GTP-binding protein bands of 24 and 26 kDa in both plasma and microsomal membranes. Immunoadsorption of microsomal membranes to anti-GLUT4 antibodies was used to isolate GLUT4-enriched membrane vesicles. This material was found to contain a much decreased amount of small G-proteins, with the exclusive presence of the 24 kDa species. Insulin treatment in vivo had no effect on the microsomal membrane content of small GTP-binding proteins, but significantly decreased the 24 kDa species in GLUT4-enriched vesicles by 36 +/- 5% (n = 3). This correlated with a decreased (30-40%) recovery of GLUT4-enriched vesicles from insulin-treated animals. Western-blot analysis of microsomal membranes with a panel of antisera against rab GTP-binding proteins indicated the presence of rab4A, with a molecular mass of 24 kDa, whereas rab1A, rab2 and rab6 were not observed. rab4A was barely detectable in GLUT4-enriched vesicles; however, insulin produced an extensive shift of rab4A from the cytosol and the microsomal fraction to the plasma membrane with a parallel increase in GLUT4. These data show that a small GTP-binding protein is co-localized with GLUT4 in an insulin-responsive intracellular compartment, and strongly suggest that this protein is involved in the exocytosis of GLUT4 in cardiac muscle. Furthermore, the observed translocation of rab4A is compatible with insulin-induced endosome recycling processes, possibly including the glucose transporters.
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Rondaij, Mariska G., Ruben Bierings, Ellen L. van Agtmaal, Karina A. Gijzen, Erica Sellink, Astrid Kragt, Stephen S. G. Ferguson, et al. "Guanine exchange factor RalGDS mediates exocytosis of Weibel-Palade bodies from endothelial cells." Blood 112, no. 1 (July 1, 2008): 56–63. http://dx.doi.org/10.1182/blood-2007-07-099309.
Full textAbstract:
Abstract The small GTP-binding protein Ral has been implicated in regulated exocytosis via its interaction with the mammalian exocyst complex. We have previously demonstrated that Ral is involved in exocytosis of Weibel-Palade bodies (WPBs). Little is known about intracellular signaling pathways that promote activation of Ral in response to ligand binding of G protein–coupled receptors. Here we show that RNAi-mediated knockdown of RalGDS, an exchange factor for Ral, results in inhibition of thrombin- and epinephrine-induced exocytosis of WPBs, while overexpression of RalGDS promotes exocytosis of WPBs. A RalGDS variant lacking its exchange domain behaves in a dominant negative manner by blocking release of WPBs. We also provide evidence that RalGDS binds calmodulin (CaM) via an amino-terminal CaM-binding domain. RalGDS association to CaM is required for Ral activation because a cell-permeable peptide comprising this RalGDS CaM-binding domain inhibits Ral activation and WPB exocytosis. Together our findings suggest that RalGDS plays a vital role in the regulation of Ral-dependent WPB exocytosis after stimulation with Ca2+- or cAMP-raising agonists.
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TODAKA, Mikio, Hideki HAYASHI, Takanobu IMANAKA, Yasumasa MITANI, Seika KAMOHARA, Kazuhiro KISHI, Keisuke TAMAOKA, et al. "Roles of insulin, guanosine 5′-[γ-thio]triphosphate and phorbol 12-myristate 13-acetate in signalling pathways of GLUT4 translocation." Biochemical Journal 315, no. 3 (May 1, 1996): 875–82. http://dx.doi.org/10.1042/bj3150875.
Full textAbstract:
Insulin, guanosine 5´-[γ-thio]triphospate (GTP[S]) and phorbol 12-myristate 13-acetate (PMA) trigger the translocation of GLUT4 (type 4 glucose transporter; insulin-sensitive glucose transporter) from an intracellular pool to the cell surface. We have developed a highly sensitive and quantitative method to detect GLUT4 immunologically on the surface of intact 3T3-L1 adipocytes and Chinese hamster ovary (CHO) cells, using c-myc epitope-tagged GLUT4 (GLUT4myc). We examined the roles of insulin, GTP[S] and PMA in the signalling pathways of GLUT4 translocation in the CHO cell system. Among small molecular GTP-binding proteins, ras, rab3D, rad and rho seem to be candidates as signal transmitters of insulin-stimulated GLUT4 translocation. Overexpression of wild-type H-ras and the dominant negative mutant H-rasS17N in our cell system respectively enhanced and blocked insulin-stimulated activation of mitogen-activated protein kinase, but did not affect insulin-stimulated GLUT4 translocation. Overexpression of rab3D or rad in the cells did not affect GLUT4 translocation triggered by insulin, GTP[S] or PMA. Treatment with Botulinum C3 exoenzyme, a specific inhibitor of rho, had no effect on GLUT4 translocation induced by insulin, GTP[S] or PMA. Therefore these small molecular GTP-binding proteins are not likely to be involved in GLUT4 translocation. In addition, insulin, GTP[S] and PMA apparently stimulate GLUT4 translocation through independent pathways.
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Tang, L. H., S. A. Stoch, I. M. Modlin, and J. R. Goldenring. "Identification of rab2 as a tubulovesicle-membrane-associated protein in rabbit gastric parietal cells." Biochemical Journal 285, no. 3 (August 1, 1992): 715–19. http://dx.doi.org/10.1042/bj2850715.
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Rab proteins, which are ras-like low-molecular-mass GTP-binding proteins, are postulated to act as specific regulators of membrane trafficking in exocytosis and endocytosis. Previously, we reported a 23 kDa tubulovesicle-associated GTP-binding protein in rabbit gastric parietal cells [Basson, Goldenring, Tang, Lewis, Padfield, Jamieson & Modlin (1991) Biochem. J. 279, 43-48]. The major component of the 23 kDa protein is now identified as rab2. Rab2 was co-localized in tubulovesicle membranes from parietal cells. Consistent with GTP-binding activity (as documented before), upon maximal stimulation of parietal cells, rab2 immunoreactivity was redistributed from a 50,000 g to a 4000 g subcellular membrane fraction. The tubulovesicle-associated rab2 behaved as an integral membrane protein, since both 0.5 M-NaCl and 0.1 M-carbonate extraction failed to remove the protein from the tubulovesicle membrane. Utilizing a PCR the rab2 cDNA sequence from rabbit parietal cells was obtained, and it showed only one amino acid difference compared with the human sequence. The results of the present study provide strong evidence that parietal cells possess a rab2 protein which is tightly associated with tubulovesicle membranes.
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Morgan-Spencer, Alex, and Daniel Greenberg. "P-REX1: A Novel RAC Activing Guanine-Nucleotide Exchange Factor in Human Platelets." Blood 110, no. 11 (November 16, 2007): 3639. http://dx.doi.org/10.1182/blood.v110.11.3639.3639.
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Abstract A major signaling pathway that regulates platelet shape change and reorganization of the cytoskeleton involves the Rho family of GTPases whose members include Rac, CDC42 and RhoA. These GTPases are converted from their inactive or GDP-loaded state to the active or GTP-loaded state by a class of enzymes called Guanine-nucleotide Exchange Factors (GEFs). GEFs are a family of multi-domain proteins that contain a GDP-GTP exchange domain (DH-PH) as well as other protein interacting domains that are regulated by the activation of receptors present on the platelet surface. We used an affinity binding technique followed by mass spectrometry analysis to identify novel Rac binding GEFs from platelet lysates. Recombinant GST-Rac fusion proteins bound to agarose beads were prepared in the GTP, GDP and nucleotide-free states and incubated with human platelet lysates. Platelet lysate proteins associated with the different GST-Rac preparations (GTP-bound, GDP-bound or nucleotide-free) were eluted and run on SDS-PAGE. Gel slices were then cut out, trypsin digested and analyzed by mass spectrometry. Platelet GEFs were identified by the presence of a DH-PH domain. Using this technique we identified three novel Rac-associated platelet GEFs including P-REX1, a G-βγ protein and phosphatidylinositol (3,4,5)-triphosphate regulated GEF previously found in neutrophils and neurons (Welch, H.C.E et al., Cell108;809–822, 2002). PREX-1 is a 196kDa peptide composed of 1659 amino that specifically activates Rac in neutrophils. In addition to the DH-PH exchange domain, PREX-1 also contains tandem PDZ and DEP domains as well as significant similarity over its C-terminal half to Inositol Polyphosphate 4-Phosphatase. Western analysis with anti-P-REX1 antibody (gift of H.C. Welch, Barbraham Institute, U.K.) showed that PREX-1 is present in purified whole platelet lysates. We have also shown that platelet PREX-1 specifically associates with GST-Rac by affinity pull-down experiments. Functional studies to characterize the exchange activity of platelet PREX-1 are ongoing. PREX-1 is the only known GEF that is directly regulated by G-βγ protein (Hill, K. et al., J Biol Chem280(6):4166–4173, 2005) and represents a novel pathway for platelet G protein coupled receptor signaling through Rac in human platelets.
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Norman, J. C., L. S. Price, A. J. Ridley, A. Hall, and A. Koffer. "Actin filament organization in activated mast cells is regulated by heterotrimeric and small GTP-binding proteins." Journal of Cell Biology 126, no. 4 (August 15, 1994): 1005–15. http://dx.doi.org/10.1083/jcb.126.4.1005.
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Rat peritoneal mast cells, both intact and permeabilized, have been used widely as model secretory cells. GTP-binding proteins and calcium play a major role in controlling their secretory response. Here we have examined changes in the organization of actin filaments in intact mast cells after activation by compound 48/80, and in permeabilized cells after direct activation of GTP-binding proteins by GTP-gamma-S. In both cases, a centripetal redistribution of cellular F-actin was observed: the content of F-actin was reduced in the cortical region and increased in the cell interior. The overall F-actin content was increased. Using permeabilized cells, we show that AIF4-, an activator of heterotrimeric G proteins, induces the disassembly of F-actin at the cortex, while the appearance of actin filaments in the interior of the cell is dependent on two small GTPases, rho and rac. Rho was found to be responsible for de novo actin polymerization, presumably from a membrane-bound monomeric pool, while rac was required for an entrapment of the released cortical filaments. Thus, a heterotrimeric G-protein and the small GTPases, rho and rac, participate in affecting the changes in the actin cytoskeleton observed after activation of mast cells.
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Ridley, A. J. "Signal transduction through the GTP-binding proteins Rac and Rho." Journal of Cell Science 1994, Supplement 18 (January 1, 1994): 127–31. http://dx.doi.org/10.1242/jcs.1994.supplement_18.19.
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Pereira-Leal, José B., and Miguel C. Seabra. "Evolution of the rab family of small GTP-binding proteins." Journal of Molecular Biology 313, no. 4 (November 2001): 889–901. http://dx.doi.org/10.1006/jmbi.2001.5072.
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Sekiguchi, Takeshi, Eiji Hirose, Nobutaka Nakashima, Miki Ii, and Takeharu Nishimoto. "Novel G Proteins, Rag C and Rag D, Interact with GTP-binding Proteins, Rag A and Rag B." Journal of Biological Chemistry 276, no. 10 (November 9, 2000): 7246–57. http://dx.doi.org/10.1074/jbc.m004389200.
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Zhang, Jing, Naava Naslavsky, and Steve Caplan. "Rabs and EHDs: alternate modes for traffic control." Bioscience Reports 32, no. 1 (September 26, 2011): 17–23. http://dx.doi.org/10.1042/bsr20110009.
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Endocytic trafficking is a highly organized process regulated by a network of proteins, including the Rab family of small GTP-binding proteins and the C-terminal EHDs (Eps15 homology-domain-containing proteins). Central roles for Rab proteins have been described in vesicle budding, delivery, tethering and fusion, whereas little is known about the functions of EHDs in membrane transport. Common effectors for these two protein families have been identified, and they facilitate regulation of sequential steps in transport. By comparing and contrasting key aspects in their modes of function, we shall promote a better understanding of how Rab proteins and EHDs regulate endocytic trafficking.
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MOYERS, Julie S., Jianhua ZHU, and C. Ronald KAHN. "Effects of phosphorylation on function of the Rad GTPase." Biochemical Journal 333, no. 3 (August 1, 1998): 609–14. http://dx.doi.org/10.1042/bj3330609.
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Rad, Gem and Kir possess unique structural features in comparison with other Ras-like GTPases, including a C-terminal 31-residue extension that lacks typical prenylation motifs. We have recently shown that Rad and Gem bind calmodulin in a Ca2+-dependent manner via this C-terminal extension, involving residues 278–297 in human Rad. This domain also contains several consensus sites for serine phosphorylation, and Rad is complexed with calmodulin-dependent protein kinase II (CaMKII) in C2C12 cells. Here we show that Rad serves as a substrate for phosphorylation by CaMKII, cAMP-dependent protein kinase (PKA), protein kinase C (PKC) and casein kinase II (CKII) with stoichiometries in vitro of 0.2–1.3 mol of phosphate/mol of Rad. By deletion and point mutation analysis we show that phosphorylation by CaMKII and PKA occurs on a single serine residue at position 273, whereas PKC and CKII phosphorylate multiple C-terminal serine residues, including Ser214, Ser257, Ser273, Ser290 and Ser299. Incubation of Rad with PKA decreases GTP binding by 60–70%, but this effect seems to be independent of phosphorylation, as it is observed with the Ser273 → Ala mutant of Rad containing a mutation at the site of PKA phosphorylation. The remainder of the serine kinases have no effect on Rad GTP binding, intrinsic GTP hydrolysis or GTP hydrolysis stimulated by the putative tumour metastasis suppressor nm23. However, phosphorylation of Rad by PKC and CKII abolishes the interaction of Rad with calmodulin. These findings suggest that the binding of Rad to calmodulin, as well as its ability to bind GTP, might be regulated by the activation of several serine kinases.
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Rezaei Adariani, Soheila, Marcel Buchholzer, Mohammad Akbarzadeh, Saeideh Nakhaei-Rad, Radovan Dvorsky, and Mohammad Reza Ahmadian. "Structural snapshots of RAF kinase interactions." Biochemical Society Transactions 46, no. 6 (October 31, 2018): 1393–406. http://dx.doi.org/10.1042/bst20170528.
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RAF (rapidly accelerated fibrosarcoma) Ser/Thr kinases (ARAF, BRAF, and CRAF) link the RAS (rat sarcoma) protein family with the MAPK (mitogen-activated protein kinase) pathway and control cell growth, differentiation, development, aging, and tumorigenesis. Their activity is specifically modulated by protein–protein interactions, post-translational modifications, and conformational changes in specific spatiotemporal patterns via various upstream regulators, including the kinases, phosphatase, GTPases, and scaffold and modulator proteins. Dephosphorylation of Ser-259 (CRAF numbering) and dissociation of 14-3-3 release the RAF regulatory domains RAS-binding domain and cysteine-rich domain for interaction with RAS-GTP and membrane lipids. This, in turn, results in RAF phosphorylation at Ser-621 and 14-3-3 reassociation, followed by its dimerization and ultimately substrate binding and phosphorylation. This review focuses on structural understanding of how distinct binding partners trigger a cascade of molecular events that induces RAF kinase activation.
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FINLIN, Brian S., Haipeng SHAO, Keiko KADONO-OKUDA, Nan GUO, and Douglas A. ANDRES. "Rem2, a new member of the Rem/Rad/Gem/Kir family of Ras-related GTPases." Biochemical Journal 347, no. 1 (March 27, 2000): 223–31. http://dx.doi.org/10.1042/bj3470223.
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Here we report the molecular cloning and biochemical characterization of Rem2 (for Rem, ad and G-related 2), a novel GTP-binding protein identified on the basis of its homology with the Rem, Rad, Gem and Kir (RGK) family of Ras-related small GTP-binding proteins. Rem2 mRNA was detected in rat brain and kidney, making it the first member of the RGK family to be expressed at relatively high levels in neuronal tissues. Recombinant Rem2 binds GTP saturably and exhibits a low intrinsic rate of GTP hydrolysis. Surprisingly, the guanine nucleotide dissociation constants for both Rem2 and Rem are significantly different than the majority of the Ras-related GTPases, displaying higher dissociation rates for GTP than GDP. Localization studies with green fluorescent protein (GFP)-tagged recombinant protein fusions indicate that Rem2 has a punctate, plasma membrane localization. Deletion of the C-terminal seven amino acid residues that are conserved in all RGK family members did not affect the cellular distribution of the GFP fusion protein, whereas a larger deletion, including much of the polybasic region of the Rem2 C-terminus, resulted in its redistribution to the cytosol. Thus Rem2 is a GTPase of the RGK family with distinctive biochemical properties and possessing a novel cellular localization signal, consistent with its having a unique role in cell physiology.
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Sethi, Tariq, Mark H. Ginsberg, Julian Downward, and Paul E. Hughes. "The Small GTP-binding Protein R-Ras Can Influence Integrin Activation by Antagonizing a Ras/Raf-initiated Integrin Suppression Pathway." Molecular Biology of the Cell 10, no. 6 (June 1999): 1799–809. http://dx.doi.org/10.1091/mbc.10.6.1799.
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The rapid modulation of ligand-binding affinity (“activation”) is a central property of the integrin family of cell adhesion receptors. The small GTP-binding protein Ras and its downstream effector kinase Raf-1 suppress integrin activation. In this study we explored the relationship between Ras and the closely related small GTP-binding protein R-Ras in modulating the integrin affinity state. We found that R-Ras does not seem to be a direct activator of integrins in Chinese hamster ovary cells. However, we observed that GTP-bound R-Ras strongly antagonizes the Ras/Raf-initiated integrin suppression pathway. Furthermore, this reversal of the Ras/Raf suppressor pathway does not seem to be via a competition between Ras and R-Ras for common downstream effectors or via an inhibition of Ras/Raf-induced MAP kinase activation. Thus, R-Ras and Ras may act in concert to regulate integrin affinity via the activation of distinct downstream effectors.
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Chuang, T. H., X. Xu, L. A. Quilliam, and G. M. Bokoch. "SmgGDS stabilizes nucleotide-bound and -free forms of the Rac1 GTP-binding protein and stimulates GTP/GDP exchange through a substituted enzyme mechanism." Biochemical Journal 303, no. 3 (November 1, 1994): 761–67. http://dx.doi.org/10.1042/bj3030761.
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The Rac proteins, Rac1 and Rac2, are essential components of the NADPH oxidase system of phagocytes and regulate the actin assembly associated with membrane ruffling. These functions are controlled by the GTP-bound form of Rac. The biochemical interaction between Rac and its only known GDP-dissociation stimulator (termed smgGDS) was characterized. SmgGDS was able to stimulate the incorporation of guanosine 5′-[gamma-thio]-triphosphate GTP[gamma S] into the RhoA, Rac2, Rac1, Rap1A and CDC42Hs GTP-binding proteins, but the activity was greatest toward RhoA and Rac2. Isoprenoid modification of these proteins was not absolutely required for the interaction with smgGDS. Interestingly, the activity of smgGDS toward Rac1 could not be observed in a [3H]GDP/GTP exchange assay under conditions where it stimulated incorporation of GTP[gamma S] into Rac1. We determined that smgGDS prevented the loss of Rac1 activity during the [3H]GDP/GTP exchange assay by demonstrating the ability of smgGDS to inhibit the loss of Rac1 GTP[gamma S]-binding during incubations at 30 degrees C. This stabilizing effect was exactly counterbalanced by the ability of smgGDS to stimulate the release of [3H]GDP from Rac1, thereby producing no net observable effect in the exchange assay. SmgGDS was able to effectively stimulate the release of GDP but not GTP[gamma S] from Rac1. SmgGDS maintains Rac1 in a nucleotide-free form after release of GDP, indicating that the reaction between Rac1 and smgGDS involves a substituted enzyme mechanism.