Dual‐Specificity Ras/Rap GTPase Activating Proteins

Abstract

The small GTP‐binding proteins Ras and Rap regulate important cellular events switching between an activated GTP‐bound form and an inactivated GDP‐bound state. Guanine exchange factors (GEFs) control the GDP to GTP‐bound cycle, while the conversion, through hydrolysis, of GTP to GDP is catalysed by GTPase‐activating proteins (GAPs). The GAPs complete Ras and Rap catalytic site for efficient GTP hydrolysis. Although Ras and Rap are highly homologous, they possess different residues in the catalytic site. In consequence, Ras and Rap GTPase‐activating proteins (RasGAPs and RapGAPs) are structurally unrelated and use different mechanisms. Surprisingly, there are several RasGAPs with dual Ras/Rap specificity: SynGAP; GAP1 family members GAP1IP4BP, Rasal and Capri; and Plexins. This characteristic was an intriguing issue for years, but today structural and biochemical studies have enlightened the overall general mechanism of these dual GAPs.

Key Concepts

  • Small GTP‐binding proteins Rap and Ras control different cellular signalling switching between inactive GDP and active GTP‐bound states.
  • Ras and Rap GTPase‐activating proteins (RasGAPs and RapGAPs) inactivate their downstream signalling catalysing the GTP hydrolysis, using RasGAP and RapGAP domains.
  • RasGAPs–Ras‐GTP interaction positions Ras Gln61, situated in switch II loop, for nucleophilic attack, while the GAP contributes an Arg (arginine finger) to neutralise unfavourable negative charges of the reaction intermediate.
  • Rap has a Thr in position 61, and RapGAPs catalyse GTP hydrolysis, contributing an Asn (Asn thumb) for nucleophilic attack.
  • There are five RasGAPs with dual Rap/Ras specificity: SynGAP, Rasal, GAP1IP4BP, Capri and Plexin.
  • Dual GAPs need extra‐GAP domains to act as RapGAPs.
  • Dual GAPs promote a specific orientation of Rap switch II, locating Gln63 as the catalytic residue.
  • Plexin‐Rap X‐ray structure showed that residues of GAP domain and an extra‐GAP motif (juxtamembrane segment) are responsible for Gln63 correct orientation.
  • SynGAP, Rasal, GAP1IP4BP, Plexin and Capri do not share the same residues, and thus they still need to be found.

Keywords: Ras; Rap; GTPase‐activating protein; dual‐specificity GAP; SynGAP; Rasal; GAP1IP4BP; Capri; Plexin

Figure 1. (a) Crystallographic structure of p120–Ras–GDP–AlFx complex (pdb 1WQ1) showing Ras in yellowish green and GAP domain in orange. A zoomed view of the catalytic site shows the residues implicated in GTP hydrolysis. (b) Crystallographic structure of the complex between the GAP domain of RapGAP1 (orange) and Rap–GDP–BeFx (yellow) (pdb 3BRW). A zoomed view of the catalytic site shows the residues implicated in GTP hydrolysis.
Figure 2. Domains and regions of human dual‐specificity Ras/RapGAPs.
Figure 3. Structure of dual‐specificity Ras/RapGAPs. (a) Model of SynGAP–Rap complex based on SynGAP C2‐GAP crystallographic structure (pdb 3BXJ). Rap in green, GAP domain in red, C2 in yellow and Rap switch II highlighted in dark blue. (b) Rasal 3D reconstruction by electron microscopy, with homologous 3D structures of the distinct domains docked inside. C2A: yellow, C2B: orange, GAP: red, PH: blue and Ct: purple. Cuellar, https://www.degruyter.com/view/j/bchm.2018.399.issue‐1/hsz‐2017‐0159/hsz‐2017‐0159.xml. (c) Crystallographic structure of PlexinC1–Rap complex (pdb 4M8N). Rap in green, GAP domain in red, juxtamembrane segment in yellow and Rap switch II highlighted in dark blue. (d) A zoomed view of the catalytic site shows the Rap and Plexin residues implicated in switch II orientation.
Figure 4. Model of common dual RAs/Rap GAP activity. Rap switch II interacts with an α‐helix of the GAP domain (α‐helix 6 of the GAP domain) and the extra‐GAP domains to achieve the catalytic orientation, with Q63 stabilising the hydrophilic water molecule for GTP hydrolysis. The GAP arginine finger completes the catalytic site neutralising negative charges at GTP γ‐phosphate.
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References

Allen M, Chu S, Brill S, Stotler C and Buckler A (1998) Restricted tissue expression pattern of a novel human rasGAP‐related gene and its murine ortholog. Gene 218: 17–25.

Battram AM, Durrant TN, Agbani EO, et al. (2017) The phosphatidylinositol 3,4,5‐trisphosphate (PI(3,4,5)P3) binder Rasa3 regulates phosphoinositide 3‐kinase (PI3K)‐dependent integrin alphaIIbbeta3 outside‐in signaling. The Journal of Biological Chemistry 292: 1691–1704.

Bernards A (2003) GAPs galore! A survey of putative Ras superfamily GTPase activating proteins in man and Drosophila. Biochimica et Biophysica Acta 1603: 47–82.

Blanc L, Ciciotte SL, Gwynn B, et al. (2012) Critical function for the Ras‐GTPase activating protein RASA3 in vertebrate erythropoiesis and megakaryopoiesis. Proceedings of the National Academy of Sciences of the United States of America 109: 12099–12104.

Bottomley JR, Reynolds JS, Lockyer PJ and Cullen PJ (1998) Structural and functional analysis of the putative inositol 1,3,4, 5‐tetrakisphosphate receptors GAP1(IP4BP) and GAP1(m). Biochemical and Biophysical Research Communications 250: 143–149.

Calvisi DF, Ladu S, Conner EA, et al. (2011) Inactivation of Ras GTPase‐activating proteins promotes unrestrained activity of wild‐type Ras in human liver cancer. Journal of Hepatology 54: 311–319.

Chen HJ, Rojas‐Soto M, Oguni A and Kennedy MB (1998) A synaptic Ras‐GTPase activating protein (p135 SynGAP) inhibited by CaM kinase II. Neuron 20: 895–904.

Chen X, Winters C, Azzam R, et al. (2008) Organization of the core structure of the postsynaptic density. Proceedings of the National Academy of Sciences of the United States of America 105: 4453–4458.

Cozier G, Sessions R, Bottomley JR, Reynolds JS and Cullen PJ (2000) Molecular modelling and site‐directed mutagenesis of the inositol 1,3,4,5‐tetrakisphosphate‐binding pleckstrin homology domain from the Ras GTPase‐activating protein GAP1IP4BP. The Biochemical Journal 349: 333–342.

Cuellar J, Valpuesta JM, Wittinghofer A and Sot B (2017) Domain topology of human Rasal. Biological Chemistry 399: 63–72.

Cullen PJ, Hsuan JJ, Truong O, et al. (1995) Identification of a specific Ins(1,3,4,5)P4‐binding protein as a member of the GAP1 family. Nature 376: 527–530.

Cullen PJ (1998) Bridging the GAP in inositol 1,3,4,5‐tetrakisphosphate signalling. Biochimica et Biophysica Acta 1436: 35–47.

Dai Y, Walker SA, de Vet E, et al. (2011) Ca2+−dependent monomer and dimer formation switches CAPRI Protein between Ras GTPase‐activating protein (GAP) and RapGAP activities. The Journal of Biological Chemistry 286: 19905–19916.

De Lisle RC (1993) A plethora of GTPases, large and small. Digestion 54: 3–8.

Gu C and Giraudo E (2013) The role of semaphorins and their receptors in vascular development and cancer. Experimental Cell Research 319: 1306–1316.

He H, Yang T, Terman JR and Zhang X (2009) Crystal structure of the plexin A3 intracellular region reveals an autoinhibited conformation through active site sequestration. Proceedings of the National Academy of Sciences of the United States of America 106: 15610–15615.

Jeyabalan N and Clement JP (2016) SYNGAP1: mind the Gap. Frontiers in Cellular Neuroscience 10: 32.

Jin H, Wang X, Ying J, et al. (2007) Epigenetic silencing of a Ca(2+)‐regulated Ras GTPase‐activating protein RASAL defines a new mechanism of Ras activation in human cancers. Proceedings of the National Academy of Sciences of the United States of America 104: 12353–12358.

Khan AQ, Kuttikrishnan S, Siveen KS, et al. (2018) RAS‐mediated oncogenic signaling pathways in human malignancies. Seminars in Cancer Biology.

Kim JH, Liao D, Lau LF and Huganir RL (1998) SynGAP: a synaptic RasGAP that associates with the PSD‐95/SAP90 protein family. Neuron 20: 683–691.

Krapivinsky G, Medina I, Krapivinsky L, Gapon S and Clapham DE (2004) SynGAP‐MUPP1‐CaMKII synaptic complexes regulate p38 MAP kinase activity and NMDA receptor‐dependent synaptic AMPA receptor potentiation. Neuron 43: 563–574.

Kupzig S, Deaconescu D, Bouyoucef D, et al. (2006) GAP1 family members constitute bifunctional Ras and Rap GTPase‐activating proteins. The Journal of Biological Chemistry 281: 9891–9900.

Kupzig S, Bouyoucef‐Cherchalli D, Yarwood S, Sessions R and Cullen PJ (2009) The ability of GAP1IP4BP to function as a Rap1 GTPase‐activating protein (GAP) requires its Ras GAP‐related domain and an arginine finger rather than an asparagine thumb. Molecular and Cellular Biology 29: 3929–3940.

Liu Q, Walker SA, Gao D, et al. (2005) CAPRI and RASAL impose different modes of information processing on Ras due to contrasting temporal filtering of Ca2+. The Journal of Cell Biology 170: 183–190.

Liu D, Yang C, Bojdani E, Murugan AK and Xing M (2013) Identification of RASAL1 as a major tumor suppressor gene in thyroid cancer. Journal of the National Cancer Institute 105: 1617–1627.

Lockyer PJ, Bottomley JR, Reynolds JS, et al. (1997) Distinct subcellular localisations of the putative inositol 1,3,4,5‐tetrakisphosphate receptors GAP1IP4BP and GAP1m result from the GAP1IP4BP PH domain directing plasma membrane targeting. Current Biology : CB 7: 1007–1010.

Lockyer PJ, Kupzig S and Cullen PJ (2001) CAPRI regulates Ca(2+)‐dependent inactivation of the Ras‐MAPK pathway. Current Biology : CB 11: 981–986.

Mao Y (2015) Hypermethylation of RASAL1: a Key for renal fibrosis. eBioMedicine 2: 7–8.

Marechal Y, Pesesse X, Jia Y, et al. (2007) Inositol 1,3,4,5‐tetrakisphosphate controls proapoptotic Bim gene expression and survival in B cells. Proceedings of the National Academy of Sciences of the United States of America 104: 13978–13983.

Mishra AK and Lambright DG (2016) Invited review: small GTPases and their GAPs. Biopolymers 105: 431–448.

Molina‐Ortiz P, Polizzi S, Ramery E, et al. (2014) Rasa3 controls megakaryocyte Rap1 activation, integrin signaling and differentiation into proplatelet. PLoS Genetics 10: e1004420.

Nakamura R, Furuno T and Nakanishi M (2006) The plasma membrane shuttling of CAPRI is related to regulation of mast cell activation. Biochemical and Biophysical Research Communications 347: 363–368.

Ohta M, Seto M, Ijichi H, et al. (2009) Decreased expression of the RAS‐GTPase activating protein RASAL1 is associated with colorectal tumor progression. Gastroenterology 136: 206–216.

Oinuma I, Ishikawa Y, Katoh H and Negishi M (2004) The Semaphorin 4D receptor Plexin‐B1 is a GTPase activating protein for R‐Ras. Science 305: 862–865.

Pannekoek WJ, Kooistra MR, Zwartkruis FJ and Bos JL (2009) Cell‐cell junction formation: the role of Rap1 and Rap1 guanine nucleotide exchange factors. Biochimica et Biophysica Acta 1788: 790–796.

Pena V, Hothorn M, Eberth A, et al. (2008) The C2 domain of SynGAP is essential for stimulation of the Rap GTPase reaction. EMBO Reports 9: 350–355.

Scheffzek K, Ahmadian MR, Kabsch W, et al. (1997) The Ras‐RasGAP complex: structural basis for GTPase activation and its loss in oncogenic Ras mutants. Science 277: 333–338.

Scrima A, Thomas C, Deaconescu D and Wittinghofer A (2008) The Rap‐RapGAP complex: GTP hydrolysis without catalytic glutamine and arginine residues. The EMBO Journal 27: 1145–1153.

Sot B, Kotting C, Deaconescu D, et al. (2010) Unravelling the mechanism of dual‐specificity GAPs. The EMBO Journal 29: 1205–1214.

Sot B, Behrmann E, Raunser S and Wittinghofer A (2013) Ras GTPase activating (RasGAP) activity of the dual specificity GAP protein Rasal requires colocalization and C2 domain binding to lipid membranes. Proceedings of the National Academy of Sciences of the United States of America 110: 111–116.

Strazza M, Azoulay‐Alfaguter I, Dun B, Baquero‐Buitrago J and Mor A (2015) CD28 inhibits T cell adhesion by recruiting CAPRI to the plasma membrane. Journal of Immunology 194: 2871–2877.

Tamagnone L, Artigiani S, Chen H, et al. (1999) Plexins are a large family of receptors for transmembrane, secreted, and GPI‐anchored semaphorins in vertebrates. Cell 99: 71–80.

Tasaka G, Negishi M and Oinuma I (2012) Semaphorin 4D/Plexin‐B1‐mediated M‐Ras GAP activity regulates actin‐based dendrite remodeling through Lamellipodin. The Journal of Neuroscience : the Official Journal of the Society for Neuroscience 32: 8293–8305.

Tong Y, Hota PK, Penachioni JY, et al. (2009) Structure and function of the intracellular region of the plexin‐b1 transmembrane receptor. The Journal of Biological Chemistry 284: 35962–35972.

Tran TS, Kolodkin AL and Bharadwaj R (2007) Semaphorin regulation of cellular morphology. Annual Review of Cell and Developmental Biology 23: 263–292.

Turner LJ, Nicholls S and Hall A (2004) The activity of the plexin‐A1 receptor is regulated by Rac. The Journal of Biological Chemistry 279: 33199–33205.

Vernoud V, Horton AC, Yang Z and Nielsen E (2003) Analysis of the small GTPase gene superfamily of Arabidopsis. Plant Physiology 131: 1191–1208.

Vigil D, Cherfils J, Rossman KL and Der CJ (2010) Ras superfamily GEFs and GAPs: validated and tractable targets for cancer therapy? Nature Reviews Cancer 10: 842–857.

Walker SA, Kupzig S, Bouyoucef D, et al. (2004) Identification of a Ras GTPase‐activating protein regulated by receptor‐mediated Ca2+ oscillations. The EMBO Journal 23: 1749–1760.

Walkup WG, Washburn L, Sweredoski MJ, et al. (2015) Phosphorylation of synaptic GTPase‐activating protein (synGAP) by Ca2+/calmodulin‐dependent protein kinase II (CaMKII) and cyclin‐dependent kinase 5 (CDK5) alters the ratio of its GAP activity toward Ras and Rap GTPases. The Journal of Biological Chemistry 290: 4908–4927.

Wang Y, He H, Srivastava N, et al. (2012) Plexins are GTPase‐activating proteins for Rap and are activated by induced dimerization. Science Signaling 5: ra6.

Wang Y, Pascoe HG, Brautigam CA, He H and Zhang X (2013) Structural basis for activation and non‐canonical catalysis of the Rap GTPase activating protein domain of plexin. eLife 2: e01279.

Worzfeld T, Swiercz JM, Senturk A, et al. (2014) Genetic dissection of plexin signaling in vivo. Proceedings of the National Academy of Sciences of the United States of America 111: 2194–2199.

Zeng M, Shang Y, Araki Y, et al. (2016) Phase transition in postsynaptic densities underlies formation of synaptic complexes and synaptic plasticity. Cell 166: 1163–1175 e1112.

Zhang J, Guo J, Dzhagalov I and He YW (2005) An essential function for the calcium‐promoted Ras inactivator in Fcgamma receptor‐mediated phagocytosis. Nature Immunology 6: 911–919.

Further Reading

Cherfils J (2014) Arf GTPases and their effectors: assembling multivalent membrane‐binding platforms. Current Opinion in Structural Biology 29: 67–76.

Haga RB and Ridley AJ (2016) Rho GTPases: regulation and roles in cancer cell biology. Small GTPases 7: 207–221.

Matchett KB, McFarlane S, Hamilton SE, et al. (2014) Ran GTPase in nuclear envelope formation and cancer metastasis. Advances in Experimental Medicine and Biology 773: 323–351.

Zhen Y and Stenmark H (2015) Cellular functions of Rab GTPases at a glance. Journal of Cell Science 128: 3171–3176.

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Sot, Begoña(Oct 2018) Dual‐Specificity Ras/Rap GTPase Activating Proteins. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0028180]