Crystal structures of p120RasGAP N-terminal SH2 domain in its apo form and in complex with a p190RhoGAP phosphotyrosine peptide


Autoři: Rachel Jaber Chehayeb aff001;  Amy L. Stiegler aff003;  Titus J. Boggon aff002
Působiště autorů: Yale College, New Haven, Connecticut, United States of America aff001;  Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, United States of America aff002;  Department of Pharmacology, Yale University, New Haven, Connecticut, United States of America aff003;  Yale Cancer Center, Yale University, New Haven, Connecticut, United States of America aff004
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article
doi: 10.1371/journal.pone.0226113

Souhrn

The Rho and Ras pathways play vital roles in cell growth, division and motility. Cross-talk between the pathways amplifies their roles in cell proliferation and motility and its dysregulation is involved in disease pathogenesis. One important interaction for cross-talk occurs between p120RasGAP (RASA1), a GTPase activating protein (GAP) for Ras, and p190RhoGAP (p190RhoGAP-A, ARHGAP35), a GAP for Rho. The binding of these proteins is primarily mediated by two SH2 domains within p120RasGAP engaging phosphorylated tyrosines of p190RhoGAP, of which the best studied is pTyr-1105. To better understand the interaction between p120RasGAP and p190RhoGAP, we determined the 1.75 Å X-ray crystal structure of the N-terminal SH2 domain of p120RasGAP in the unliganded form, and its 1.6 Å co-crystal structure in complex with a synthesized phosphotyrosine peptide, EEENI(p-Tyr)SVPHDST, corresponding to residues 1100–1112 of p190RhoGAP. We find that the N-terminal SH2 domain of p120RhoGAP has the characteristic SH2 fold encompassing a central beta-sheet flanked by two alpha-helices, and that peptide binding stabilizes specific conformations of the βE-βF loop and arginine residues R212 and R231. Site-directed mutagenesis and native gel shifts confirm phosphotyrosine binding through the conserved FLVR motif arginine residue R207, and isothermal titration calorimetry finds a dissociation constant of 0.3 ± 0.1 μM between the phosphopeptide and SH2 domain. These results demonstrate that the major interaction between two important GAP proteins, p120RasGAP and p190RhoGAP, is mediated by a canonical SH2-pTyr interaction.

Klíčová slova:

Arginine – Crystal structure – Crystals – Phosphorylation – Protein interactions – SH2 domains – Ras signaling – Isothermal titration calorimetry


Zdroje

1. Barbacid M. ras genes. Annu Rev Biochem. 1987;56:779–827. Epub 1987/01/01. doi: 10.1146/annurev.bi.56.070187.004023 3304147.

2. Hall A. Rho GTPases and the actin cytoskeleton. Science. 1998;279(5350):509–14. doi: 10.1126/science.279.5350.509 9438836.

3. Bryant SS, Briggs S, Smithgall TE, Martin GA, McCormick F, Chang JH, et al. Two SH2 domains of p120 Ras GTPase-activating protein bind synergistically to tyrosine phosphorylated p190 Rho GTPase-activating protein. J Biol Chem. 1995;270(30):17947–52. doi: 10.1074/jbc.270.30.17947 7629101.

4. Wildenberg GA, Dohn MR, Carnahan RH, Davis MA, Lobdell NA, Settleman J, et al. p120-catenin and p190RhoGAP regulate cell-cell adhesion by coordinating antagonism between Rac and Rho. Cell. 2006;127(5):1027–39. doi: 10.1016/j.cell.2006.09.046 17129786.

5. Kulkarni SV, Gish G, van der Geer P, Henkemeyer M, Pawson T. Role of p120 Ras-GAP in directed cell movement. J Cell Biol. 2000;149(2):457–70. Epub 2000/04/18. doi: 10.1083/jcb.149.2.457 10769036.

6. van der Geer P, Henkemeyer M, Jacks T, Pawson T. Aberrant Ras regulation and reduced p190 tyrosine phosphorylation in cells lacking p120-Gap. Mol Cell Biol. 1997;17(4):1840–7. Epub 1997/04/01. doi: 10.1128/mcb.17.4.1840 9121432.

7. Chang JH, Gill S, Settleman J, Parsons SJ. c-Src regulates the simultaneous rearrangement of actin cytoskeleton, p190RhoGAP, and p120RasGAP following epidermal growth factor stimulation. J Cell Biol. 1995;130(2):355–68. doi: 10.1083/jcb.130.2.355 7542246.

8. Sfakianos MK, Eisman A, Gourley SL, Bradley WD, Scheetz AJ, Settleman J, et al. Inhibition of Rho via Arg and p190RhoGAP in the postnatal mouse hippocampus regulates dendritic spine maturation, synapse and dendrite stability, and behavior. J Neurosci. 2007;27(41):10982–92. doi: 10.1523/JNEUROSCI.0793-07.2007 17928439.

9. Bradley WD, Hernandez SE, Settleman J, Koleske AJ. Integrin signaling through Arg activates p190RhoGAP by promoting its binding to p120RasGAP and recruitment to the membrane. Mol Biol Cell. 2006;17(11):4827–36. doi: 10.1091/mbc.E06-02-0132 16971514.

10. Arthur WT, Burridge K. RhoA inactivation by p190RhoGAP regulates cell spreading and migration by promoting membrane protrusion and polarity. Mol Biol Cell. 2001;12(9):2711–20. doi: 10.1091/mbc.12.9.2711 11553710.

11. Ridley AJ, Self AJ, Kasmi F, Paterson HF, Hall A, Marshall CJ, et al. rho family GTPase activating proteins p190, bcr and rhoGAP show distinct specificities in vitro and in vivo. EMBO J. 1993;12(13):5151–60. 8262058.

12. Stiegler AL, Boggon TJ. PseudoGTPase domains in p190RhoGAP proteins: a mini-review. Biochem Soc Trans. 2018;46(6):1713–20. Epub 2018/12/06. doi: 10.1042/BST20180481 30514771.

13. Stiegler AL, Boggon TJ. The N-terminal GTPase domain of p190RhoGAP proteins is a pseudoGTPase. Structure. 2018;26(11):1451–61. doi: 10.1016/j.str.2018.07.015 30174148.

14. Stiegler AL, Boggon TJ. p190RhoGAP proteins contain pseudoGTPase domains. Nature communications. 2017;8(1):506. Epub 2017/09/13. doi: 10.1038/s41467-017-00483-x 28894085.

15. Burbelo PD, Miyamoto S, Utani A, Brill S, Yamada KM, Hall A, et al. p190-B, a new member of the Rho GAP family, and Rho are induced to cluster after integrin cross-linking. J Biol Chem. 1995;270(52):30919–26. doi: 10.1074/jbc.270.52.30919 8537347.

16. LeClerc S, Palaniswami R, Xie BX, Govindan MV. Molecular cloning and characterization of a factor that binds the human glucocorticoid receptor gene and represses its expression. J Biol Chem. 1991;266(26):17333–40. 1894621.

17. Settleman J, Narasimhan V, Foster LC, Weinberg RA. Molecular cloning of cDNAs encoding the GAP-associated protein p190: implications for a signaling pathway from ras to the nucleus. Cell. 1992;69(3):539–49. doi: 10.1016/0092-8674(92)90454-k 1581965.

18. Roof RW, Haskell MD, Dukes BD, Sherman N, Kinter M, Parsons SJ. Phosphotyrosine (p-Tyr)-dependent and -independent mechanisms of p190 RhoGAP-p120 RasGAP interaction: Tyr 1105 of p190, a substrate for c-Src, is the sole p-Tyr mediator of complex formation. Mol Cell Biol. 1998;18(12):7052–63. doi: 10.1128/mcb.18.12.7052 9819392.

19. Haskell MD, Nickles AL, Agati JM, Su L, Dukes BD, Parsons SJ. Phosphorylation of p190 on Tyr1105 by c-Src is necessary but not sufficient for EGF-induced actin disassembly in C3H10T1/2 fibroblasts. J Cell Sci. 2001;114(Pt 9):1699–708. 11309200.

20. Hu KQ, Settleman J. Tandem SH2 binding sites mediate the RasGAP-RhoGAP interaction: a conformational mechanism for SH3 domain regulation. EMBO J. 1997;16(3):473–83. doi: 10.1093/emboj/16.3.473 9034330.

21. Hernandez SE, Settleman J, Koleske AJ. Adhesion-dependent regulation of p190RhoGAP in the developing brain by the Abl-related gene tyrosine kinase. Curr Biol. 2004;14(8):691–6. doi: 10.1016/j.cub.2004.03.062 15084284.

22. Hornbeck PV, Zhang B, Murray B, Kornhauser JM, Latham V, Skrzypek E. PhosphoSitePlus, 2014: mutations, PTMs and recalibrations. Nucleic Acids Res. 2015;43(Database issue):D512–20. doi: 10.1093/nar/gku1267 25514926.

23. Pamonsinlapatham P, Hadj-Slimane R, Lepelletier Y, Allain B, Toccafondi M, Garbay C, et al. p120-Ras GTPase activating protein (RasGAP): a multi-interacting protein in downstream signaling. Biochimie. 2009;91(3):320–8. doi: 10.1016/j.biochi.2008.10.010 19022332.

24. McCormick F, Adari H, Trahey M, Halenbeck R, Koths K, Martin GA, et al. Interaction of ras p21 proteins with GTPase activating protein. Cold Spring Harbor symposia on quantitative biology. 1988;53 Pt 2:849–54. Epub 1988/01/01. doi: 10.1101/sqb.1988.053.01.097 2855502.

25. Adari H, Lowy DR, Willumsen BM, Der CJ, McCormick F. Guanosine triphosphatase activating protein (GAP) interacts with the p21 ras effector binding domain. Science. 1988;240(4851):518–21. Epub 1988/04/22. doi: 10.1126/science.2833817 2833817.

26. Wang J, Tian X, Han R, Zhang X, Wang X, Shen H, et al. Downregulation of miR-486-5p contributes to tumor progression and metastasis by targeting protumorigenic ARHGAP5 in lung cancer. Oncogene. 2014;33(9):1181–9. Epub 2013/03/12. doi: 10.1038/onc.2013.42 23474761.

27. Lawrence MS, Stojanov P, Mermel CH, Robinson JT, Garraway LA, Golub TR, et al. Discovery and saturation analysis of cancer genes across 21 tumour types. Nature. 2014;505(7484):495–501. doi: 10.1038/nature12912 24390350.

28. Fang Y, Zhu X, Wang J, Li N, Li D, Sakib N, et al. MiR-744 functions as a proto-oncogene in nasopharyngeal carcinoma progression and metastasis via transcriptional control of ARHGAP5. Oncotarget. 2015;6(15):13164–75. Epub 2015/05/12. doi: 10.18632/oncotarget.3754 25961434.

29. Sordella R, Classon M, Hu KQ, Matheson SF, Brouns MR, Fine B, et al. Modulation of CREB activity by the Rho GTPase regulates cell and organism size during mouse embryonic development. Dev Cell. 2002;2(5):553–65. doi: 10.1016/s1534-5807(02)00162-4 12015964.

30. Su L, Pertz O, Mikawa M, Hahn K, Parsons SJ. p190RhoGAP negatively regulates Rho activity at the cleavage furrow of mitotic cells. Exp Cell Res. 2009;315(8):1347–59. Epub 2009/03/04. doi: 10.1016/j.yexcr.2009.02.014 19254711.

31. Moran MF, Polakis P, McCormick F, Pawson T, Ellis C. Protein-tyrosine kinases regulate the phosphorylation, protein interactions, subcellular distribution, and activity of p21ras GTPase-activating protein. Mol Cell Biol. 1991;11(4):1804–12. Epub 1991/04/01. doi: 10.1128/mcb.11.4.1804 2005883.

32. Bernards A, Settleman J. GAP control: regulating the regulators of small GTPases. Trends Cell Biol. 2004;14(7):377–85. Epub 2004/07/13. doi: 10.1016/j.tcb.2004.05.003 15246431.

33. Bos JL, Rehmann H, Wittinghofer A. GEFs and GAPs: critical elements in the control of small G proteins. Cell. 2007;129(5):865–77. doi: 10.1016/j.cell.2007.05.018 17540168.

34. Trahey M, McCormick F. A cytoplasmic protein stimulates normal N-ras p21 GTPase, but does not affect oncogenic mutants. Science. 1987;238(4826):542–5. Epub 1987/10/23. doi: 10.1126/science.2821624 2821624.

35. Campbell JD, Alexandrov A, Kim J, Wala J, Berger AH, Pedamallu CS, et al. Distinct patterns of somatic genome alterations in lung adenocarcinomas and squamous cell carcinomas. Nat Genet. 2016;48(6):607–16. Epub 2016/05/10. doi: 10.1038/ng.3564 27158780.

36. Chan PC, Chen HC. p120RasGAP-mediated activation of c-Src is critical for oncogenic Ras to induce tumor invasion. Cancer Res. 2012;72(9):2405–15. Epub 2012/03/14. doi: 10.1158/0008-5472.CAN-11-3078 22411953.

37. Berndt SI, Wang Z, Yeager M, Alavanja MC, Albanes D, Amundadottir L, et al. Two susceptibility loci identified for prostate cancer aggressiveness. Nature communications. 2015;6:6889. Epub 2015/05/06. doi: 10.1038/ncomms7889 25939597.

38. Antoine-Bertrand J, Duquette PM, Alchini R, Kennedy TE, Fournier AE, Lamarche-Vane N. p120RasGAP Protein Mediates Netrin-1 Protein-induced Cortical Axon Outgrowth and Guidance. J Biol Chem. 2016;291(9):4589–602. Epub 2015/12/30. doi: 10.1074/jbc.M115.674846 26710849.

39. Wang DZ, Nur EKMS, Tikoo A, Montague W, Maruta H. The GTPase and Rho GAP domains of p190, a tumor suppressor protein that binds the M(r) 120,000 Ras GAP, independently function as anti-Ras tumor suppressors. Cancer Res. 1997;57(12):2478–84. 9192829.

40. Boon LM, Mulliken JB, Vikkula M. RASA1: variable phenotype with capillary and arteriovenous malformations. Curr Opin Genet Dev. 2005;15(3):265–9. doi: 10.1016/j.gde.2005.03.004 15917201.

41. Eerola I, Boon LM, Mulliken JB, Burrows PE, Dompmartin A, Watanabe S, et al. Capillary malformation-arteriovenous malformation, a new clinical and genetic disorder caused by RASA1 mutations. Am J Hum Genet. 2003;73(6):1240–9. Epub 2003/11/26. doi: 10.1086/379793 14639529.

42. Revencu N, Boon LM, Mulliken JB, Enjolras O, Cordisco MR, Burrows PE, et al. Parkes Weber syndrome, vein of Galen aneurysmal malformation, and other fast-flow vascular anomalies are caused by RASA1 mutations. Hum Mutat. 2008;29(7):959–65. Epub 2008/05/01. doi: 10.1002/humu.20746 18446851.

43. Revencu N, Boon LM, Mendola A, Cordisco MR, Dubois J, Clapuyt P, et al. RASA1 mutations and associated phenotypes in 68 families with capillary malformation-arteriovenous malformation. Hum Mutat. 2013;34(12):1632–41. Epub 2013/09/17. doi: 10.1002/humu.22431 24038909.

44. Otwinowski Z, Minor W. Processing of X-ray diffraction data collected in oscillation mode. In: Carter CW, Sweet RM, editors. Methods in Enzymology. 276, Part A. San Diego: Academic Press (New York); 1997. p. 307–26.

45. Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N, et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr. 2010;66(Pt 2):213–21. doi: 10.1107/S0907444909052925 20124702.

46. McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ. Phaser crystallographic software. Journal of applied crystallography. 2007;40(Pt 4):658–74. doi: 10.1107/S0021889807021206 19461840.

47. Terwilliger TC, Grosse-Kunstleve RW, Afonine PV, Moriarty NW, Zwart PH, Hung LW, et al. Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard. Acta Crystallogr D Biol Crystallogr. 2008;64(Pt 1):61–9. Epub 2007/12/21. doi: 10.1107/S090744490705024X 18094468.

48. Lebedev AA, Isupov MN. Space-group and origin ambiguity in macromolecular structures with pseudo-symmetry and its treatment with the program Zanuda. Acta Crystallogr D Biol Crystallogr. 2014;70(Pt 9):2430–43. doi: 10.1107/S1399004714014795 25195756.

49. Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development of Coot. Acta Crystallogr D Biol Crystallogr. 2010;66(Pt 4):486–501. doi: 10.1107/S0907444910007493 20383002.

50. Chen VB, Arendall WB 3rd, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr. 2010;66(Pt 1):12–21. doi: 10.1107/S0907444909042073 20057044.

51. McNicholas S, Potterton E, Wilson KS, Noble ME. Presenting your structures: the CCP4mg molecular-graphics software. Acta Crystallogr D Biol Crystallogr. 2011;67(Pt 4):386–94. doi: 10.1107/S0907444911007281 21460457.

52. Krissinel E, Henrick K. Inference of macromolecular assemblies from crystalline state. J Mol Biol. 2007;372(3):774–97. doi: 10.1016/j.jmb.2007.05.022 17681537.

53. Morin A, Eisenbraun B, Key J, Sanschagrin PC, Timony MA, Ottaviano M, et al. Cutting edge: Collaboration gets the most out of software. eLife. 2013;2:e01456. http://dx.doi.org/10.7554/eLife.01456 24040512

54. Zhang ZY, Maclean D, Thieme-Sefler AM, Roeske RW, Dixon JE. A continuous spectrophotometric and fluorimetric assay for protein tyrosine phosphatase using phosphotyrosine-containing peptides. Anal Biochem. 1993;211(1):7–15. Epub 1993/05/15. doi: 10.1006/abio.1993.1224 7686722.

55. Waksman G, Kominos D, Robertson SC, Pant N, Baltimore D, Birge RB, et al. Crystal structure of the phosphotyrosine recognition domain SH2 of v-src complexed with tyrosine-phosphorylated peptides. Nature. 1992;358(6388):646–53. doi: 10.1038/358646a0 1379696.

56. Waksman G, Kuriyan J. Structure and specificity of the SH2 domain. Cell. 2004;116(2 Suppl):S45–8, 3 p following S8. doi: 10.1016/s0092-8674(04)00043-1 15055581.

57. Eck MJ, Shoelson SE, Harrison SC. Recognition of a high-affinity phosphotyrosyl peptide by the Src homology-2 domain of p56lck. Nature. 1993;362:87–91. doi: 10.1038/362087a0 7680435

58. Overduin M, Rios CB, Mayer BJ, Baltimore D, Cowburn D. Three-Dimensional Solution Structure of the src Homology 2 Domain of c-abl. Cell. 1992;70:697–704. doi: 10.1016/0092-8674(92)90437-h 1505033

59. Booker GW, Breeze AL, Downing AK, Panayotou G, Gout I, Waterfield MD, et al. Structure of SH2 domain of the p85a subunit of phosphatidylinositol-3-OH kinase. Nature. 1992;358:684–7. doi: 10.1038/358684a0 1323062

60. Songyang Z, Shoelson SE, McGlade J, Olivier P, Pawson T, Bustelo XR, et al. Specific motifs recognized by the SH2 domains of Csk 3BP2, fps/fes, Grb-2, HCP, SHC, Syk and Vav. Mol Cell Biol. 1994;14:2777–85. doi: 10.1128/mcb.14.4.2777 7511210

61. Huang H, Li L, Wu C, Schibli D, Colwill K, Ma S, et al. Defining the specificity space of the human SRC homology 2 domain. Mol Cell Proteomics. 2008;7(4):768–84. Epub 2007/10/25. doi: 10.1074/mcp.M700312-MCP200 17956856.

62. Songyang Z, Shoelson SE, Chaudhuri M, Gish G, Pawson T, Haser WG, et al. SH2 domains recognize specific phosphopeptide sequences. Cell. 1993;72(5):767–78. Epub 1993/03/12. doi: 10.1016/0092-8674(93)90404-e 7680959.

63. Hidaka M, Homma Y, Takenawa T. Highly conserved eight amino acid sequence in SH2 is important for recognition of phosphotyrosine site. Biochem Biophys Res Commun. 1991;180(3):1490–7. Epub 1991/11/14. doi: 10.1016/s0006-291x(05)81364-6 1719984.

64. Boggon TJ, Shapiro L. Screening for phasing atoms in protein crystallography. Structure. 2000;8(7):R143–9. doi: 10.1016/s0969-2126(00)00168-4 10903954 ot required.

65. Heraud C, Pinault M, Lagree V, Moreau V. p190RhoGAPs, the ARHGAP35- and ARHGAP5-Encoded Proteins, in Health and Disease. Cells. 2019;8(4). Epub 2019/04/25. doi: 10.3390/cells8040351 31013840.

66. Holm L, Rosenstrom P. Dali server: conservation mapping in 3D. Nucleic Acids Res. 2010;38(Web Server issue):W545–9. doi: 10.1093/nar/gkq366 20457744.

67. Liu BA, Engelmann BW, Nash PD. The language of SH2 domain interactions defines phosphotyrosine-mediated signal transduction. FEBS Lett. 2012;586(17):2597–605. Epub 2012/05/10. doi: 10.1016/j.febslet.2012.04.054 22569091.

68. McKercher MA, Guan X, Tan Z, Wuttke DS. Multimodal Recognition of Diverse Peptides by the C-Terminal SH2 Domain of Phospholipase C-gamma1 Protein. Biochemistry. 2017;56(16):2225–37. Epub 2017/04/05. doi: 10.1021/acs.biochem.7b00023 28376302.

69. Meyer PA, Socias S, Key J, Ransey E, Tjon EC, Buschiazzo A, et al. Data publication with the structural biology data grid supports live analysis. Nature communications. 2016;7:10882. doi: 10.1038/ncomms10882 26947396.


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