A conserved, N-terminal tyrosine signal directs Ras for inhibition by Rabex-5


Autoři: Chalita Washington aff001;  Rachel Chernet aff001;  Rewatee H. Gokhale aff001;  Yesenia Martino-Cortez aff001;  Hsiu-Yu Liu aff006;  Ashley M. Rosenberg aff001;  Sivan Shahar aff001;  Cathie M. Pfleger aff001
Působiště autorů: Department of Oncological Sciences, The Icahn School of Medicine at Mount Sinai, New York, New York, United States of America aff001;  University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America aff002;  The Graduate School of Biomedical Sciences, The Icahn School of Medicine at Mount Sinai, New York, New York, United States of America aff003;  The Tisch Cancer Institute, The Icahn School of Medicine at Mount Sinai, New York, New York, United States of America aff004;  Tufts University School of Medicine, Boston, Massachusetts, United States of America aff005;  Memorial Sloan Kettering Cancer Center, New York, New York, United States of America aff006;  Columbia University, New York, New York, United States of America aff007;  New York Medical College, Valhalla, New York, United States of America aff008
Vyšlo v časopise: A conserved, N-terminal tyrosine signal directs Ras for inhibition by Rabex-5. PLoS Genet 16(6): e32767. doi:10.1371/journal.pgen.1008715
Kategorie: Research Article
doi: 10.1371/journal.pgen.1008715

Souhrn

Dysregulation of the Ras oncogene in development causes developmental disorders, “Rasopathies,” whereas mutational activation or amplification of Ras in differentiated tissues causes cancer. Rabex-5 (also called RabGEF1) inhibits Ras by promoting Ras mono- and di-ubiquitination. We report here that Rabex-5-mediated Ras ubiquitination requires Ras Tyrosine 4 (Y4), a site of known phosphorylation. Ras substitution mutants insensitive to Y4 phosphorylation did not undergo Rabex-5-mediated ubiquitination in cells and exhibited Ras gain-of-function phenotypes in vivo. Ras Y4 phosphomimic substitution increased Rabex-5-mediated ubiquitination in cells. Y4 phosphomimic substitution in oncogenic Ras blocked the morphological phenotypes associated with oncogenic Ras in vivo dependent on the presence of Rabex-5. We developed polyclonal antibodies raised against an N-terminal Ras peptide phosphorylated at Y4. These anti-phospho-Y4 antibodies showed dramatic recognition of recombinant wild-type Ras and RasG12V proteins when incubated with JAK2 or SRC kinases but not of RasY4F or RasY4F,G12V recombinant proteins suggesting that JAK2 and SRC could promote phosphorylation of Ras proteins at Y4 in vitro. Anti-phospho-Y4 antibodies also showed recognition of RasG12V protein, but not wild-type Ras, when incubated with EGFR. A role for JAK2, SRC, and EGFR (kinases with well-known roles to activate signaling through Ras), to promote Ras Y4 phosphorylation could represent a feedback mechanism to limit Ras activation and thus establish Ras homeostasis. Notably, rare variants of Ras at Y4 have been found in cerebellar glioblastomas. Therefore, our work identifies a physiologically relevant Ras ubiquitination signal and highlights a requirement for Y4 for Ras inhibition by Rabex-5 to maintain Ras pathway homeostasis and to prevent tissue transformation.

Klíčová slova:

Carcinogenesis – Drosophila melanogaster – Eyes – Phenotypes – Phosphorylation – Ras signaling – RNA interference – Ubiquitination


Zdroje

1. Hancock JF, Paterson H, Marshall CJ. A polybasic domain or palmitoylation is required in addition to the CAAX motif to localize p21ras to the plasma membrane. Cell 1990; 63: 133–139. doi: 10.1016/0092-8674(90)90294-o 2208277

2. Quinlan MP, Settleman J. Isoform-specific ras functions in development and cancer. Future Oncol. 2009; 5: 105–116. doi: 10.2217/14796694.5.1.105 19243303

3. Prior IA, Hancock JF. Ras trafficking, localization and compartmentalized signalling. Semin. Cell Dev. Biol. 2012; 23: 145–153. doi: 10.1016/j.semcdb.2011.09.002 21924373

4. Tartaglia M, Gelb BD. Disorders of dysregulated signal traffic through the RAS-MAPK pathway: phenotypic spectrum and molecular mechanisms. Ann. N. Y. Acad. Sci. 2010; 1214: 99–121. doi: 10.1111/j.1749-6632.2010.05790.x 20958325

5. Rauen KA. The RASopathies. Annu. Rev. Genomics Hum. Genet. 2013; 14: 355–369. doi: 10.1146/annurev-genom-091212-153523 23875798

6. Niemeyer CM. RAS diseases in children. Haematologica. 2014; 99: 1653–1662. doi: 10.3324/haematol.2014.114595 25420281

7. Bezniakow N, Gos M, Obersztyn E. The RASopathies as an example of RAS/MAPK pathway disturbances—clinical presentation and molecular pathogenesis of selected syndromes. Dev. Period Med. 2014; 18: 285–296. 25182392

8. Aoki Y, Niihori T, Inoue S, Matsubara Y. Recent advances in RASopathies. J. Hum. Genet. 2016; 61: 33–39. doi: 10.1038/jhg.2015.114 26446362

9. Bustelo XR, Crespo P, Fernández-Pisonero I, Rodríguez-Fdez S. RAS GTPase-dependent pathways in developmental diseases: old guys, new lads, and current challenges. Curr. Opin. Cell Biol. 2018; 55: 42–51. doi: 10.1016/j.ceb.2018.06.007 30007125

10. Stout MC, Campbell PM. RASpecting the oncogene: New pathways to therapeutic advances. Biochem. Pharmacol. 2018; 158: 217–228. doi: 10.1016/j.bcp.2018.10.022 30352234

11. Khan AQ, Kuttikrishnan S, Siveen KS, Prabhu KS, Shanmugakonar M, Al-Naemi HA, et al. RAS-mediated oncogenic signaling pathways in human malignancies. Semin. Cancer Biol. 2018; pii: S1044-579X: 30002–30006.

12. Simanshu DK, Nissley DV, McCormick F. RAS Proteins and Their Regulators in Human Disease. Cell 2017; 170: 17–33. doi: 10.1016/j.cell.2017.06.009 28666118

13. Yan H, Jahanshahi M, Horvath EA, Liu H-Y, Pfleger CM. Rabex-5 ubiquitin ligase activity restricts Ras signaling to establish pathway homeostasis in vivo in Drosophila. Curr. Biol. 2010; 20: 1378–1382. doi: 10.1016/j.cub.2010.06.058 20655224

14. Xu L, Lubkov V, Taylor LJ, Bar-Sagi D. Feedback regulation of Ras signaling by Rabex-5-mediated ubiquitination. Curr. Biol. 2010; 20: 1372–1377. doi: 10.1016/j.cub.2010.06.051 20655225

15. Jura N, Scotto-Lavino E, Sobczyk A, Bar-Sagi D. Differential modification of Ras proteins by ubiquitination. Mol. Cell. 2006; 21: 679–687. doi: 10.1016/j.molcel.2006.02.011 16507365

16. Yan H, Chin M-L, Horvath EA, Kane EA, Pfleger CM. Impairment of ubiquitylation by mutation in Drosophila E1 promotes both cell-autonomous and non-cell-autonomous Ras-ERK activation in vivo. J Cell Sci 2009; 122: 1461–1470. doi: 10.1242/jcs.042267 19366732

17. Zeng T, Wang Q, Fu J, Lin Q, Bi J, Ding W, et al. Impeded Nedd4-1-mediated Ras degradation underlies Ras-driven tumorigenesis. Cell Rep. 2014; 7: 871–82. doi: 10.1016/j.celrep.2014.03.045 24746824

18. Kim SE, Yoon JY, Jeong WJ, Jeon SH, Park Y, Yoon JB, et al. (2009). H-Ras is degraded by Wnt/beta-catenin signaling via beta-TrCP-mediated polyubiquitylation. J Cell Sci. 2009; 122: 842–848. doi: 10.1242/jcs.040493 19240121

19. Bigenzahn JW, Collu GM, Kartnig F, Pieraks M, Vladimer GI, Heinz LX, et al. LZTR1 is a regulator of RAS ubiquitination and signaling. Science 2018; 362: 1171–1177. doi: 10.1126/science.aap8210 30442766

20. Steklov M, Pandolfi S, Baietti MF, Batiuk A, Carai P, Najm P, et al. Mutations in LZTR1 drive human disease by dysregulating RAS ubiquitination. Science 2018; 362: 1177–1182. doi: 10.1126/science.aap7607 30442762

21. Scheele JS, Rhee JM, Boss GR. Determination of absolute amounts of GDP and GTP bound to Ras in mammalian cells: comparison of parental and Ras-overproducing NIH 3T3 fibroblasts. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1097–1100. doi: 10.1073/pnas.92.4.1097 7862641

22. Khrenova MG, Mironov VA, Grigorenko BL, Nemukhin AV. Modeling the role of G12V and G13V Ras mutations in the Ras-GAP-catalyzed hydrolysis reaction of guanosine triphosphate. Biochemistry 2014; 53: 7093–7099. doi: 10.1021/bi5011333 25339142

23. Mayya V, Lundgren DH, Hwang SI, Rezaul K, Wu L, Eng JK, et al. Quantitative phosphoproteomic analysis of T cell receptor signaling reveals system-wide modulation of protein-protein interactions. Sci. Signal. 2009; 2: ra46. doi: 10.1126/scisignal.2000007 19690332

24. Bunda S, Heir P, Srikumar T, Cook JD, Burrell K, Kano Y, et al. Src promotes GTPase activity of Ras via tyrosine 32 phosphorylation. Proc. Natl. Acad. Sci. U. S. A. 2014; 111: E3785–E3794. doi: 10.1073/pnas.1406559111 25157176

25. Stokes MP, Farnsworth CL, Moritz A, Silva JC, Jia X, Lee KA. et al. PTMScan direct: identification and quantification of peptides from critical signaling proteins by immunoaffinity enrichment coupled with LC-MS/MS. Mol. Cell Proteomics 2012; 11: 187–201. doi: 10.1074/mcp.M111.015883 22322096

26. Santamaria A, Wang B, Elowe S, Malik R, Zhang F, Bauer M, et al. The Plk1-dependent phosphoproteome of the early mitotic spindle. Mol. Cell Proteomics 2011; 10: M110.004457.

27. Zhou H, Di Palma S, Preisinger C, Peng M, Polat AN, Heck AJ, et al. Toward a comprehensive characterization of a human cancer cell phosphoproteome. J. Proteome Res. 2013; 12: 260–271. doi: 10.1021/pr300630k 23186163

28. Ting PY, Johnson CW, Fang C, Cao X, Graeber TG, Mattos C, et al. Tyrosine phosphorylation of RAS by ABL allosterically enhances effector binding. FASEB J. 2015; 29: 3750–3761. doi: 10.1096/fj.15-271510 25999467

29. Moritz A, Li Y, Guo A, Villén J, Wang Y, MacNeill J, et al. Akt-RSK-S6 kinase signaling networks activated by oncogenic receptor tyrosine kinases. Sci Signal. 2010; 3: ra64. doi: 10.1126/scisignal.2000998 20736484

30. Takahashi T, Serada S, Ako M, Fujimoto M, Miyazaki Y, et al. New findings of kinase switching in gastrointestinal stromal tumor under imatinib using phosphoproteomic analysis. Int. J. Cancer. 2013; 133: 2737–2743. doi: 10.1002/ijc.28282 23716303

31. Reimels TA, Pfleger CM. Drosophila Rabex-5 restricts Notch activity in hematopoietic cells and maintains hematopoietic homeostasis. J Cell Sci. 2015; 128: 4512–4525. doi: 10.1242/jcs.174433 26567216

32. Argetsinger LS, Kouadio JL, Steen H, Stensballe A, Jensen ON, Carter-Su C. Autophosphorylation of JAK2 on tyrosines 221 and 570 regulates its activity. Mol Cell Biol. 2004; 24: 4955–4967. doi: 10.1128/MCB.24.11.4955-4967.2004 15143187

33. Songyang Z, Carraway KL 3rd, Eck MJ, Harrison SC, Feldman RA, Mohammadi M, et al. Catalytic specificity of protein-tyrosine kinases is critical for selective signalling. Nature. 1995; 373: 536–539. doi: 10.1038/373536a0 7845468

34. Lindquist JA, Simeoni L, Schraven B. Transmembrane adapters: attractants for cytoplasmic effectors. Immunol Rev. 2003; 191: 165–182. doi: 10.1034/j.1600-065x.2003.00007.x 12614359

35. Songyang Z, Cantley LC. Recognition and specificity in protein tyrosine kinase-mediated signalling. Trends Biochem Sci. 1995; 20: 470–475. doi: 10.1016/s0968-0004(00)89103-3 8578591

36. Skowyra D, Craig KL, Tyers M, Elledge SJ, Harper JW. F-box proteins are receptors that recruit phosphorylated substrates to the SCF ubiquitin-ligase complex. Cell 1997; 91: 209–219. doi: 10.1016/s0092-8674(00)80403-1 9346238

37. Skaar J R, Pagan J K, Pagano M. Mechanisms and function of substrate recruitment by F-box proteins. Nature Rev. Mol. Cell Biol. 2013; 14: 369–381.

38. Filipčík P, Curry JR, Mace PD. When Worlds Collide-Mechanisms at the Interface between Phosphorylation and Ubiquitination. J Mol Biol. 2017; 429: 1097–1113. doi: 10.1016/j.jmb.2017.02.011 28235544

39. Wagner SA, Beli P, Weinert BT, Schölz C, Kelstrup CD, Young C, et al. Proteomic analyses reveal divergent ubiquitylation site patterns in murine tissues. Mol Cell Proteomics. 2012; 11:1578–1585. doi: 10.1074/mcp.M112.017905 22790023

40. Sasaki AT, Carracedo A, Locasale JW, Anastasiou D, Takeuchi K, Kahoud ER, et al. Ubiquitination of K-Ras enhances activation and facilitates binding to select downstream effectors. Sci Signal. 2011; 4: ra13. doi: 10.1126/scisignal.2001518 21386094

41. Baker R, Wilkerson EM, Sumita K, Isom DG, Sasaki AT, Dohlman HG, et al. Differences in the regulation of K-Ras and H-Ras isoforms by monoubiquitination. J Biol Chem. 2013; 288: 36856–36862. doi: 10.1074/jbc.C113.525691 24247240

42. Mertins P, Qiao JW, Patel J, Udeshi ND, Clauser KR, Mani DR, et al. Integrated proteomic analysis of post-translational modifications by serial enrichment. Nat Methods. 2013;10: 634–637. doi: 10.1038/nmeth.2518 23749302

43. Udeshi ND, Svinkina T, Mertins P, Kuhn E, Mani DR, Qiao JW, et al. Refined preparation and use of anti-diglycine remnant (K-ε-GG) antibody enables routine quantification of 10,000s of ubiquitination sites in single proteomics experiments. Mol Cell Proteomics. 2013; 12: 825–831. doi: 10.1074/mcp.O112.027094 23266961

44. Wagner SA, Beli P, Weinert BT, Nielsen ML, Cox J, Mann M, et al. A proteome-wide, quantitative survey of in vivo ubiquitylation sites reveals widespread regulatory roles. Mol Cell Proteomics. 2011; 10: M111.013284.

45. Povlsen LK, Beli P, Wagner SA, Poulsen SL, Sylvestersen KB, Poulsen JW, et al. Systems-wide analysis of ubiquitylation dynamics reveals a key role for PAF15 ubiquitylation in DNA-damage bypass. Nat Cell Biol. 2012; 14: 1089–1098. doi: 10.1038/ncb2579 23000965

46. Danielsen JM, Sylvestersen KB, Bekker-Jensen S, Szklarczyk D, Poulsen JW, Horn H, et al. Mass spectrometric analysis of lysine ubiquitylation reveals promiscuity at site level. Mol Cell Proteomics. 2011; 10: M110.003590.

47. Hobbs GA, Gunawardena HP, Baker R, Campbell SL. Site-specific monoubiquitination activates Ras by impeding GTPase-activating protein function. Small GTPases. 2013; 4: 186–192. doi: 10.4161/sgtp.26270 24030601

48. Milinkovic VP, Skender Gazibara MK, Manojlovic Gacic EM, Gazibara TM, Tanic NT. The impact of TP53 and RAS mutations on cerebellar glioblastomas. Exp. Mol. Pathol. 2014; 97: 202–207. doi: 10.1016/j.yexmp.2014.07.009 25036404


Článek vyšel v časopise

PLOS Genetics


2020 Číslo 6

Nejčtenější v tomto čísle

Tomuto tématu se dále věnují…


Kurzy

Zvyšte si kvalifikaci online z pohodlí domova

Nová éra v léčbě migrény
nový kurz
Autoři: MUDr. Eva Medová, MUDr. Tomáš Nežádal, Ph.D.

Imunitní trombocytopenie (ITP) u dospělých pacientů
Autoři: prof. MUDr. Tomáš Kozák, Ph.D., MBA

Pěnová skleroterapie
Autoři: MUDr. Marek Šlais

White paper - jak vidíme optimální péči o zubní náhrady
Autoři: MUDr. Jindřich Charvát, CSc.

Hemofilie - série kurzů

Všechny kurzy
Kurzy Doporučená témata Časopisy
Přihlášení
Zapomenuté heslo

Nemáte účet?  Registrujte se

Zapomenuté heslo

Zadejte e-mailovou adresu se kterou jste vytvářel(a) účet, budou Vám na ni zaslány informace k nastavení nového hesla.

Přihlášení

Nemáte účet?  Registrujte se

VIRTUÁLNÍ ČEKÁRNA ČR Jste praktický lékař nebo pediatr? Zapojte se! Jste praktik nebo pediatr? Zapojte se!

×