The C-terminus of H-Ras as a target for the covalent binding of reactive compounds modulating Ras-dependent pathways.
Ras proteins are crucial players in differentiation and oncogenesis and constitute important drug targets. The localization and activity of Ras proteins are highly dependent on posttranslational modifications at their C-termini. In addition to an isoprenylated cysteine, H-Ras, but not other Ras proteins, possesses two cysteine residues (C181 and C184) in the C-terminal hypervariable domain that act as palmitoylation sites in cells. Cyclopentenone prostaglandins (cyPG) are reactive lipidic mediators that covalently bind to H-Ras and activate H-Ras dependent pathways. Dienone cyPG, such as 15-deoxy-Δ(12,14)-PGJ(2) (15d-PGJ(2)) and Δ(12)-PGJ(2) selectively bind to the H-Ras hypervariable domain. Here we show that these cyPG bind simultaneously C181 and C184 of H-Ras, thus potentially altering the conformational tendencies of the hypervariable domain. Based on these results, we have explored the capacity of several bifunctional cysteine reactive small molecules to bind to the hypervariable domain of H-Ras proteins. Interestingly, phenylarsine oxide (PAO), a widely used tyrosine phosphatase inhibitor, and dibromobimane, a cross-linking agent used for cysteine mapping, effectively bind H-Ras hypervariable domain. The interaction of PAO with H-Ras takes place in vitro and in cells and blocks modification of H-Ras by 15d-PGJ(2). Moreover, PAO treatment selectively alters H-Ras membrane partition and the pattern of H-Ras activation in cells, from the plasma membrane to endomembranes. These results identify H-Ras as a novel target for PAO. More importantly, these observations reveal that small molecules or reactive intermediates interacting with spatially vicinal cysteines induce intramolecular cross-linking of H-Ras C-terminus potentially contributing to the modulation of Ras-dependent pathways.
Department of Chemical and Physical Biology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
Clara L
CL
Oeste
Beatriz
B
Díez-Dacal
Francesca
F
Bray
Mario
M
García de Lacoba
Beatriz G
BG
de la Torre
David
D
Andreu
Antonio J
AJ
Ruiz-Sánchez
Ezequiel
E
Pérez-Inestrosa
Carlota A
CA
García-Domínguez
José M
JM
Rojas
Dolores
D
Pérez-Sala
D000818:Animals; D001152:Arsenicals / Q000378:metabolism; D001665:Binding Sites; D001643:Bridged Bicyclo Compounds / Q000378:metabolism; D002460:Cell Line; D003432:Cross-Linking Reagents; D003517:Cyclopentanes; D003545:Cysteine / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D011453:Prostaglandins / Q000378:metabolism*; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection; D018631:ras Proteins / Q000378:metabolism
D001152:Arsenicals; D001643:Bridged Bicyclo Compounds; D003432:Cross-Linking Reagents; D003517:Cyclopentanes; D004791:Enzyme Inhibitors; D011453:Prostaglandins; C029341:oxophenylarsine; C037094:dibromobimane; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins; D003545:Cysteine; C013905:cyclopentenone
Biochem J. 2001 Apr 1;355(Pt 1):145-53
11256959
Science. 2005 Mar 18;307(5716):1746-52
15705808
Proc Natl Acad Sci U S A. 2007 Dec 26;104(52):20979-84
18077391
ScientificWorldJournal. 2010 Apr 13;10:655-75
20419278
Bioinformatics. 2006 Jan 15;22(2):195-201
16301204
Int J Pept Protein Res. 1990 Mar;35(3):161-214
2191922
J Biol Chem. 2003 Jul 11;278(28):26046-54
12709421
Chem Res Toxicol. 2004 Oct;17(10):1313-22
15487891
Biochim Biophys Acta. 2005 Dec 30;1746(3):274-83
16039730
Bioinformatics. 2003 May 1;19(7):874-81
12724298
J Mol Biol. 2005 Mar 11;346(5):1423-40
15713491
Nat Rev Cancer. 2003 Jun;3(6):459-65
12778136
Mol Pharmacol. 2003 Jun;63(6):1349-55
12761345
J Mol Biol. 2004 Nov 5;343(5):1391-408
15491620
J Biol Chem. 1997 Feb 14;272(7):4323-6
9020151
J Biol Chem. 1981 Feb 25;256(4):1604-7
6257680
Mol Pharmacol. 2010 Oct;78(4):723-33
20631055
J Biol Chem. 2000 Sep 22;275(38):29894-9
10896662
J Biol Chem. 2003 Nov 28;278(48):47731-43
14504278
J Am Chem Soc. 2007 Oct 10;129(40):12280-6
17880077
Nat Cell Biol. 2002 May;4(5):343-50
11988737
Methods Enzymol. 1997;277:396-404
9379925
Proc Natl Acad Sci U S A. 2003 Apr 15;100(8):4772-7
12684535
Traffic. 2010 Sep;11(9):1221-33
20573066
Cell Death Differ. 1999 Aug;6(8):722-8
10467345
PLoS One. 2008;3(12):e4015
19107210
J Biol Chem. 2009 May 8;284(19):12609-21
19276070
J Cell Biol. 2010 Oct 4;191(1):23-9
20876282
Cell Mol Life Sci. 2007 Oct;64(19-20):2575-89
17628742
PLoS One. 2009 Dec 02;4(12):e8117
19956591
Front Biosci. 2007 May 01;12:4456-72
17485388
Chem Res Toxicol. 2008 Jan;21(1):138-44
18052108
J Biol Chem. 2001 Sep 21;276(38):35530-6
11466314
Biochemistry. 2007 Jun 5;46(22):6607-16
17489560
Dev Biol. 2008 Apr 1;316(1):135-48
18281031
J Biol Chem. 2003 Dec 19;278(51):51251-60
14532268
Cell. 1989 Jun 30;57(7):1167-77
2661017
J Biol Chem. 2000 Jul 21;275(29):22037-47
10801823
Annu Rev Biochem. 1987;56:779-827
3304147
J Mass Spectrom. 2007 Nov;42(11):1474-84
17960581
J Biol Chem. 1991 Nov 5;266(31):21186-9
1939160
Biochemistry. 2001 Jul 3;40(26):7903-13
11425319
J Cell Sci. 2009 Mar 15;122(Pt 6):842-8
19240121
J Biol Chem. 2005 Oct 14;280(41):34997-5010
16079139