Human Ran cysteine 112 oxidation by pervanadate regulates its binding to keratins.
We used a proteomic approach to identify proteins that associate with keratins 8 or 18 (K8/K18) in a pervanadate-dependent manner. Pervanadate triggers Ran-K8/K18 binding and a gel-migration-shift of Ran from 25 to 27 kDa, which does not occur upon exposure to H2O2 or vanadate or if pervanadate is excluded during cell solubilization. Generation of 27-kDa Ran is not related to hyperphosphorylation, is heat-insensitive, but occurs upon conversion of Ran cysteines to cysteic acid. The pervanadate-mediated Ran cysteine --> cysteic acid oxidation and its related gel migration shift affects other proteins including actin. Mutation of the three Ran cysteines (Cys-85, -112, and -120) showed that Ran Cys-112 oxidation generates 27-kDa Ran and accounts for its keratin binding. Proteasome inhibition accentuates Ran-keratin binding after cell exposure to pervanadate. Therefore, cell-free exposure to pervanadate causes cysteine to cysteic acid oxidation of Ran and several other proteins and Ran-K8/K18 association. In cells, stabilization of oxidized Ran by proteasome inhibition promotes Ran-keratin interaction. Keratin sequestration of oxidized Ran may provide a back-up protective mechanism in some cases of oxidative injury.
Tao
Guo-Zhong
GZ
Palo Alto Veterans Affairs Medical Center, Palo Alto, California 94304, USA. guozhongtao@stanford.edu
Zhou
Qin
Q
Strnad
Pavel
P
Salemi
Michelle R
MR
Lee
Young Moo
YM
Omary
M Bishr
MB
D000199:Actins / Q000737:chemistry; D045744:Cell Line, Tumor; D002465:Cell Movement; D002470:Cell Survival; D002474:Cell-Free System; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D047468:Immunoprecipitation; D007633:Keratins / Q000737:chemistry*; D013058:Mass Spectrometry; D008954:Models, Biological; D009154:Mutation; D010100:Oxygen / Q000378:metabolism*; D010766:Phosphorylation; D061988:Proteasome Inhibitors; D011485:Protein Binding; D040901:Proteomics; D014443:Tyrosine / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology*; D020931:ran GTP-Binding Protein / Q000737:chemistry* / Q000378:metabolism
D000199:Actins; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D061988:Proteasome Inhibitors; C076715:pervanadate; D014638:Vanadates; D014443:Tyrosine; D007633:Keratins; D020931:ran GTP-Binding Protein; D003545:Cysteine; D010100:Oxygen