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  current news   Press   selected story    
     
  11th October 2010  
 

ATM and NEMO-dependent ELKS ubiquitination coordinates TAK1-mediated IKK activation in response to genotoxic stress.

 
 




Authors:
Zhao-Hui Wu1,2,*, Ee Tsin Wong3*, Yuling Shi1,2, Jixiao Niu1,2, Zhijian Chen4, Shigeki Miyamoto5 and Vinay Tergaonkar3.

1 - Department of Pathology and Laboratory Medicine
2 - Center for Cancer Research, University of Tennessee Health Science Center, Memphis, TN 38163
3 - Institute of molecular and Cell Biology. Biopolis A*STAR, Singapore 138673, Singapore
4 - Howard Hughes Medical Institute, Department of molecular biology, University of Texas Southwestern Medical Center, Dallas, TX75390
5 - Department of Pharmacology, University of Wisconsin-Madison, Madison, WI 53705
* These authors contributed equally.

Published in Molecular Cell, Oct 8, 2010 Epub ahead of print

Abstract
Activation of the transcription factor NF-κB by multiple genotoxic stimuli modulates cancer cell survival.This response is mediated by a conserved pathway involving the nuclear ATM kinase and cytoplasmic IκB kinase (IKK); however the molecular link between them remains incompletely understood. Here we show that ATM activates the IKK kinase TAK1 in a manner dependent on IKKγ/NEMO and ELKS (a protein rich in glutamate, leucine, lysine and serine). K63-linked polyubiquitination of ELKS, dependent on the ubiquitin ligase XIAP and the conjugating enzyme UBC13, allows ELKS association with TAK1 via its ubiquitin-binding subunits TAB2/3. Although NEMO mutants defective in ubiquitin binding permit ATM-dependent TAK1 activation, they block NEMO association with ELKS and IKK activation. Thus, ATM and NEMO dependent ubiquitination of ELKS leads to the ubiquitin-dependent assembly of TAK1/TAB2/3 and NEMO/IKK complexes, resulting in IKK and NF-κB activation following genotoxic stimuli.

 
 


 
 


Figure legend: XIAP regulates genotoxic agent-induced NF-κB activation through facilitating polyubiquitination of ELKS.
(A)
Hela S3 cells were treated with CPT (10 uM, 2 h) and analyzed using anti-ELKS antibody for IP. (B) HEK293 cells were transfected with HA-XIAP and ELKS-Myc constructs. Cells were treated with VP16 or left untreated. Total cell extracts were subjected to IP with anti-HA followed by immunoblotting with anti-Myc antibody. (C) Wild-type (+/+) and XIAPdeficient (-/-) MEFs were treated with TNFα (T), CPT (C), Doxorubicin (D) or left untreated. Whole cell lysates were analyzed by EMSA and immunoblotting as indicated. (D) Wild-type (+/+) and XIAPdeficient (-/-) MEFs were treated as in (C) and TAK1 activation was analyzed with a TAK1 kinase assay. (E) HEK293 cells were mock transfected or transfected with XIAP siRNA. ELKS ubiquitination upon VP16 treatment was visualized with anti-ELKS IP followed by immunoblotting with anti-Ub antibody. (F) 31 Wild-type (+/+) and XIAP-deficient (-/-) MEFs were treated with Doxorubicin or left untreated. ELKS ubiquitination was determined as in (E). (G) HEK293 cells were transfected with HA-TRAF2 along with either Flag-XIAP wildtype or HA-XIAP delRING mutant as indicated. ELKS ubiquitination were examined as in (E). Expressions of TRAF2 and XIAP in input were determined by immunoblotting as shown. (H) HEK293 cells were transfected with siRNAs targeting various E3 ligases as shown and NF-κB activation by VP16 (V) or TNFα (T) was measured by EMSA. (I) A diagram model depicting TAK1-mediated IKK and NF-κB activation upon genotoxic stress.

For more information on Assistant Professor Vinay Tergaonkar’s lab, please click here.