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Molecular and Cellular Biology, January 2007, p. 497-509, Vol. 27, No. 2
0270-7306/07/$08.00+0     doi:10.1128/MCB.01772-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Calcium-Dependent Regulation of NEMO Nuclear Export in Response to Genotoxic Stimuli{triangledown}

Craig M. Berchtold,1 Zhao-Hui Wu,1 Tony T. Huang,2 and Shigeki Miyamoto1*

Department of Pharmacology, 301 SMI, 1300 University Avenue, University of Wisconsin, Madison, Wisconsin 53706,1 Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, M642, Boston, Massachusetts 021152

Received 19 September 2006/ Accepted 20 October 2006

The mechanisms involved in activation of the transcription factor NF-{kappa}B by genotoxic agents are not well understood. Previously, we provided evidence that a regulatory subunit of the I{kappa}B kinase (IKK) complex, NF-{kappa}B essential modulator (NEMO)/IKK{gamma}, is a component of a nuclear signal that is generated after DNA damage to mediate NF-{kappa}B activation. Here, we found that etoposide (VP16) and camptothecin induced increases in intracellular free calcium levels at 60 min after stimulation of CEM T leukemic cells. Inhibition of calcium increases by calcium chelators, BAPTA-AM and EGTA-AM, abrogated NF-{kappa}B activation by these agents in several cell types examined. Conversely, thapsigargin and ionomycin attenuated the BAPTA-AM effects and promoted NF-{kappa}B activation by the genotoxic stimuli. Analyses of nuclear NEMO levels in VP16-treated cells suggested that calcium was required for nuclear export of NEMO. Inhibition of the nuclear exporter CRM1 by leptomycin B did not interfere with NEMO nuclear export. Similarly, deficiency of a plausible calcium-dependent nuclear export receptor, calreticulin, failed to prevent NF-{kappa}B activation by VP16. However, temperature inactivation of the Ran guanine nucleotide exchange factor RCC1 in the tsBN2 cell line harboring a temperature-sensitive mutant of RCC1 blocked NF-{kappa}B activation induced by genotoxic stimuli. Overexpression of Ran in this cell model showed that DNA damage stimuli induced formation of a complex between Ran and NEMO, suggesting that RCC1 regulated NF-{kappa}B activation through the modulation of RanGTP. Indeed, evidence for VP16-inducible interaction between Ran-GTP and NEMO could be obtained by means of glutathione S-transferase (GST) pull-down assays using GST fused to the Ran binding domain of RanBP2, which specifically interacts with the GTP-bound form of Ran. BAPTA-AM did not alter these interactions, suggesting that calcium is a necessary step beyond the formation of a Ran-GTP-NEMO complex in the nucleus. These results suggest that calcium has a unique role in genotoxic stress-induced NF-{kappa}B signaling by regulating nuclear export of NEMO subsequent to the formation of a nuclear export complex composed of Ran-GTP, NEMO, and presumably, an undefined nuclear export receptor.

* Corresponding author. Mailing address: Department of Pharmacology, 301 SMI, 1300 University Avenue, University of Wisconsin, Madison, WI 53706. Phone: (608) 262-9281. Fax. (608) 262-1257. E-mail: smiyamot{at}wisc.edu.

{triangledown} Published ahead of print on 30 October 2006.

Molecular and Cellular Biology, January 2007, p. 497-509, Vol. 27, No. 2
0270-7306/07/$08.00+0     doi:10.1128/MCB.01772-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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