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Mol. Cell. Biol. doi:10.1128/MCB.01772-06
Copyright (c) 2006, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Calcium-dependent regulation of NEMO nuclear export in response to genotoxic stimuli

Department of Pharmacology, 301 SMI, 1300 University Avenue, University of Wisconsin, Madison, WI 53706, USA; Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, M642 Boston, MA 02115

^* To whom correspondence should be addressed. Email: smiyamot{at}wisc.edu.

	Abstract

The mechanisms involved in activation of the transcription factor NF-B by genotoxic agents are not well understood. Previously, we provided evidence that a regulatory subunit of the IB kinase (IKK) complex, NF-B essential modulator (NEMO)/IKK, is a component of a nuclear signal that is generated after DNA damage to mediate NF-B activation. Here, we found that etoposide (VP16) and camptothecin (CPT) induced increases in intracellular free calcium levels at 60 minutes after stimulation of CEM T leukemic cells. Inhibition of calcium increases by calcium chelators, BAPTA-AM and EGTA-AM, abrogated NF-B activation by these agents in several cell types examined. Conversely, thapsigargin and ionomycin attenuated the BAPTA-AM effects and promoted NF-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-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-B activation induced by genotoxic stimuli. Over-expression 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-B activation through the modulation of RanGTP. Indeed, evidence for VP16-inducible interaction between Ran-GTP and NEMO could be obtained by means of 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-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.