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

Craig M. Berchtold, Zhaohui Wu, Tony T. Huang, Shigeki Miyamoto

Research output: Contribution to journalArticle

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Abstract

The mechanisms involved in activation of the transcription factor NF-κB by genotoxic agents are not well understood. Previously, we provided evidence that a regulatory subunit of the IκB kinase (IKK) complex, NF-κB essential modulator (NEMO)/IKKγ, is a component of a nuclear signal that is generated after DNA damage to mediate NF-κB 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-κB activation by these agents in several cell types examined. Conversely, thapsigargin and ionomycin attenuated the BAPTA-AM effects and promoted NF-κB 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-κB 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-κB 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-κB 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-κB 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.

Original languageEnglish (US)
Pages (from-to)497-509
Number of pages13
JournalMolecular and cellular biology
Volume27
Issue number2
DOIs
StatePublished - Jan 1 2007

Fingerprint

Cell Nucleus Active Transport
Guanosine Triphosphate
Calcium
DNA Damage
Cytoplasmic and Nuclear Receptors
Glutathione Transferase
Calreticulin
Guanine Nucleotide Exchange Factors
Camptothecin
Ionomycin
Temperature
Thapsigargin
Etoposide
Transcription Factors
Phosphotransferases
Cell Line
1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cell Biology

Cite this

Calcium-dependent regulation of NEMO nuclear export in response to genotoxic stimuli. / Berchtold, Craig M.; Wu, Zhaohui; Huang, Tony T.; Miyamoto, Shigeki.

In: Molecular and cellular biology, Vol. 27, No. 2, 01.01.2007, p. 497-509.

Research output: Contribution to journalArticle

Berchtold, Craig M. ; Wu, Zhaohui ; Huang, Tony T. ; Miyamoto, Shigeki. / Calcium-dependent regulation of NEMO nuclear export in response to genotoxic stimuli. In: Molecular and cellular biology. 2007 ; Vol. 27, No. 2. pp. 497-509.
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