Biochemical and Biophysical Research Communications
Intermolecular disulfide bond formation in the NEMO dimer requires Cys54 and Cys347
Section snippets
Materials and methods
Plasmids and site-directed mutagenesis. pcDNA3-based vectors for expression of FLAG-NEMO and Myc-NEMO were obtained from S. Ghosh (Yale University) and S. Miyamoto (University of Wisconsin), respectively. Site-directed mutations in NEMO were generated by overlapping polymerase chain reaction-based mutagenesis and were subcloned into the relevant vectors. Retroviral vectors for wild-type NEMO or NEMO-C54/347A were created in pBABE-puro. All constructs were confirmed by DNA sequencing. Details of
NEMO dimerization is mediated through a disulfide bond
A recent study [8] reported that NEMO could be detected as a dimer on SDS–polyacrylamide gels, but that these NEMO dimers disappeared upon extended heating of protein samples in SDS sample buffer containing β-mercaptoethanaol (β-Me). These results suggested to us that NEMO was forming dimers though a disulfide bond(s) under normal protein isolation conditions. To further investigate NEMO dimer formation, we prepared cell extracts from NEMO-deficient fibroblasts overexpressing FLAG-NEMO using
Discussion
Exposure to oxidizing conditions can induce protein disulfides in many cytosolic proteins, which can sometimes affect protein function [18]. In this report, we present evidence that NEMO forms disulfide-bonded homodimers in vivo. In addition, we show that Cys54 and Cys347 of NEMO are required for disulfide bond formation by NEMO, and our predicted structural model of the NEMO dimer indicates that Cys54-Cys54 and Cys347-Cys347 disulfide bonds can form. We cannot distinguish whether there are
Acknowledgments
This research was supported by a grant from the NIH (CA47763 to TDG). MH was supported by a Pre-doctoral Fellowship from the Natural Sciences & Engineering Research Council of Canada. SC was supported by NIH grant ES03775 (to E. Loechler). WC, TE, KC and SY received funding from the Boston University Undergraduate Research Opportunities Program, and BA was supported by an NSF Undergraduate Research Grant. We thank U. Hansen for LPS, S. Bardhan and J. Porco for EqM, A. Hoffmann for
References (21)
- et al.
IKKγ/NEMO facilitates the recruitment of the IκBα proteins into the IκB kinase complex
J. Biol. Chem.
(2001) - et al.
Posttranslational modifications of NEMO and its partners in NF-κB signaling
Trends Cell Biol.
(2006) - et al.
NEMO trimerizes through its coiled-coil C-terminal domain
J. Biol. Chem.
(2002) - et al.
The trimerization domain of NEMO is composed of the interacting C-terminal CC2 and LZ coiled-coil subdomains
J. Biol. Chem.
(2004) - et al.
A point mutation in NEMO associated with anhidrotic ectodermal dysplasia with immunodeficiency pathology results in destabilization of the oligomer and reduces lipopolysaccharide- and tumor necrosis factor-mediated NF-κB activation
J. Biol. Chem.
(2006) - et al.
Inhibition of transcription factor NF-κB signaling proteins IKKβ and p65 through specific cysteine residues by epoxyquinone A monomer: correlation with its anti-cancer cell growth activity
Biochem. Pharmacol.
(2006) - et al.
Phosphorylation of serine 68 in the IκB kinase (IKK)-binding domain of NEMO interferes with the structure of the IKK complex and tumor necrosis factor-α-induced NF-κB activity
J. Biol. Chem.
(2008) - et al.
Cytokine-induced activation of NF-κB is inhibited by hydrogen peroxide through oxidative inactivation of IκB kinase
J. Biol. Chem.
(2001) Post-translational modifications regulating the activity and function of the NF-κB pathway
Oncogene
(2006)- et al.
NEMO oligomerization in the dynamic assembly of the IκB kinase core complex
FEBS J.
(2007)
Cited by (0)
- 1
Present address: Lineberger Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA.
- 2
Present address: Department of Molecular Cancer Biology, Duke University, Durham, NC 27710, USA.
- 3
Present address: Hematology/Oncology-Cancer Biology Program, Beth Israel Deaconess Medical Center, Boston, MA 02215, USA.