Quantification of cellular NEMO content and its impact on NF-κB activation by genotoxic stress.
NF-κB essential modulator, NEMO, plays a key role in canonical NF-κB signaling induced by a variety of stimuli, including cytokines and genotoxic agents. To dissect the different biochemical and functional roles of NEMO in NF-κB signaling, various mutant forms of NEMO have been previously analyzed. However, transient or stable overexpression of wild-type NEMO can significantly inhibit NF-κB activation, thereby confounding the analysis of NEMO mutant phenotypes. What levels of NEMO overexpression lead to such an artifact and what levels are tolerated with no significant impact on NEMO function in NF-κB activation are currently unknown. Here we purified full-length recombinant human NEMO protein and used it as a standard to quantify the average number of NEMO molecules per cell in a 1.3E2 NEMO-deficient murine pre-B cell clone stably reconstituted with full-length human NEMO (C5). We determined that the C5 cell clone has an average of 4 x 10(5) molecules of NEMO per cell. Stable reconstitution of 1.3E2 cells with different numbers of NEMO molecules per cell has demonstrated that a 10-fold range of NEMO expression (0.6-6x10(5) molecules per cell) yields statistically equivalent NF-κB activation in response to the DNA damaging agent etoposide. Using the C5 cell line, we also quantified the number of NEMO molecules per cell in several commonly employed human cell lines. These results establish baseline numbers of endogenous NEMO per cell and highlight surprisingly normal functionality of NEMO in the DNA damage pathway over a wide range of expression levels that can provide a guideline for future NEMO reconstitution studies.
Department of Oncology, University of Wisconsin, Madison, Wisconsin, United States of America.
Byounghoon
B
Hwang
Department of Oncology, University of Wisconsin, Madison, Wisconsin, United States of America; Integrated Program in Biochemistry, University of Wisconsin, Madison, Wisconsin, United States of America.
Funita P
FP
Phan
Department of Oncology, University of Wisconsin, Madison, Wisconsin, United States of America; Molecular and Cellular Pharmacology, University of Wisconsin, Madison, Wisconsin, United States of America.
Kevin
K
McCool
Department of Oncology, University of Wisconsin, Madison, Wisconsin, United States of America.
Eun Young
EY
Choi
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.
Jinsam
J
You
Integrated Program in Biochemistry, University of Wisconsin, Madison, Wisconsin, United States of America; Department of Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin, United States of America.
Adam
A
Johnson
Department of Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin, United States of America.
Anjon
A
Audhya
Department of Oncology, University of Wisconsin, Madison, Wisconsin, United States of America; Integrated Program in Biochemistry, University of Wisconsin, Madison, Wisconsin, United States of America; Molecular and Cellular Pharmacology, University of Wisconsin, Madison, Wisconsin, United States of America.
Shigeki
S
Miyamoto
D000818:Animals; D002460:Cell Line; D004249:DNA Damage / Q000502:physiology*; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism*; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism*; D054448:Precursor Cells, B-Lymphoid / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology
C491731:IKBKG protein, human; D016328:NF-kappa B; D051550:I-kappa B Kinase