I kappaB alpha physically interacts with a cytoskeleton-associated protein through its signal response domain.
The I kappaB alpha protein is a key molecular target involved in the control of NF-kappaB/Rel transcription factors during viral infection or inflammatory reactions. This NF-kappaB-inhibitory factor is regulated by posttranslational phosphorylation and ubiquitination of its amino-terminal signal response domain that targets I kappaB alpha for rapid proteolysis by the ... 26S proteasome. In an attempt to identify regulators of the I kappaB alpha inhibitory activity, we undertook a yeast two-hybrid genetic screen, using the amino-terminal end of I kappaB alpha as bait, and identified 12 independent interacting clones. Sequence analysis identified some of these cDNA clones as Dlc-1, a sequence encoding a small, 9-kDa human homolog of the outer-arm dynein light-chain protein. In the two-hybrid assay, Dlc-1 also interacted with full-length I kappaB alpha protein but not with N-terminal-deletion-containing versions of I kappaB alpha. I kappaB alpha interacted in vitro with a glutathione S-transferase-Dlc-1 fusion protein, and RelA(p65) did not displace this association, demonstrating that p65 and Dlc-1 contact different protein motifs of I kappaB alpha. Importantly, in HeLa and 293 cells, endogenous and transfected I kappaB alpha coimmunoprecipitated with Myc-tagged or endogenous Dlc-1. Indirect immunofluorescence analyzed by confocal microscopy indicated that Dlc-1 and I kappaB alpha colocalized with both nuclear and cytoplasmic distribution. Furthermore, Dlc-1 and I kappaB alpha were found to associate with the microtubule organizing center, a perinuclear region from which microtubules radiate. Likewise, I kappaB alpha colocalized with alpha-tubulin filaments. Taken together, these results highlight an intriguing interaction between the I kappaB alpha protein and the human homolog of a member of the dynein family of motor proteins and provide a potential link between cytoskeleton dynamics and gene regulation.
Mesh Terms:
Amino Acid Sequence, Animals, Base Sequence, Binding Sites, Cell Line, Cell Nucleus, Cytoplasm, Cytoskeletal Proteins, DNA Primers, DNA-Binding Proteins, Dyneins, Fluorescent Antibody Technique, Indirect, Humans, I-kappa B Proteins, Microscopy, Confocal, Microtubules, Molecular Sequence Data, Polymerase Chain Reaction, Protein Sorting Signals, Saccharomyces cerevisiae, Sequence Homology, Amino Acid, Transfection
Amino Acid Sequence, Animals, Base Sequence, Binding Sites, Cell Line, Cell Nucleus, Cytoplasm, Cytoskeletal Proteins, DNA Primers, DNA-Binding Proteins, Dyneins, Fluorescent Antibody Technique, Indirect, Humans, I-kappa B Proteins, Microscopy, Confocal, Microtubules, Molecular Sequence Data, Polymerase Chain Reaction, Protein Sorting Signals, Saccharomyces cerevisiae, Sequence Homology, Amino Acid, Transfection
Mol. Cell. Biol.
Date: Dec. 01, 1997
PubMed ID: 9372968
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