Site-specific tyrosine phosphorylation of IkappaBalpha negatively regulates its inducible phosphorylation and degradation.
The transcription factor NF-kappaB is retained in the cytoplasm by its interaction with the inhibitory subunit known as IkappaB. Signal-induced serine phosphorylation and subsequent ubiquitination of IkappaBalpha target it for degradation by the 26 S proteasome. Recently, pervanadate, a protein-tyrosine phosphatase inhibitor, was shown to block the degradation of IkappaBalpha, thus inhibiting NF-kappaB activation. We investigated the mechanism by which pervanadate inhibits the degradation of IkappaBalpha. Western blot analysis of IkappaBalpha from tumor necrosis factor-treated cells revealed a slower migrating IkappaBalpha species that was subsequently degraded. However, pervanadate-treated cells also revealed a slower migrating species of IkappaBalpha that appeared in a time- and dose-dependent manner and was not degraded by tumor necrosis factor. The slower migrating species of IkappaBalpha from pervanadate-treated cells was tyrosine-phosphorylated as revealed by cross-reactivity with anti-phosphotyrosine antibodies, by the ability of the specific tyrosine phosphatase PTP1B to dephosphorylate it, and by phosphoamino acid analysis of IkappaBalpha immunoprecipitated from 32P-labeled cells. By site-specific mutagenesis and deletion analysis, we identified Tyr-42 on IkappaBalpha as the phosphoacceptor site. Furthermore, in an in vitro reconstitution system, tyrosine-phosphorylated IkappaBalpha was protected from degradation. Our results demonstrate that inducible phosphorylation and degradation of IkappaBalpha are negatively regulated by phosphorylation at Tyr-42, thus preventing NF-kappaB activation.
Singh
S
S
Cytokine Research Laboratory, Department of Molecular Oncology, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
Darnay
B G
BG
Aggarwal
B B
BB
D000595:Amino Acid Sequence; D015153:Blotting, Western; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004790:Enzyme Induction; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D020672:I-kappa B Proteins; D008969:Molecular Sequence Data; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors*; D010766:Phosphorylation; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured; D014443:Tyrosine; D014638:Vanadates / Q000494:pharmacology
D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; C076715:pervanadate; D000072000:NF-KappaB Inhibitor alpha; D014638:Vanadates; D014443:Tyrosine; D017027:Protein Tyrosine Phosphatases