Constitutive phosphorylation of IkappaBalpha by casein kinase II occurs preferentially at serine 293: requirement for degradation of free IkappaBalpha.
IkappaBalpha is a phosphoprotein that sequesters the NF-kappaB/Rel transcription factors in the cytoplasm by physical association. Following induction by a wide variety of agents, IkappaBalpha is further phosphorylated and degraded, allowing NF-kappaB/Rel proteins to translocate to the nucleus and induce transcription. We have previously reported that the constitutive phosphorylation site resides in the C-terminal PEST region of IkappaBalpha and is phosphorylated by casein kinase II (CKII). Here we show that serine 293 is the preferred CKII phosphorylation site. Additionally, we show compensatory phosphorylation by CKII at neighboring serine and threonine residues. Thus, only when all five of the serine and threonine residues in the C-terminal region of IkappaBalpha are converted to alanine (MutF), is constitutive phosphorylation abolished. Finally, we show that constitutive phosphorylation is required for efficient degradation of free IkappaBalpha, in that unassociated Mutf has a half-life two times longer than wild-type IkappaBalpha. A serine residue alone at position 293, as well as aspartic acid at this position, can revert the Mutf phenotype. Therefore, the constitutive CKII phosphorylation site is an integral part of the PEST region of IkappaBalpha, and this phosphorylation is required for rapid proteolysis of the unassociated protein.
Schwarz
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EM
Laboratory of Genetics, The Salk Institute, San Diego, California, 92186, USA.
Van Antwerp
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Verma
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D000595:Amino Acid Sequence; D000818:Animals; D047390:Casein Kinase II; D002645:Chickens; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D005982:Glutathione Transferase; D006801:Humans; D020672:I-kappa B Proteins; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine; D013552:Swine
D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse; C000606206:Nfkbia protein, rat; D010748:Phosphopeptides; D011993:Recombinant Fusion Proteins; D000072000:NF-KappaB Inhibitor alpha; D012694:Serine; D005982:Glutathione Transferase; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases