Phosphorylation of IκBα in the C-Terminal PEST Domain by Casein Kinase II Affects Intrinsic Protein Stability

• Published in: Molecular and cellular biology 1996-04, Vol.16 (4), p.1401-1409
• Main Authors: Lin, Rongtuan, Beauparlant, Pierre, Makris, Constantin, Meloche, Sylvain, Hiscott, John
• Format: Article
• Language: English
• ISSN: 1098-5549; 0270-7306; 1098-5549
• DOI: 10.1128/MCB.16.4.1401

The NF-κB/Rel transcription factors participate in the activation of immune system regulatory genes and viral early genes including the human immunodeficiency virus type 1 long terminal repeat. NF-κB/Rel proteins are coupled to inhibitory molecules, collectively termed IκB, which are responsible for cytoplasmic retention of NF-κB. Cell activation leads to the phosphorylation and degradation of IκBα, permitting NF-κB/Rel translocation to the nucleus and target gene activation. To further characterize the signaling events that contribute to IκBα phosphorylation, a kinase activity was isolated from Jurkat T cells that specifically interacted with IκBα in an affinity chromatography step and phosphorylated IκBα with high specificity in vitro. By using an in-gel kinase assay with recombinant IκBα as substrate, two forms of the kinase (43 and 38 kDa) were identified. Biochemical criteria and immunological cross-reactivity identified the kinase activity as the alpha catalytic subunit of casein kinase II (CKII). Deletion mutants of IκBα (Δ1 to Δ4) localized phosphorylation to the C-terminal PEST domain of IκBα. Point mutation of residues T-291, S-283, and T-299 dramatically reduced phosphorylation of IκBα by the kinase in vitro. NIH-3T3 cells that stably expressed wild-type IκBα (wtIκB), double-point-mutated IκBα(T291A, S283A), or triple-point-mutated IκBα(T291A, S283A, T299A) under the control of the tetracycline-responsive promoter were generated. Constitutive phosphorylation of the triple point mutant was eliminated in vivo, although tumor necrosis factor-inducible IκBα degradation was unaffected. In cell lines and in transiently transfected cells, mutation of the CKII sites in IκBα resulted in a protein with increased intrinsic stability. Together with results demonstrating a role for N-terminal sites in inducer-mediated phosphorylation and degradation of IκBα, these studies indicate that CKII sites in the C-terminal PEST domain are important for constitutive phosphorylation and intrinsic stability of IκBα.