Phosphorylation of I kappa B alpha in the C-terminal PEST domain by casein kinase iu affects intrinsic protein stability

The NF-kappa B/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-kappa B/Rel proteins are coupled to inhibitory molecules, collectively termed I kappa B, which are responsible for cytoplasmic retention of NF-kappa B. Cell activation leads to the phosphorylation and degradation of I kappa B alpha, permitting NF-kappa B/Rel translocation to the nucleus and target gene activation. To further characterize the signaling events that contribute to I kappa B alpha phosphorylation, a kinase activity was isolated from Jurkat T cells that specifically interacted with I kappa B alpha in an affinity chromatography step and phosphorylated I kappa B alpha with high specificity in vitro. By using an in-gel kinase assay with recombinant I kappa B alpha 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 I kappa B alpha (Delta 1 to Delta 4) localized phosphorylation to the C-terminal PEST domain of I kappa B alpha. Point mutation of residues T-291, S-283, and T-299 dramatically reduced phosphorylation of I kappa B alpha by the kinase in vitro. NIH-3T3 cells that stably expressed wild-type I kappa B alpha (wtI kappa B), double-point-mutated I kappa B alpha(T291A, S283A), or triple-point-mutated I kappa B alpha(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 I kappa B alpha degradation was unaffected. In cell lines and in transiently transfected cells, mutation of the CKII sites in I kappa B alpha 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 IKB alpha, these studies indicate that CKII sites in the C-terminal PEST domain are important for constitutive phosphorylation and intrinsic stability of I kappa B alpha.