BASAL PHOSPHORYLATION OF THE PEST DOMAIN IN I-KAPPA-B-BETA REGULATES ITS FUNCTIONAL INTERACTION WITH THE C-REL PROTOONCOGENE PRODUCT

The product of the c-rel proto-oncogene (c-Rel) belongs to the NF-kapp a B/Rel family of polypeptides and has been implicated in the transcri ptional control of cell proliferation and immune function, In human T lymphocytes, c-Rel is sequestered in the cytoplasmic compartment by co nstitutively phosphorylated inhibitors, including I kappa B alpha and I kappa B beta. Studies with bacterially expressed forms of these inhi bitory proteins revealed that unphosphorylated I kappa alpha but not I KB beta assembles with c-Rel and inhibits its DIC'A binding activity, Furthermore, latent I kappa B beta-c-Rel complexes derived from mammal ian cells were sensitive to phosphatase treatment, whereas I kappa B a lpha-c-Rel complexes mere resistant. We have identified a constitutive protein kinase in unstimulated T cells that associates with and phosp horylates I kappa B beta in vitro. The substrate specificity, electrop horetic mobility, and antigenic propel ties of this I kappa beta B-ass ociated kinase (BAK) suggest identity with casein kinase II (CKII), an enzyme known to mediate basal phosphorylation of I kappa B alpha. Pho sphorylation of recombinant I kappa B beta by either BAK or CKII resto red the capacity of this inhibitor to antagonize the DNA binding activ ity of c-Rel. Peptide mapping and mutational analyses localized the bu lk of the basal phosphorylation sites in I kappa B beta to the C-termi nal PEST domain, which contains two potential acceptors for CKII-media ted phosphoryl group transfer (Ser-313 and Ser-315). Point mutations i ntroduced into the full-length inhibitor at Ser-313 and Ser-315 led to a significant reduction in the phosphorylation of I kappa B beta and severely impaired its c-Rel inhibitory function in vivo. Taken togethe r, these findings strongly suggest that basal phosphorylation of the P EST domain of I kappa B beta at consensus CKII sites is required for t he efficient formation of latent I kappa B beta-c-Rel complexes.