ACTIVATION OF NF-KAPPA-B BY PHOSPHATASE INHIBITORS INVOLVES THE PHOSPHORYLATION OF I-KAPPA-B-ALPHA AT PHOSPHATASE 2A-SENSITIVE SITES

Activation of NF-kappa B by various cellular stimuli involves the phos phorylation and subsequent degradation of its inhibitor, I kappa B alp ha, although the underlying mechanism remains unclear. In the present study, the role of serine/threonine phosphatases in the regulation of I kappa B alpha phosphorylation was investigated. Our studies demonstr ate that incubation of human T cells with low concentrations (similar to 1-5 nM) of calyculin A or okadaic acid, potent inhibitors of protei n phosphatase type 1 (PP-1) and type 2A (PP-2A), induces the phosphory lation of I kappa B alpha even in the absence of any cellular stimulus . This action of the phosphatase inhibitors, which is associated with the activation of the RelA . p50 NF-kappa B heterodimer, is not affect ed by agents that block the induction of I kappa B alpha phosphorylati on by tumor necrosis factor alpha (TNF-alpha). Furthermore, the phosph orylated I kappa B alpha from calyculin A-treated cells, but not that from TNF-alpha-stimulated cells, is sensitive to PP-BA in vitro, sugge sting the existence of fundamental differences in the phosphorylation of I kappa B alpha induced by the two different NF-kappa B inducers. H owever, induction of I kappa B alpha phosphorylation by both TNF-alpha and the phosphatase inhibitors is associated with the subse quent deg radation of I kappa B alpha. We further demonstrate that TNF-alpha- an d calyculin A-induced I kappa B alpha degradation exhibits similar but not identical sensitivities to a proteasome inhibitor, Together, thes e results suggest that phosphorylation of I kappa B alpha, mediated th rough both the TMF-alpha-inducible and the PP-2A-opposing kinases, may serve to target I kappa B alpha for proteasome-mediated degradation.