PURIFIED I-KAPPA-B-BETA IS INACTIVATED UPON DEPHOSPHORYLATION

In uninduced cells, the NF-kappa-B transcription factor resides in the cytoplasm in complex with an inhibitory protein, I-kappa-B. I-kappa-B is a specific inhibitor of DNA binding and apparently prevents nuclear uptake of NF-kappa-B. Stimulation of cells, for instance with the cytokine tumor necrosis factor, releases I-kappa-B and allows nuclear translocation and DNA binding of NF-kappa-B to regulatory DNA sequences in many genes. We recently reported on the purification of a major form of I-kappa-B, referred to as I-kappa-B-alpha, with a molecular size of 37 kDa. Here, we purified and characterized I-kappa-B-beta, a chromatographically distinct second form of I-kappa-B. I-kappa-B-beta has a size of 43 kDa and, as I-kappa-B-alpha, an acidic isoelectric point between 4.8 and 5.0. Both forms of I-kappa-B were inactivated by a treatment with protein kinases A and C in vitro. In contrast to I-kappa-B-alpha, I-kappa-B-beta lost its inhibiting activity upon a treatment with phosphatase. Phosphatase treatment also released active NF-kappa-B from its inactive complex with I-kappa-B-beta suggesting that the activation of NF-kappa-B in intact cells might not only rely on phosphate transfer onto I-kappa-B but also on phosphate removal from one form of I-kappa-B.