Phosphorylation of threonine 10 on CKBBP1/SAG/ROC2/Rbx2 by protein kinase CKII promotes the degradation of IκBα and p27^Kip1

In eukaryotic cells, protein kinase CKII is required for progression through the cell division cycle. We recently reported that CKBBP1/SAG/ROC2/Rbx2 associates with the β-subunit of CKII and is phosphorylated by purified CKII in the presence of ATP in vitro. In this report, we demonstrate that CKBBP1 is efficiently phosphorylated in vitro by purified CKII in the presence of GTP and by heparin-sensitive protein kinase in HeLa cell extract. Mutational analysis indicates that CKII phosphorylates threonine at residue 10 within CKBBP1. Furthermore, CKBBP1 is phosphorylated in vivo and threonine to alanine mutation at residue 10 abrogates the phosphorylation of CKBBP1 observed in vivo, indicating that CKII is a major kinase that is responsible for in vivo phosphorylation of CKBBP1. As compared with the wild-type CKBBP1 or CKBBP1^T10E (in which threonine 10 is replaced by glutamate), overexpression of nonphosphorylatable CKBBP1 (CKBBP1^T10A) results in accumulation of IκBα and p27^Kip1. Experiments using proteasome inhibitor MG132 and CKII inhibitor 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole suggest that the accumulation of IκBα and p27^Kip1 results primarily from the reduction of proteasomal degradation in cells expressing CKBBP1^T10A, and that CKII-mediated CKBBP1 phosphorylation is required for efficient degradation of IκBα and p27^Kip1. Overexpression of CKBBP1^T10A in HeLa cells suppresses cell proliferation and causes accumulation of G₁/G₀ peak of the cell cycle. Taken together, our results indicate that CKII may control IκBα and p27^Kip1 degradation and thereby G₁/S phase transition through the phosphorylation of threonine 10 within CKBBP1.