Phosphorylation of threonine 10 on CKBBP1/SAG/ROC2/Rbx2 by protein kinase CKII promotes the degradation of IkappaBalpha and p27Kip1.
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 beta-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 CKBBP1T10E (in which threonine 10 is replaced by glutamate), overexpression of nonphosphorylatable CKBBP1 (CKBBP1T10A) results in accumulation of IkappaBalpha and p27Kip1. Experiments using proteasome inhibitor MG132 and CKII inhibitor 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole suggest that the accumulation of IkappaBalpha and p27Kip1 results primarily from the reduction of proteasomal degradation in cells expressing CKBBP1T10A, and that CKII-mediated CKBBP1 phosphorylation is required for efficient degradation of IkappaBalpha and p27Kip1. Overexpression of CKBBP1T10A 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 IkappaBalpha and p27Kip1 degradation and thereby G1/S phase transition through the phosphorylation of threonine 10 within CKBBP1.
Kim
Yun-Sook
YS
Department of Biochemistry, College of Natural Sciences, Kyungpook National University, Daegu 702-701, Korea.
Lee
Jae-Yong
JY
Son
Mi-Young
MY
Park
Wan
W
Bae
Young-Seuk
YS
D000409:Alanine; D047390:Casein Kinase II; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002455:Cell Division; D050760:Cyclin-Dependent Kinase Inhibitor p27; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016193:G1 Phase; D015870:Gene Expression; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism*; D007976:Leupeptins / Q000494:pharmacology; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D000072000:NF-KappaB Inhibitor alpha; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012313:RNA / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016196:S Phase; D013329:Structure-Activity Relationship; D013912:Threonine / Q000378:metabolism*; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases
D018797:Cell Cycle Proteins; D004791:Enzyme Inhibitors; D020672:I-kappa B Proteins; D007976:Leupeptins; D009097:Multienzyme Complexes; C000606204:NFKBIA protein, human; D016601:RNA-Binding Proteins; D025521:Tumor Suppressor Proteins; D000072000:NF-KappaB Inhibitor alpha; D050760:Cyclin-Dependent Kinase Inhibitor p27; D013912:Threonine; D012313:RNA; D006160:Guanosine Triphosphate; C118307:RNF7 protein, human; D044767:Ubiquitin-Protein Ligases; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D000409:Alanine; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde