The Ring-H2 finger motif of CKBBP1/SAG is necessary for interaction with protein kinase CKII and optimal cell proliferation.
Protein kinase CKII (CKII) is required for progression through the cell division cycle. We recently reported that the beta subunit of protein kinase CKII (CKIIbeta) associates with CKBBP1 that contains the Ring-H2 finger motif in the yeast two-hybrid system. We demonstrate here that the Ring-H2 finger-disrupted mutant of CKBBP1 does not interact with purified CKIIbeta in vitro, which shows that the Ring-H2 finger motif is critical for direct interaction with CKIIbeta. The CKII holoenzyme is efficiently co-precipitated with the wild-type CKBBP1, but not with the Ring-H2 finger-disrupted CKBBP1, from whole cell extracts when epitope-tagged CKBBP1 is transiently expressed in HeLa cells. Disruption of the Ring-H2 finger motif does not affect the cellular localization of CKBBP1 in HeLa cells. The increased expression of either the wild-type CKBBP1 or Ring-H2 finger-disrupted CKBBP1 does not modulate the protein or the activity levels of CKII in HeLa cells. However, the stable expression of Ring-H2 finger-disrupted CKBBP1 in HeLa cells suppresses cell proliferation and causes the accumulation of the G1/G0 peak of the cell cycle. The Ring-H2 finger motif is required for maximal CKBBP1 phosphorylation by CKII, suggesting that the stable binding of CKBBP1 to CKII is necessary for its efficient phosphorylation. Taken together, these results suggest that the complex formation of CKIIbeta with CKBBP1 and/or CKII-mediated CKBBP1 phosphorylation is important for the G1/S phase transition of the cell cycle.
Kim
Yun-Sook
YS
Department of Biochemistry, College of Natural Sciences, Kyungpook National University, Daegu 702-701, Korea.
Ha
Kwon-Soo
KS
Kim
Young-Ho
YH
Bae
Young-Seuk
YS
D020816:Amino Acid Motifs; D015153:Blotting, Western; D047390:Casein Kinase II; D002455:Cell Division; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D005434:Flow Cytometry; D016193:G1 Phase; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D016196:S Phase; D013997:Time Factors; D044767:Ubiquitin-Protein Ligases; D016335:Zinc Fingers
D016601:RNA-Binding Proteins; C118307:RNF7 protein, human; D044767:Ubiquitin-Protein Ligases; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases