Identification of ubiquitin ligase activity of RBCK1 and its inhibition by splice variant RBCK2 and protein kinase Cbeta.
We previously identified a RING-IBR protein, RBCK1, as a protein kinase C (PKC) beta- and zeta-interacting protein, and its splice variant, RBCK2, lacking the C-terminal half including the RING-IBR domain. RBCK1 has been shown to function as a transcriptional activator whose nuclear translocation is prevented by interaction with the cytoplasmic RBCK2. We here demonstrate that RBCK1, like many other RING proteins, also possesses a ubiquitin ligase (E3) activity and that its E3 activity is inhibited by interaction with RBCK2. Moreover, RBCK1 has been found to undergo efficient phosphorylation by PKCbeta. The phosphorylated RBCK1 shows no self-ubiquitination activity in vitro. Overexpression of PKCbeta leads to significant increases in the amounts of intracellular RBCK1, presumably suppressing the proteasomal degradation of RBCK1 through self-ubiquitination, whereas coexpression with PKCalpha, PKCepsilon, and PKCzeta shows no or little effect on the intracellular amount of RBCK1. Taken together, the E3 activity of RBCK1 is controlled by two distinct manners, interaction with RBCK2 and phosphorylation by PKCbeta. It is possible that other RING proteins, such as Parkin, BRCA1, and RNF8, having the E3 activity, are also down-regulated by interaction with their RING-lacking splice variants and/or phosphorylation by protein kinases.
Tatematsu
Kenji
K
Department of Structural Molecular Biology, The Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan. kenji44@sanken.osaka-u.ac.jp
Yoshimoto
Nobuo
N
Okajima
Toshihide
T
Tanizawa
Katsuyuki
K
Kuroda
Shun'ichi
S
D021581:Active Transport, Cell Nucleus; D017398:Alternative Splicing; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D005786:Gene Expression Regulation; D006801:Humans; D008954:Models, Biological; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D064546:Protein Kinase C beta; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism
D011500:Protein Synthesis Inhibitors; D014157:Transcription Factors; D025801:Ubiquitin; D003513:Cycloheximide; C504507:RBCK1 protein, human; D044767:Ubiquitin-Protein Ligases; D011493:Protein Kinase C; D064546:Protein Kinase C beta