Identification of ubiquitin ligase activity of RBCK1 and its inhibition by splice variant RBCK2 and protein kinase C beta
ABSTRACT 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.
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ABSTRACT: The RING-in-between-RING (RBR) E3s are a curious family of ubiquitin E3-ligases, whose mechanism of action is unusual in several ways. Their activities are auto-inhibited, causing a requirement for activation by protein-protein interactions or posttranslational modifications. They catalyse ubiquitin conjugation by a concerted RING/HECT-like mechanism in which the RING1 domain facilitates E2-discharge to directly form a thioester intermediate with a cysteine in RING2. This short-lived, HECT-like intermediate then modifies the target. Uniquely, the RBR ligase HOIP makes use of this mechanism to target the ubiquitin amino-terminus, by presenting the target ubiquitin for modification using its distinctive LDD region.EMBO Reports 02/2014; 15(2). DOI:10.1002/embr.201338166 · 7.86 Impact Factor
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ABSTRACT: Recently, we developed a novel type of phosphate-affinity gel electrophoresis. The phosphate-affinity site is a polyacrylamide-bound dinuclear manganese(II) complex of a phosphate-binding tag nanomolecule, Phos-tag, which enables the mobility shift detection of phosphorylated proteins from their nonphosphorylated counterparts in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the quantitative analysis of protein kinase and phosphatase reactions on a polyacrylamide gel without any special apparatuses, radioactive isotopes, or chemical labels. This review article summarizes four applications of protein phosphorylation profiling using a type of affinity electrophoresis, Mn2+-Phos-tag SDS-PAGE, as follows: i) in vitro kinase activity profiling for the analysis of the phosphoprotein isotypes derived from various kinase reactions, ii) in vivo kinase activity profiling for the analysis of extracellular signal-dependent protein phosphorylation, iii) in vitro kinase inhibition profiling for the quantitative analysis of a kinase-specific inhibitor, and iv) a two-dimensional mobility-shifting procedure using Mn2+-Phos-tag SDS-PAGE for the detailed analysis of phosphoprotein isotypes. In addition, we describe the significant advantages, including a higher resolution power for the separation of protein phosphoisotypes compared with the conventional gel-based electrophoresis methods. Protein phosphorylation profiling can provide the basis for understanding the molecular origins of diseases and potentially developing tools toward therapeutic intervention. Therefore, the phosphate-affinity gel electrophoresis methodologies established by using Phos-tag can greatly facilitate the phosphoproteomics for the determination of protein phosphorylation status in life science laboratories worldwide.Current Proteomics 06/2009; 6(2):104-121. DOI:10.2174/157016409788680965 · 0.44 Impact Factor
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ABSTRACT: The completion of the Human Genome Project aroused renewed interest in alternative splicing, an efficient and widespread mechanism that generates multiple protein isoforms from individual genes. Although our knowledge about alternative splicing is growing exponentially, its real impact on cellular life is still to be clarified. Connecting all splicing features (genes, splice transcripts, isoforms, and relative functions) may be useful to resolve this tangle. Herein, we will start from the case of a single gene, Parkinson protein 2, E3 ubiquitin protein ligase (PARK2), one of the largest in our genome. This gene is implicated in the pathogenesis of autosomal recessive juvenile Parkinsonism and it has been recently linked to cancer, leprosy, autism, type 2 diabetes mellitus and Alzheimer's disease. PARK2 primary transcript undergoes an extensive alternative splicing, which enhances transcriptomic diversification and protein diversity in tissues and cells. This review will provide an update of all human PARK2 alternative splice transcripts and isoforms presently known, and correlate them to those in rat and mouse, two common animal models for studying human disease genes. Alternative splicing relies upon a complex process that could be easily altered by both cis and trans-acting mutations. Although the contribution of PARK2 splicing in human disease remains to be fully explored, some evidences show disruption of this versatile form of genetic regulation may have pathological consequences.Current Genomics 06/2014; 15(3):203-16. DOI:10.2174/1389202915666140426003342 · 2.87 Impact Factor