We previously identified a RING-IBR protein, RBCK1, as a protein kinase C (PKC) β- and ζ-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 PKCβ. The phosphorylated RBCK1 shows no
self-ubiquitination activity in vitro. Overexpression of PKCβ leads to significant increases in the amounts of intracellular RBCK1, presumably suppressing the
proteasomal degradation of RBCK1 through self-ubiquitination, whereas coexpression with PKCα, PKCϵ, and PKCζ 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 PKCβ. 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.
"Alternative splicing events on intrinsically disordered protein regions could regulate interactions of PARK2 isoforms with specific cellular targets. In addition, PARK2 isoforms generated by different alternative splice transcripts could interact with each other mutually regulating their functions, as it has been reported for RBCK1, a protein with IBR and E3 ubiquitin ligase domains, whose migration in the nucleus is inhibited by interaction with RBCK2, an isoform lacking IBR domain . "
[Show abstract][Hide abstract] 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 · 2.34 Impact Factor
"As with the use of any fusion tag, it is important to show the tagged protein behaves similar to the endogenous protein. Previous studies with HaloTag fusion proteins have demonstrated proper physiology, including DNA binding and localization [19,40-45]. In order to demonstrate specific DNA binding of the HaloTag-CREB fusion protein, HaloCHIP assays were performed in triplicates using transiently expressed HaloTag-CREB as the experimental sample and untransfected HeLa cells as a control. "
[Show abstract][Hide abstract] ABSTRACT: Regulation of gene expression is essential for normal development and cellular growth. Transcriptional events are tightly controlled both spatially and temporally by specific DNA-protein interactions. In this study we finely map the genome-wide targets of the CREB protein across all known and predicted human promoters, and characterize the functional consequences of a subset of these binding events using high-throughput reporter assays. To measure CREB binding, we used HaloCHIP, an antibody-free alternative to the ChIP method that utilizes the HaloTag fusion protein, and also high-throughput promoter-luciferase reporter assays, which provide rapid and quantitative screening of promoters for transcriptional activation or repression in living cells.
In analysis of CREB genome-wide binding events using a comprehensive DNA microarray of human promoters, we observe for the first time that CREB has a strong preference for binding at bidirectional promoters and unlike unidirectional promoters, these binding events often occur downstream of transcription start sites. Comparison between HaloCHIP-chip and ChIP-chip data reveal this to be true for both methodologies, indicating it is not a bias of the technology chosen. Transcriptional data obtained from promoter-luciferase reporter arrays also show an unprecedented, high level of activation of CREB-bound promoters in the presence of the co-activator protein TORC1.
These data suggest for the first time that TORC1 provides directional information when CREB is bound at bidirectional promoters and possible pausing of the CREB protein after initial transcriptional activation. Also, this combined approach demonstrates the ability to more broadly characterize CREB protein-DNA interactions wherein not only DNA binding sites are discovered, but also the potential of the promoter sequence to respond to CREB is evaluated.
"du et al . , 2008 ; Ydenberg and Rose , 2009 ) , plants ( Uhrig et al . , 2008 ) , insects ( Oh and Irvine , 2008 ; Sakai et al . , 2009 ) , and higher animals ( Miyata and Nishida , 2007 ; Gallon , 2008 ; Gui et al . , 2008 ; Igarashi et al . , 2008 ; Ishiai et al . , 2008 ; Kubota et al . , 2008 ; Masaoka et al . , 2008 ; Takeya et al . , 2008 ; Tatematsu et al . , 2008 ; Grob et al . , 2009 ; Ihara et al . , 2009 ; Kawashima et al . , 2009 ; Matsui et al . , 2009 ; Oyama et al . , 2009 ; Sumara et al . , 2009 ; Ta - numa et al . , 2009 ) ."
[Show abstract][Hide abstract] 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 · 0.64 Impact Factor
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