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.
- SourceAvailable from: Eiji Kinoshita[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 · 0.44 Impact Factor
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ABSTRACT: Mutations in the human EYA1 gene are associated with several congenital disorders, as for example BOR (branchio-oto-renal) syndrome. BOR patients suffer from severe malformations of the ear, the branchial arches and the kidneys. The mechanisms by which EYA1 mutations cause human disease are only poorly understood. Several disease-associated EYA1 mutations were characterized in this work regarding their effect on Eya1 protein function. Some of the mutations lead to enhanced proteasomal degradation of the protein in mammalian cells. Loss of Eya1 activity due to loss of Eya1 protein might represent a so far unknown mechanism for the onset of EYA1-associated diseases. Further analyses revealed that ubiquitination occurs in the C-terminus of Eya1 and is inhibited by interaction with Six1. These findings indicate that Six1 is involved in the regulation of Eya1 protein stability. A central aim of this work was the identification of novel Eya1-interacting proteins. Using yeast two-hybrid analysis two novel interaction partners were identified: Sipl1 and Rbck1. Binding studies demonstrated that the interaction is mediated via the C-terminus of Eya1 and the Ubl domain of Sipl1 or Rbck1, respectively. Furthermore, orthologs of Sipl1 and Rbck1 were identified in zebrafish. Sipl1 and Rbck1 are co-expressed with Eya1 in several organs during embryogenesis of both mouse and zebrafish. Interestingly, knockdown of one Sipl1 ortholog in zebrafish led to a BOR syndrome-like phenotype. The results of expression studies and knockdown analyses indicate that, indeed, the Eya1-Sipl1/Rbck1 interaction is of physiological relevance in the context of organ development. This hypothesis was underlined by the identification of SIPL1 and RBCK1 mutations in patients suffering from BOR syndrome. A first mechanistic basis was provided by results from transactivation studies showing that Sipl1 and Rbck1 enhance the function of Eya proteins to act as co-activators for the Six transcription factors.
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ABSTRACT: Plant pathogenic bacteria secrete effector proteins that attack the host signaling machinery to suppress immunity. Effectors can be recognized by hosts leading to immunity. One such effector is AvrPtoB of Pseudomonas syringae, which degrades host protein kinases, such as tomato Fen, through an E3 ligase domain. Pto kinase, which is highly related to Fen, recognizes AvrPtoB in conjunction with the resistance protein Prf. Here we show that Pto is resistant to AvrPtoB-mediated degradation because it inactivates the E3 ligase domain. AvrPtoB ubiquitinated Fen within the catalytic cleft, leading to its breakdown and loss of the associated Prf protein. Pto avoids this by phosphorylating and inactivating the AvrPtoB E3 domain. Thus, inactivation of a pathogen virulence molecule is one mechanism by which plants resist disease.Science 06/2009; 324(5928):784-7. DOI:10.1126/science.1169430 · 31.48 Impact Factor