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The genome of eukaryotic cells is frequently exposed to damage by various genotoxins. Phosphorylation of histone H2AX at Serine 139 (γ-H2AX) is a hallmark of DNA damage. RNF8 monoubiquitinates γ-H2AX with the Lys63-linked ubiquitin chain to tether DNA repair molecules at DNA lesions. A high-throughput screening identified RNF8 as a binding partner of dual-specificity tyrosine phosphorylation-regulated kinase 2 (DYRK2). Notably, DNA damage-induced monoubiquitination of γ-H2AX is impaired in DYRK2-depleted cells. The foci formation of p53-binding protein 1 at DNA double-strand break sites is suppressed in DYRK2 knockdown cells, which fail to repair the DNA damage. A homologous recombination assay showed decreased repair efficiency in DYRK2-depleted cells. Our findings indicate direct interaction of DYRK2 with RNF8 in regulating response to DNA damage., (© 2017 Federation of European Biochemical Societies.)

The Cdk-related protein kinase Pctaire1/Cdk16 is abundantly expressed in brain, testis and skeletal muscle. Functional roles of Pctaire1 such as regulation of neuron migration and neurite outgrowth thus far have been mainly elucidated in the field of nervous system development. Although these regulations based on cytoskeletal rearrangements evoke a possible role of Pctaire1 in the development of skeletal muscle, little is known in this regard. In this study, we demonstrated that myogenic differentiation and subsequent fusion is promoted in Pctaire1 overexpressing cells, and conversely, is inhibited in the knockdown cells. Furthermore, our findings suggest that Pctaire1 exerts promyogenic effects by regulating myoblast migration and process formation during skeletal myogenesis., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Accumulating evidence suggests that caspase-3-mediated cleavage of protein kinase could be a key event to regulate cell differentiation. In this study, we investigated the role of Nek5 kinase, identified as a novel substrate for caspase-3, in skeletal muscle differentiation. Up-regulation of Nek5 mRNA expression was accompanied by cell differentiation. Myotube formation was promoted in Nek5 expressing cells, and was conversely inhibited in Nek5 knockdown cells. Furthermore, we found that caspase-3 activity, an important factor for myogenic differentiation, was enhanced by Nek5 cleavage. Although caspase-3-cleaved Nek5 partially exerted a promyogenic effect, it tended to induce apoptotic cell death. In summary, our findings suggest that Nek5 promotes myogenic differentiation through up-regulation of caspase activity., (Copyright © 2013. Published by Elsevier B.V.)

Plant genome possesses over 100 protein phosphatase (PPase) genes that are key regulators of signal transduction via phosphorylation/dephosphorylation event. Here we report a comprehensive functional analysis of protein serine/threonine, dual-specificity and tyrosine phosphatases using recombinant PPases produced by wheat cell-free protein synthesis system. Eighty-two recombinant PPases were successfully produced using Arabidopsis full-length cDNA as templates. In vitro PPase assay was performed using phosphorylated myelin basic protein as substrate. Among the AtPPases examined, 26 serine/threonine, three dual-specificity and one tyrosine PPases exhibited catalytic activity, including 20 serine/threonine and one dual-specificity PPases that showed in vitro activities for the first time. Our study demonstrates genome-wide biochemical analysis of AtPPases using wheat cell-free system, and provides new information and insights on enzyme activities., (Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

We screened a library of human single-transmembrane proteins (sTMPs), produced by a cell-free system, using a luminescent assay to identify those that can be cleaved by caspase-8 (CASP8). Of the 407 sTMPs screened, only the interleukin-21 receptor (IL21R), vezatin (VEZT), and carbonic anhydrase XIV were cleaved at Asp344, Asp655 and Asp53, respectively. We confirmed that IL21R and VEZT were also cleaved in apoptotic HeLa cells with the cleavage sites. Interestingly, IL21R was cleaved within 30 min after apoptosis induction. Furthermore the CASP8-cleaved form of IL21R did not induce phosphorylation at Tyr705 of STAT3. Our results suggest that the interleukin-21 signaling cascade is negatively regulated by CASP8., (Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Suppressor of cytokine signaling 1 (SOCS1) is a recently identified host factor that positively regulates the intracellular trafficking and stability of HIV-1 Gag. We here examine the molecular mechanism by which SOCS1 regulates intercellular Gag trafficking and virus particle production. We find that SOCS1 colocalizes with Gag along the microtubule network and promotes microtubule stability. SOCS1 also increases the amount of Gag associated with microtubules. Both nocodazole treatment and the expression of the microtubule-destabilizing protein, stathmin, inhibit the enhancement of HIV-1 particle production by SOCS1. SOCS1 facilitates Gag ubiquitination and the co-expression of a dominant-negative ubiquitin significantly inhibits the association of Gag with microtubules. We thus propose that the microtubule network plays a role in SOCS1-mediated HIV-1 Gag transport and virus particle formation.

The nuclear hormone receptors (NHRs), a family of transcription factors, bind directly to the hormone response elements (HREs) to regulate gene expression. In this study, we describe a comprehensive NHR-HRE profiling analysis with a new high-throughput DNA binding assay system utilizing wheat germ cell-free protein production and fluorescence correlation spectroscopy (FCS). This approach revealed NHR binding to natural response elements and new heterodimeric NHR-HRE bindings. We analyzed 408 possible binding combinations between 34 human NHRs and 12 different HREs, and identified 205 NHR-HRE binding combinations, 124 of which have not been previously reported. Thus, this study provides a novel biochemical classification of the human NHRs, as well as describing a novel approach to the large-scale analysis of DNA-protein interactions.

Calcium/calmodulin-dependent protein kinase II (CaMKII) plays a crucial role in mediating calcium signaling. Here, we demonstrate a method for screening substrates phosphorylated by human CaMKII delta using a wheat cell-free system. The cell-free mixture expressing CaMKII delta was incubated with HeLa extracts and radiolabeled ATP. From analysis of two-dimensional electrophoresis gels and mass spectrometry, two proteins were found. The cell-free based in vitro kinase assay revealed that CaMKII delta phosphorylates eukaryotic translation initiation factor 4B and stress-induced phosphoprotein 1 (STIP1), the latter on Ser189. Furthermore, constitutively-active CaMKII delta phosphorylated STIP1 in HeLa cells and dramatically promoted nuclear localization of STIP1, suggesting that calcium signals via CaMKII delta may regulate subcellular localization of STIP1. This approach may be a useful tool for target screening of protein kinases.

Protein microarray is considered to be one of the key analytical tools for high-throughput protein function analysis. Here, we report that the Arabidopsis HY5 functions as a novel DNA-binding tag (DBtag) for proteins. We also demonstrate that the DBtagged proteins could be immobilized and purified on a newly designed agarose/DNA microplate. Furthermore, we show three applications using the microarray: (1) detection of autophosphorylation activity of DBtagged human protein kinases and inhibition of their activity by staurosporine, (2) specific cleavage of DBtagged proteins by a virus protease and caspase 3, and (3) detection of a protein-protein interaction between the DBtagged UBE2N and UBE2v1. Thus, this method may facilitate rapid functional analysis of a wide range of proteins.

Solanum chacoense ovule receptor kinase 28 (ScORK28) was found among 30 receptor kinases from an ovule cDNA library enriched for weakly expressed mRNAs. This LRR-RLK displayed high level of tissue specificity at the RNA and protein levels and was predominantly expressed in female reproductive tissues. Protein expression analyses in planta revealed that ScORK28 was N-glycosylated and ScORK28::GFP fusion analyses showed that it was localized at the plasma membrane. Bacterial expression of ScORK28 catalytic domain followed by kinase activity assays revealed that ScORK28 is an active Mg2+-dependent protein kinase and that the juxtamembrane domain is necessary for kinase activity.

We report a morphological study of functioning ribosomes in a efficient and robust cell-free protein synthesis system prepared from wheat embryos. Sucrose density gradient analysis of translated mixtures programmed with luciferase mRNAs having different 5' and 3' untranslated regions showed formation of large polysomes. Electron microscopic examination of translation mixtures programmed with those of capped and polyadenylated mRNA revealed that ribosomes assemble into a circular-type polysome in vitro. Furthermore, a series of experiments using mRNAs lacking either cap, poly(A) tail or both also resulted in the formation of circular polysomes, which are indistinguishable from those with the original mRNA. The wheat germ cell-free system may provide a good experimental system for understanding functional ribosomes at the molecular level.

Plant ribosomal RNA apurinic site specific lyase (RALyase) cleaves the phosphodiester bond at the depurinated site produced by ribosome-inactivating protein, while the biological role of this enzyme is not clear. As the depurinated ribosomes retain weak translation elongation activities, it was suggested that RALyase completes the ribosome inactivation. To confirm this point, we measured the effects of the phosphodiester cleavage using a fusion of wheat RALyase produced with a cell-free protein synthesis system from wheat germ. The results indicated that RALyase diminishes the residual elongation activities of the depurinated ribosomes.

A high-throughput cell-free protein synthesis method has been described. The methodology is based on a bilayer diffusion system that enables the continuous supply of substrates, together with the continuous removal of small byproducts, through a phase between the translation mixture and substrate mixture. With the use of a multititer plate the system was functional for a prolonged time, and as a consequence yielded more than 10 times that of the similar batch-mode reaction. Combining this method with a wheat germ cell-free translation system developed by us, the system could produce a large amount of protein sufficient for carrying out functional analyses. This novel bilayer-based cell-free protein synthesis system with its simplicity, minimum time and low cost may be useful practical methodology in the post-genome era.