Rice serine/threonine kinase 1 is required for the stimulation of OsNug2 GTPase activity |
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Authors: | Jae Bok Heo Yun Mi Lee Hee Rang Yun Chak Han Im Yong-Suk Lee Young Byong Yi Chian Kwon Jun Lim Jeong Dong Bahk |
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Institution: | 1. Department of Molecular Biotechnology, Dong-A University, Busan 604-714, South Korea;2. Eco-friendliness Research Department, Gyeongsangnam-do Agricultural Research and Extension Services, Jinju 660-360, South Korea;3. Department of Biotechnology, Dong-A University, Busan 604-714, South Korea;4. Department of Molecular Biology, Dankook University, Yongin 448-701, South Korea;5. Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, South Korea;6. Department of Biochemistry, Gyeongsang National University, Jinju 660-701, South Korea |
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Abstract: | Several GTPases are required for ribosome biogenesis and assembly. We recently identified rice (Oryza sativa) nuclear/nucleolar GTPase 2 (OsNug2), a YlqF/YawG family GTPase, as having a role in pre-60S ribosomal subunit maturation. To investigate the potential factors involved in regulating OsNug2 function, yeast two-hybrid screens were performed using OsNug2 as bait. Rice serine/threonine kinase 1 (OsSTK1) was identified as a candidate interacting protein. OsSTK1 appeared to interact with OsNug2 both in vitro and in vivo. OsSTK1 was found to have no effect on the GTP-binding activity of OsNug2; however, the presence of recombinant OsSTK1 in OsNug2 assay reaction mixtures increased OsNug2 GTPase activity. A kinase assay showed that OsSTK1 had weak autophosphorylation activity and strongly phosphorylated serine 209 of OsNug2. Using yeast complementation testing, we identified a GAL::OsNug2(S209N) mutation-harboring yeast strain that exhibited a growth-defective phenotype on galactose medium at 39 °C, which was divergent from that of a yeast strain harboring GAL::OsNug2. The intrinsic GTPase activity of OsNug2(S209N), which was found to be similar to that of OsNug2, was not fully enhanced upon weak binding of OsSTK1. Our findings indicate that OsSTK1 functions as a positive regulator of OsNug2 by enhancing OsNug2 GTPase activity. In addition, phosphorylation of OsNug2 serine 209 is essential for its complete function in biological functional pathway. |
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Keywords: | BiFC bimolecular fluorescence complementation cDNA complementary DNA GFP green fluorescent protein GST glutathione sepharose transferase GTP guanosine-5&prime -triphosphate GDP guanosine-5&prime -diphosphate OsNug2 Oryza sativa nuclear/nucleolar GTPase 2 PEG polyethylene glycol RFP red fluorescent protein rRNA ribosomal RNA |
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