In vitro phosphorylation of the movement protein of tomato mosaic tobamovirus by a cellular kinase

• Published in: Journal of general virology 2000-08, Vol.81 (8), p.2095-2102
• Main Authors: Matsushita, Yasuhiko, Hanazawa, Kohtaro, Yoshioka, Kuniaki, Oguchi, Taichi, Kawakami, Shigeki, Watanabe, Yuichiro, Nishiguchi, Masamichi, Nyunoya, Hiroshi
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Casein Kinase II; Lycopersicon esculentum - virology; Molecular Sequence Data; movement protein; Phosphorylation; Plant Viral Movement Proteins; Protein Kinases - physiology; Protein-Serine-Threonine Kinases - physiology; Recombinant Fusion Proteins - metabolism; Tobamovirus - chemistry; Tomato mosaic virus; Viral Proteins - metabolism
• ISSN: 0022-1317; 1465-2099
• DOI: 10.1099/0022-1317-81-8-2095

Gene Research Center, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan 1 Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Meguro-ku, Tokyo 153-8902, Japan 2 National Institute of Agrobiological Resources, 2-1-2 Kan-nondai, Tsukuba, Ibaraki 305-8602, Japan 3 Author for correspondence: Hiroshi Nyunoya. Fax [phone]. e-mail nyunoya{at}cc.tuat.ac.jp The movement protein (MP) of tomato mosaic virus (ToMV) was produced in E . coli as a soluble fusion protein with glutathione S -transferase. When immobilized on glutathione affinity beads, the recombinant protein was phosphorylated in vitro by incubating with cell extracts of Nicotiana tabacum and tobacco suspension culture cells (BY-2) in the presence of [ - 32 P]ATP. Phosphorylation occurred even after washing the beads with a detergent-containing buffer, indicating that the recombinant MP formed a stable complex with some protein kinase(s) during incubation with the cell extract. Phosphoamino acid analysis revealed that the MP was phosphorylated on serine and threonine residues. Phosphorylation of the MP was decreased by addition of kinase inhibitors such as heparin, suramin and quercetin, which are known to be effective for casein kinase II (CK II). The phosphorylation level was not changed by other types of inhibitor. In addition, as shown for animal and plant CK II, [ - 32 P]GTP was efficiently used as a phosphoryl donor. Phosphorylation was not affected by amino acid replacements at serine-37 and serine-238, but was completely inhibited by deletion of the carboxy-terminal 9 amino acids, including threonine-256, serine-257, serine-261 and serine-263. These results suggest that the MP of ToMV could be phosphorylated in plant cells by a host protein kinase that is closely related to CK II.