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

Y. Matsushita, K. Hanazawa, K. Yoshioka, T. Oguchi, S. Kawakami, Y. Watanabe, M. Nishiguchi, H. Nyunoya

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Abstract

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 [γ-32P]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, [γ-32P]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.

Original languageEnglish
Pages (from-to)2095-2102
Number of pages8
JournalJournal of General Virology
Volume81
Issue number8
DOIs
Publication statusPublished - 2000 Jan 1
Externally publishedYes

ASJC Scopus subject areas

  • Virology

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    Matsushita, Y., Hanazawa, K., Yoshioka, K., Oguchi, T., Kawakami, S., Watanabe, Y., Nishiguchi, M., & Nyunoya, H. (2000). In vitro phosphorylation of the movement protein of tomato mosaic tobamovirus by a cellular kinase. Journal of General Virology, 81(8), 2095-2102. https://doi.org/10.1099/0022-1317-81-8-2095