In vitro phosphorylation of the movement protein of tomato mosaic tobamovirus by a cellular kinase.
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 [gamma-(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, [gamma-(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.
Matsushita
Yasuhiko
Y
Gene Research Center, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan1.
Hanazawa
Kohtaro
K
Gene Research Center, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan1.
Yoshioka
Kuniaki
K
Gene Research Center, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan1.
Oguchi
Taichi
T
Gene Research Center, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan1.
Kawakami
Shigeki
S
Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Meguro-ku, Tokyo 153-8902, Japan2.
Watanabe
Yuichiro
Y
Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Meguro-ku, Tokyo 153-8902, Japan2.
Nishiguchi
Masamichi
M
National Institute of Agrobiological Resources, 2-1-2 Kan-nondai, Tsukuba, Ibaraki 305-8602, Japan3.
Nyunoya
Hiroshi
H
Gene Research Center, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan1.
D000595:Amino Acid Sequence; D047390:Casein Kinase II; D018551:Solanum lycopersicum / Q000821:virology*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D053830:Plant Viral Movement Proteins; D011494:Protein Kinases / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017898:Tobamovirus / Q000737:chemistry*; D014764:Viral Proteins / Q000378:metabolism*
D053830:Plant Viral Movement Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D011494:Protein Kinases; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases