Further studies on the hyperphosphorylated form (p40) of the rabies virus nominal phosphoprotein (P).
We investigated possible mechanisms involved in production of a hyperphosphorylated form (p40) of rabies virus P protein, to which two dimensional (2-D) gel electrophoresis was applied. The P gene products produced in Escherichia coli cells could be detected as a single spot of unphosphorylated 37-kDa form (termed as p37-0) in a 2-D gel. The 37-kDa proteins in the virus-infected cells are composed of some phosphorylated forms, including a major p37-1 and more phosphorylated minor forms (e.g., p37-2, p37-3, etc.), but little p37-0 is detected (Eriguchi et al., 2002). When the E. coli -produced P protein analogues were incubated with BHK-21 cell lysates, heparin-sensitive phosphorylation occurred as described previously (Takamatsu et al., 1998), giving an additional 40-kDa spot. However, such a p40-like derivative displayed a little more basic pI value than that of the authentic p40 produced in the infected cells; hence, the former was termed p40-0 (pI=4.78), while the latter, p40-1 (pI=4.73). In contrast, p40 produced in the P cDNAtransfected animal cell was detected at the p40-1 position. In addition, staurosporine did not affect the p40-1 production in virus-infected nor the P cDNA-transfected animal cells, while the agent reduced production of hyperphosphorylated forms of p37, resulting in accumulation of p37-1, but not of p37-0. These results suggest that, although p37-0 may become a substrate for the heparin-sensitive protein kinase (PK) in vitro, only p37-1 is a substrate for p40 production catalyzed by heparin-sensitive PK in animal cells, and staurosporine-sensitive PK is involved in the production of more phosphorylated forms of p37, but not in p37-1 production from p37-0.
Toriumi
Harufusa
H
Department of Molecular Microbiology, Graduate School of Pharmaceutical Sciences, Kyoto University, Japan.
Eriguchi
Yoshiro
Y
Takamatsu
Fumihiko
F
Kawai
Akihiko
A
D000818:Animals; D019556:COS Cells; D002460:Cell Line; D006224:Cricetinae; D018076:DNA, Complementary; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism*; D011820:Rabies virus / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity*; D019311:Staurosporine / Q000494:pharmacology; D014162:Transfection; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D018076:DNA, Complementary; D014764:Viral Proteins; C087087:nominal phosphoprotein, rabies virus; D006493:Heparin; D011494:Protein Kinases; D019311:Staurosporine