| Abstrakt: |
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 colicells 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 cDNA‐transfected 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. |
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