Abstract
The regulation of protein synthesis was studied in KRC-7 cells (rat hepatoma) grown in complete medium, during serum starvation, and mitogen activation. Upon serum starvation, the cells lost almost completely p67 mRNA, p67 protein, and protein synthesis activity. After phorbol 12-myristate 13-acetate addition, the same serum-starved cells regained p67 mRNA, p67 protein, and protein synthesis activity. Also, the extracts from the serum-starved cells phosphorylated the eukaryotic initiation factor-2 (eIF-2) alpha-subunit. This eIF-2 alpha-subunit phosphorylation was not observed when the extracts from either the cells grown in complete medium or mitogen-activated cells were used (Gupta, S., Wu, S., Chatterjee, N., Ilan, J., Ilan, J., Osterman, J. C., and Gupta, N. K. (1995) Gene Expr. 5, 113-122). We now report the following. 1) The eIF-2 kinase activity was the same in the cells grown in complete medium, after serum starvation, and subsequent mitogen stimulation. However, the eIF-2 kinase in the cells grown in complete medium and also after mitogen activation of the serum-starved cells cannot phosphorylate eIF-2 alpha-subunit as these cells contain p67. After removal of endogenous p67 by p67 antibodies, the extracts from all these cells similarly phosphorylated exogenously added eIF-2. 2) None of the cell extracts showed p67 deglycosylase activity. 3) The p67 mRNA was synthesized in serum-starved cells by expression of a p67 cDNA. The appearance of p67 mRNA in the serum-starved cells was accompanied by the appearance of p67 protein. Also, the rates of protein synthesis in the serum-starved cells were restored nearly to the level observed in the confluent cells. The expression of p67 cDNA also significantly increased protein synthesis rates in the cells grown in complete medium and in mitogen-activated cells. These results show that the loss of protein synthesis activity in serum-starved cells was due to loss of p67 mRNA. The expressed p67 mRNA was stable in serum-starved cells. These results, therefore, suggest that the loss of p67 mRNA in serum-starved cells is due to loss of p67 transcription. The p67 transcription regulates translation.
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