P67 TRANSCRIPTION REGULATES TRANSLATION IN SERUM-STARVED AND MITOGEN-ACTIVATED KRC-7 CELLS

The regulation of protein synthesis was studied in KRC-7 cells (rat he patoma) grown in complete medium, during serum starvation, and mitogen activation. Upon serum starvation, the cells lost almost completely p 67 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 extra cts from the serum-starved cells phosphorylated the eukaryotic initiat ion factor-2 (eIF-2) alpha-subunit. This eIF-2 alpha-subunit phosphory lation 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) T he eIF-2 kinase activity was the same in the cells grown in complete m edium, after serum starvation, and subsequent mitogen stimulation. How ever, the eIF-2 kinase in the cells grown in complete medium and also after mitogen activation of the serum-starved cells cannot phosphoryla te eIF-2 alpha-subunit as these cells contain p67. After removal of en dogenous p67 by p67 antibodies, the extracts from all these cells simi larly phosphorylated exogenously added eIF-2. 2) None of the cell extr acts showed p67 deglycosylase activity. 3) The p67 mRNA was synthesize d 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 sta rved cells were restored nearly to the level observed in the confluent cells. The expression of p67 cDNA also significantly increased protei n synthesis rates in the cells grown in complete medium and in mitogen -activated cells. These results show that the loss of protein synthesi s activity in serum-starved cells was due to loss of p67 mRNA. The exp ressed p67 mRNA was stable in serum-starved cells. These results, ther efore, suggest that the loss of p67 mRNA in serum-starved cells is due to loss of p67 transcription. The p67 transcription regulates transla tion.