Carbon source-dependent regulation of cell growth by murine protein kinaseC epsilon expression in Saccharomyces cerevisiae

Protein kinase C is known to play a role in cell cycle regulation in both l ower and higher eucaryotic cells. Since mutations in yeast proteins involve d in cell cycle regulation can often be rescued by the mammalian homolog an d since significant conservation exists between PKC-signalling pathways in yeast and mammalian cells, cell cycle regulation by mammalian PKC isoforms may be effectively studied in a simpler genetically-accessible model system such as Saccharomyces cerevisiae. With this objective in mind, we transfec ted S. cerevisiae cells with a plasmid (pYEC epsilon) coding for the expres sion of murine protein kinase C epsilon (PKC epsilon) under the control of a galactose-inducible promoter. Unlike mock-transfected cells, yeast cells transformed with pYEC epsilon expressed, in a galactose-dependent manner, a n 89 kDa protein that was recognized by a human PKC epsilon antibody. Extra cts from these pYEC epsilon-transfected cells could phosphorylate a PKC eps ilon substrate peptide in a phospholipid/phorbol ester-dependent manner. Mo reover, this catalytic activity could be inhibited by a fusion protein in w hich the regulatory domain of murine PKC epsilon was fused in frame with GS T (GST-R epsilon), further confirming the successful expression of murine P KC epsilon. Induction of PKC epsilon expression by galactose in cells trans formed with pVEC epsilon increased Ca++ uptake by the cells approximately 5 -fold and resulted in a dramatic inhibition of cell growth in glycerol. How ever, when glucose was used as the carbon source, PKC epsilon expression ha d no effect on cell growth. This was in contrast to what was observed upon bovine PKC alpha or PKC beta-I expression in yeast, where expression of the se PKC isoforms strongly and moderately inhibited growth in glucose, respec tively. Visualization of the cells by phase contrast microscopy indicated t hat murine PKC epsilon expression in the presence of glycerol resulted in a significant increase in the number of yeast cells exhibiting very small bu ds. Since overall growth of the cells was dramatically decreased, the data suggests that PKC epsilon expression potently inhibits the progression of y east cells through the cell cycle after the initiation of budding. In addit ion, a small amount of the PKC epsilon-expressing yeast cells (1-2%) exhibi ted gross alterations in cell morphology and defects in both chromosome seg regation and septum formation. This suggests that for those cells which do complete DNA synthesis, murine PKC epsilon expression may nevertheless inhi bit yeast cell growth by retarding and/or imparing cell division. Taken tog ether, the data suggests murine PKC epsilon expression potently reduces the growth of yeast cells in a carbon source-dependent fashion by affecting pr ogression through multiple points within the cell cycle. This murine PKC ep silon-expressing yeast strain may serve as a very useful tool in the elucid ation of mechanism(s) by which external environmental signals (possibly thr ough specific PKC isoforms) regulate cell cycle progression in both yeast a nd mammalian cells. J Cell Physiol 178.216-226, 1999. (C) 1999 Wiley-Liss, Inc.