Carbon source‐dependent regulation of cell growth by murine protein kinase C epsilon expression in Saccharomyces cerevisiae

Abstract

Protein kinase C is known to play a role in cell cycle regulation in both lower and higher eucaryotic cells. Since mutations in yeast proteins involved in cell cycle regulation can often be rescued by the mammalian homolog and 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 transfected S. cerevisiae cells with a plasmid (pYECε) coding for the expression of murine protein kinase C epsilon (PKCε) under the control of a galactose-inducible promoter. Unlike mock-transfected cells, yeast cells transformed with pYECε expressed, in a galactose-dependent manner, an 89 kDa protein that was recognized by a human PKCε antibody. Extracts from these pYECε-transfected cells could phosphorylate a PKCε substrate peptide in a phospholipid/phorbol ester-dependent manner. Moreover, this catalytic activity could be inhibited by a fusion protein in which the regulatory domain of murine PKCε was fused in frame with GST (GST-Rε), further confirming the successful expression of murine PKCε. Induction of PKCε expression by galactose in cells transformed with pYECε increased Ca++ uptake by the cells approximately 5-fold and resulted in a dramatic inhibition of cell growth in glycerol. However, when glucose was used as the carbon source, PKCε expression had no effect on cell growth. This was in contrast to what was observed upon bovine PKCα or PKCβ-I expression in yeast, where expression of these PKC isoforms strongly and moderately inhibited growth in glucose, respectively. Visualization of the cells by phase contrast microscopy indicated that murine PKCε expression in the presence of glycerol resulted in a significant increase in the number of yeast cells exhibiting very small buds. Since overall growth of the cells was dramatically decreased, the data suggests that PKCε expression potently inhibits the progression of yeast cells through the cell cycle after the initiation of budding. In addition, a small amount of the PKCε-expressing yeast cells (1–2%) exhibited gross alterations in cell morphology and defects in both chromosome segregation and septum formation. This suggests that for those cells which do complete DNA synthesis, murine PKCε expression may nevertheless inhibit yeast cell growth by retarding and/or imparing cell division. Taken together, the data suggests murine PKCε expression potently reduces the growth of yeast cells in a carbon source-dependent fashion by affecting progression through multiple points within the cell cycle. This murine PKCε-expressing yeast strain may serve as a very useful tool in the elucidation of mechanism(s) by which external environmental signals (possibly through specific PKC isoforms) regulate cell cycle progression in both yeast and mammalian cells. J Cell Physiol 178:216–226, 1999. © 1999 Wiley-Liss, Inc.