Carbon source-dependent regulation of cell growth by murine protein kinase C epsilon expression in Saccharomyces cerevisiae.
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 (pYECepsilon) coding for the expression of murine protein kinase C epsilon (PKCepsilon) under the control of a galactose-inducible promoter. Unlike mock-transfected cells, yeast cells transformed with pYECepsilon expressed, in a galactose-dependent manner, an 89 kDa protein that was recognized by a human PKCepsilon antibody. Extracts from these pYECepsilon-transfected cells could phosphorylate a PKCepsilon 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 PKCepsilon was fused in frame with GST (GST-Repsilon), further confirming the successful expression of murine PKCepsilon. Induction of PKCepsilon expression by galactose in cells transformed with pYECepsilon 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, PKCepsilon expression had no effect on cell growth. This was in contrast to what was observed upon bovine PKCalpha or PKCbeta-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 PKCepsilon 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 PKCepsilon expression potently inhibits the progression of yeast cells through the cell cycle after the initiation of budding. In addition, a small amount of the PKCepsilon-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 PKCepsilon expression may nevertheless inhibit yeast cell growth by retarding and/or imparing cell division. Taken together, the data suggests murine PKCepsilon 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 PKCepsilon-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.
Department of Research, Northeastern Ontario Regional Cancer Centre, Sudbury, Canada. aparissenti@cancercare.on.ca
A M
AM
Parissenti
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D
Villeneuve
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Kirwan-Rhude
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D
Busch
D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002455:Cell Division / Q000502:physiology*; D005690:Galactose / Q000378:metabolism; D015870:Gene Expression; D005947:Glucose / Q000378:metabolism; D005990:Glycerol / Q000378:metabolism; D006801:Humans; D017136:Ion Transport; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D010957:Plasmids / Q000235:genetics; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D051744:Protein Kinase C-epsilon; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D007527:Isoenzymes; C497256:Prkce protein, mouse; D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon; D005947:Glucose; D005990:Glycerol; D002118:Calcium; D005690:Galactose