Abstract
Ca2+ plays essential roles as a second messenger often in synergism with the calcium- and phospholipid-dependent phorbol ester receptor, protein kinase C (PKC), which stimulates Ca2+ influx in various cell types in a potential positive feedback mechanism. To address the compatibility of these mechanisms between lower eukaryotes and mammals, we have stably expressed bovine PKC alpha in the yeast Saccharomyces cerevisiae. We find that phorbol ester binding sites are created which stimulate a specific calcium- and phospholipid-dependent catalytic activity in vitro. Phorbol ester activation in vivo stimulates PKC down-regulation, uptake of extracellular Ca2+, Ca2+ dependence of cell viability, and changes in cell morphology. This may represent activation of a putative PKC-mediated signaling pathway utilized by functional yeast homologs of mammalian PKC isoforms. These are suggested by some protein data; however, their genes have not yet been characterized (Simon, A. J., Milner, Y., Saville, S. P., Dvir, A., Mochly-Rosen, D., and Orr, E. (1991) Proc. R. Soc. Lond. B 243, 165-171). Our findings indicate that bovine PKC alpha is functional in yeast and stimulates calcium uptake in a manner similar to some of its responses in mammalian cells, which suggests compatible aspects of higher and lower eukaryotic signaling pathways and the feasibility of dissecting parts of the action of common signaling mediators in a simple genetic model.
Highlights
G1 events in mammalian cells (reviewed by Alkon and Rasmussen (1988) and Campbell (1983))
Down-regulation, uptake of extracellular Ca2+, Ca2+dependence of cell viability, and changes in cell morphology, suggesting that bovine PKCa-expressing cells (PKCa) is fully functional in yeast and
Our findings suggest that bovine PKCa fractions
Summary
0 1993 hy The American Society for Biochemistry and Molecular Biology, Inc. Vol 268, No 5, Issue of February 15, pp. 3456-3462,1993 Printed in U.S.A. In S. cereuisiae a PKC-related, diacylglycerol-stimulated but phorbol cellviability, and changes in cell morphology This ester-unresponsiveproteinactivity with distinctsubstrate may represent activation of a putative PKC-mediated specificity has been described (Ogita et al, 1990; Iwai et al, signaling pathway utilized by functional yeast homo- 1992), as well as putative mammalian-like PKCisoforms in a logs of mammalian PKC isoforms. Phorbol ester activation in uiuo stimulates PKC down-regulation, uptake of extracellular Ca2+, Ca2+dependence of cell viability, and changes in cell morphology, suggesting that bovine PKCa is fully functional in yeast and. The costs of publication of this article were defrayed in part by the payment of page charges This including 10 base pairs of the 3’-untranslated sequence (PKCa) and a truncated cDNA lacking coding sequences for 149 (aa 524-672) carboxyl-terminal aa (CD149)were inserted intothe Hind111 and XbaI sites under control of galactose-inducible transcriptional elements in the high copy number yeast episomal expression plasmid YEp52 containing the LEU2 gene for selection (Broach et al, 1983). Cells weretransferred to Multiscreen filtration microtiter plates (Millipore) and washed four times with 200 pl of 10 mM MES, 10 mM Tris, 35 mM CaC12pH, 6.0, filters were dried, and cell-associated radioactivity was determined by liquid scintillation spectroscopy
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