A protein kinase antigenically related to pp60v-src possibly involved in yeast cell cycle control: positive in vivo regulation by sterol.

Abstract

The effects of ergosterol, yeast's natural sterol, on cell cycling and a protein kinase antigenically related to pp6O' were examined in a sterol auxotroph ofSaccharomyces cerevisiae. Sterol-depleted cells accumulate in an unbudded, G1 state. Cell budding and proliferation are reinitiated upon addition of nonlimiting ergosterol or cholesterol with trace ergosterol, whereas cholesterol or trace ergosterol alone is less effective. Stimulation of a protein kinase associated with immune complexes of yeast protein and anti-pp60v1 shows a positive correlation with exit from the G1 phase following ergosterol addition. Ergosterol-stimulated cells also demonstrate an increase in phosphatidylinositol kinase activity. The data suggest that hormonal levels of ergosterol (effective concentration, 1 nM) participate in a signaling process associated with a protein kinase possibly involved in yeast cell cycle control.In the budding yeast Saccharomyces cerevisiae nutritional signals normally control cell growth and division. When yeast cells stop growing in response to nutritional deprivation they arrest in the unbudded, G1 phase of the cell cycle (1). The isolation of many temperature-sensitive cell division cycle (cdc) mutants has led to a better understanding of the regulation of yeast cell proliferation and has resulted in the description of a G1-phase "start" event that is controlled by a number of genes (2). One of these genes, CDC28, has been cloned and sequenced (3). The CDC28 gene product shares homology with a number of mammalian protein kinases, among these a vertebrate cAMP-dependent protein kinase, as well as viral oncogenes such as v-mos and v-src (3). The CDC28-encoded protein has now been shown to possess a protein kinase activity, as has a related cdc2+-encoded protein in the fission yeast (4, 5). Moreover, a decrease in the activity ofthe cdc2-encoded protein correlates with cell cycle arrest upon nitrogen starvation in Schizosaccharomyces pombe (5). cAMP-dependent protein kinase and the RAS gene product have also been implicated in growth control in yeast (6). Interestingly, an association has been demonstrated between the activities of the pp60vsrc tyrosine kinase and phosphatidylinositol (PtdIns) kinase in Rous sarcoma virustransformed chicken embryo fibroblasts (7). Although the role of the polyphosphatidylinositol phosphates in yeast physiology has yet to be determined (8-10), we have observed a marked ergosterol-mediated stimulation of the turnover of these lipids that correlates with an enhanced rate of cell proliferation (11).Studies Gl-phase cell cycle arrest (sterol starvation) or G1 release and cell proliferation (sterol addition). This analysis has led to the identification of a protein kinase having antigenic homology with the transforming protein of the Rous sarcoma virus and whose activity is positively regulated by a trace amount of ergosterol. To our knowledge, evidence for the existence of a protein kinase possibly involved in yeast cell cycle control, whose activity is exquisitely sens...

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