The protein kinase C (PKC)-MAPK signaling cascade is activated and is essential for viability when cells are starved for the phospholipid precursor inositol. In this study, we report that inhibiting inositol-containing sphingolipid metabolism, either by inositol starvation or treatment with agents that block sphingolipid synthesis, triggers PKC signaling independent of sphingoid base accumulation. Under these same growth conditions, a fluorescent biosensor that detects the necessary PKC signaling intermediate, phosphatidylinositol (PI)-4-phosphate (PI4P), is enriched on the plasma membrane. The appearance of the PI4P biosensor on the plasma membrane correlates with PKC activation and requires the PI 4-kinase Stt4p. Like other mutations in the PKC-MAPK pathway, mutants defective in Stt4p and the PI4P 5-kinase Mss4p, which generates phosphatidylinositol 4,5-bisphosphate, exhibit inositol auxotrophy, yet fully derepress INO1, encoding inositol-3-phosphate synthase. These observations suggest that inositol-containing sphingolipid metabolism controls PKC signaling by regulating access of the signaling lipids PI4P and phosphatidylinositol 4,5-bisphosphate to effector proteins on the plasma membrane.The protein kinase C (PKC) pathway, also known in yeast as the cell wall integrity pathway, is a highly conserved signal transduction pathway that is activated in Saccharomyces cerevisiae during periods of polarized cell growth (1, 2) as well as by numerous environmental stresses, including elevated temperature (3), entry into stationary growth phase (4), and treatment with agents that interfere with cell wall biogenesis (5, 6). Signals produced on the cell surface are amplified and relayed by PKC to downstream targets through a three-component MAPK phosphorylation cascade composed of the MEK kinase Bck1p, the redundant MEKs Mkk1p and Mkk2p, and the MAPK Slt2p (Fig. 1A). Pkc1p homologs in mammals are directly regulated by lipids, including diacylglycerol (DAG) 2 produced by hydrolysis of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ), anionic phospholipids, and sphingolipids (7,8). Nevertheless, these lipid metabolites do not appear to play identical roles in yeast PKC activation.In yeast, the phosphorylated derivatives of phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PI4P) and PI(4,5)P 2 , are essential for PKC signaling during heat stress (9). PI4P is produced on the plasma membrane by the PI 4-kinase Stt4p and is subsequently phosphorylated to PI(4,5)P 2 by the PI4P 5-kinase Mss4p. Stt4p was originally identified in a genetic screen for mutants that are hypersensitive to staurosporine, a specific inhibitor of PKC (10). Mutations in both STT4 and MSS4 cause cell lysis phenotypes associated with defects in cell wall integrity signaling, and these defects are suppressed by overexpression of PKC1 (10, 11). Stt4p-dependent pools of PI4P and PI(4,5)P 2 are proposed to regulate PKC signaling by plasma membrane recruitment of the guanine nucleotide exchange factor Rom2p, where it carries out multiple function...