Copyright © 1998 by the Genetics Society of America An Essential Function of a Phosphoinositide-Specific Phospholipase C Is Relieved by Inhibition of a Cyclin-Dependent Protein Kinase in the Yeast Saccharomyces cerevisiae Jeffrey S. Flick* and Jeremy Thorner† *Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146 and †Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, California 94720-3202 Manuscript received July 24, 1997 Accepted for publication September 19, 1997 ABSTRACT The PLC1 gene product of Saccharomyces cerevisiae is a homolog of the d isoform of mammalian phospho- inositide-specific phospholipase C (PI-PLC). We found that two genes (SPL1 and SPL2), when overex- pressed, can bypass the temperature-sensitive growth defect of a plc1D cell. SPL1 is identical to the PHO81 gene, which encodes an inhibitor of a cyclin (Pho80p)-dependent protein kinase (Pho85p) complex (Cdk). In addition to overproduction of Pho81p, two other conditions that inactivate this Cdk, a cyclin (pho80D) mutation and growth on low-phosphate medium, also permitted growth of plc1D cells at the re- strictive temperature. Suppression of the temperature sensitivity of plc1D cells by pho80D does not depend upon the Pho4p transcriptional regulator, the only known substrate of the Pho80p/Pho85p Cdk. The sec- ond suppressor, SPL2, encodes a small (17-kD) protein that bears similarity to the ankyrin repeat regions present in Pho81p and in other known Cdk inhibitors. Both pho81D and spl2D show a synthetic phenotype in combination with plc1D. Unlike single mutants, plc1D pho81D and plc1D spl2D double mutants were un- able to grow on synthetic complete medium, but were able to grow on rich medium. RANSMEMBRANE signaling in eukaryotes fre- Cellular responses after PIP2 hydrolysis and produc- quently involves activation of a phosphoinositide- T tion of IP3 and DAG depend on the cell type and in- specific phospholipase C (PI-PLC), which hydrolyzes clude proliferation, differentiation, and secretion (Ber- ridge Valius Kazlauskas Weiss phosphatidylinositol 4,5-bisphosphate (PIP2) and gener- 1993; and 1993; and Littman ates two second messengers, inositol 1,4,5-trisphosphate 1994). In mammalian cells, IP3 binds to intra- Lee Rhee (IP3) and 1,2-diacylglycerol (DAG; and 1995). cellular receptors, stimulating the release of sequestered Three classes of PI-PLC (b, g, and d) have been charac- Ca21, thereby activating Ca21- and calmodulin-regu- terized at the molecular level; each contains a con- lated protein kinases and phosphoprotein phosphatases served catalytic domain comprised of two segments, (Clapham 1995). DAG remains in the membrane, designated X and Y, as well as nonconserved segments where it can activate members of the protein kinase C that confer distinct modes of regulation. Members of (PKC) family (Nishizuka 1992). Both second messen- the PI-PLC b family can be stimulated by binding of the ger pathways can elicit changes in gene expression Ga (Lee et al. 1992) and Gbg (Touhara et al. 1994; Wu (Clipstone and Crabtree 1992; Franz et al. 1994; et al. 1993) subunits released upon activation of G pro- Hill and Treisman 1995; O’Keefe et al. 1992). tein–coupled receptors. Stimulation of the PI-PLC g In animal cells, the role of any given PI-PLC isoform family depends on SH2 domains, which mediate inter- is difficult to assess because of the multiplicity of PI- action with and phosphorylation by receptor-tyrosine PLC isotypes present. Moreover, the precise function of Koch kinases ( et al. 1991). In contrast, regulation of PIP2 turnover in any given cellular response after re- the PI-PLC d family is less well characterized, although ceptor activation is complicated because of the simulta- it has been reported that a mammalian PI-PLC d can be neous recruitment of additional distinct signaling sys- stimulated in vitro by a GTPase-activating protein (GAP) tems, for example, the Ras- and PI-3 kinase–dependent for the small G protein Rho (Homma and Emori 1995) pathways (Valius and Kazlauskas 1993). Genetic ap- Feng and by interaction with a novel Gah GTPase ( et al. proaches have revealed cell type–specific requirements 1996). for particular PI-PLC isozymes. A Drosophila mutant (norpA) deficient in a PI-PLC b4 isoform lacks light- stimulated membrane potential in its photoreceptor cells and is blind (Bloomquist et al. 1988), defining a Corresponding author: Jeffrey S. Flick, Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232-0146. role for PIP2 mobilization by this PI-PLC b isoform in E-mail: fl[email protected] insect phototransduction. Disruption of the mouse PI- Genetics 148: 33–47 (January, 1998) 34 J. S. Flick and J. Thorner PLC g1 gene results in embryonic lethality, indicating growth at 358. One of the genes, PHO81, is a critical an essential requirement for this isoform in multicellu- regulatory factor of the PHO regulon (Schneider et al. lar development (Ji et al. 1997). In contrast, loss of a PI- 1994). The second gene, SPL2, encodes a novel protein PLC d homolog in Dictyostelium discoideum did not result that is regulated in part by the PHO regulon. In S. cerevi- in any apparent phenotype (Drayer et al. 1994); conse- siae, response to phosphate starvation involves the quently, the role of phosphoinositide turnover by PI- PHO81-dependent inhibition (Schneider et al. 1994) PLC d in this organism remains unknown. of a cyclin-dependent protein kinase (Cdk) comprised As a means to elucidate cellular functions depen- of the PHO80 and PHO85 gene products (Kaffman et dent on the activity of a PI-PLC d, we have also under- al. 1994; Santos et al. 1995). Pho81p action reduces taken a genetic analysis using the budding yeast Saccha- the inhibitory phosphorylation of the Pho4p transcrip- romyces cerevisiae. We (Flick and Thorner 1993) and tion factor, thereby allowing high-level expression of others (Payne and Fitzgerald-Hayes 1993; Yoko-o et genes encoding secreted acid phosphatase (PHO5), al. 1993) isolated a yeast gene, PLC1, that encodes a ho- other phosphatases, phosphate transporters, and other molog of mammalian PI-PLC d isoforms. Moreover, we products required for efficient phosphate assimilation (Flick and Thorner 1993) demonstrated that Plc1p is (Johnston and Carlson 1992). a PI-PLC, that its activity in vitro is Ca21-dependent and The findings presented here suggest, first, that Plc1p that, as observed for the mammalian enzymes, its sub- (or, more likely, the products of the reaction it cata- strate selectivity is influenced by the concentration of lyzes) acts to antagonize, directly or indirectly, either Ca21. Yeast cells lacking Plc1p are viable, but grow the Pho80p/Pho85p Cdk or one of the downstream slowly at 308 (and below), and display several other substrates of this protein kinase. Second, our results in- phenotypes, including temperature-sensitive lethality dicate that the target of Plc1p and Pho80p/Pho85p ac- (at 348 and above; Flick and Thorner 1993; Payne tion is a novel factor, not the Pho4p transactivator. Fi- and Fitzgerald-Hayes 1993), sensitivity to hyperos- nally, the genetic interactions we have uncovered motic conditions (Flick and Thorner 1993), missegre- among PLC1, SPL2, PHO81, and PHO80/PHO85 show gation of chromosomes during mitosis (Payne and that the products of these genes participate in overlap- Fitzgerald-Hayes 1993), and poor utilization of car- ping regulatory pathways necessary for adaptation to bon sources other than glucose (Flick and Thorner changing nutrient and temperature conditions. 1993; Payne and Fitzgerald-Hayes 1993). In at least one genetic background, a plc1 null allele is lethal (Yoko-o et al. 1993). MATERIALS AND METHODS As one approach for identifying the roles that Plc1p Strains, growth conditions, and general methods: The yeast plays in cellular physiology, we isolated and character- strains used in this study are listed in Table 1. Strain construc- ized two genes, which, when present in high-copy num- tion followed standard methods (Rose et al. 1990). Yeast was ber, bypass the requirement for Plc1p activity for grown on agar plates containing either YPGlc medium (1% TABLE 1 Yeast strains used in this study Straina Genotypeb Source YPH499 MATa Sikorski and Hieter 1989 YPH500 MATa Sikorski and Hieter 1989 YSS5 MATa pho80D::HIS3 Salama et al. 1994 YJF32 MATa plc1D::HIS3 Flick and Thorner 1993 YJF132 MATa plc1D::LEU2 Flick and Thorner 1993 YJF133 MATa plc1D::LEU2 Flick and Thorner 1993 YJF251 MATa plcD::LEU2 pho80D::HIS3 This work YJF252 MATa plc1D::LEU2 pho80D::HIS3 This work YJF277 MATa spl2D::HIS3 This work YJF306 MATa plc1D::LEU2 spl2D::HIS3 This work YJF386 MATa plc1D::LEU2 pho80D::HIS3 spl2D::HIS3 This work YJF552 MATa pho81D::TRP1 This work YJF555 MATa plc1D::HIS3 pho81D::TRP1 This work YJF567 MATa plc1D::LEU2 pho80D::HIS3 pho4D::TRP1 This work BJ3501 MATa pep4::HIS3 prb1-D1 can1 gal2 ADE2 LYS2 TRP1 LEU2 Jones 1991 a All strains are derived from YPH499 or YPH500, except for the protease-deficient strain BJ3501. b Except for strain BJ3501, all strains listed also carry the following mutations: ade2-101oc his3-D200 leu2-D1 lys2-801am trp1-D1 and ura3-52. Plc1p Function and the Pho80p/Pho85p Cdk in Yeast 35 yeast extract, 2% peptone, 2% glucose) or a synthetic com- BamHI and SalI to yield plasmid pJF181. The complete nucle- plete medium (SCGlc) that contained (per liter): 1.7 g Yeast otide sequence on both strands of this 1.8-kb genomic seg- Nitrogen Base without amino acids or ammonium sulfate ment was determined using a Sequenase kit (United States (Difco, Detroit, MI); 5 g (NH4)2SO4, 20 g glucose, 20 mg each Biochemical, Cleveland, OH) and conditions recommended uracil and l-Arg, 30 mg each l-Tyr and l-Ile, 40 mg adenine by the supplier from a series of nested deletions that were sulfate, 50 mg l-Phe, 60 mg l-His, 100 mg each L-Glu and generated by a procedure described previously (Henikoff l-Asp, 150 mg each l-Met and l-Val, 200 mg of l-Thr, 260 mg 1984).