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Pil1p and Lsp1p Negatively Regulate the 3-Phosphoinositide-dependent Protein Kinase-like Kinase Pkh1p and Downstream Signaling Pathways Pkc1p and Ypk1p*

The Saccharomyces cerevisiae homologs, Pkh1/2p, of the mammalian 3-phosphoinositide-dependent protein kinase 1 (PDK1) regulate the Pkc1-MAP kinase cascade and the partially parallel Ypk1/2p pathway(s) that control growth and cell integrity. Mammalian PDK1 is regulated by 3-phosphoinositides, whereas Pkh1/2p are regulated by sphingolipid long-chain bases (LCBs). Recently Pkh1/2p were found to complex with two related proteins, Pil1p (Ygr086) and Lsp1p (Ypl004). Because these two proteins are not related to any known protein we sought to characterize their functions. We show that Pkh1p phosphorylates both proteins in vitro in a reaction that is only weakly regulated by LCBs. In contrast, LCBs inhibit phosphorylation of Pil1p by Pkh2p, whereas LCBs stimulate phosphorylation of Lsp1p by Pkh2p. We find that Pil1p and Lsp1p down-regulate resistance to heat stress and, specifically, that they down-regulate the activity of the Pkc1p-MAP and Ypk1p pathways during heat stress. Pil1p and Lsp1p are thus the first proteins identified as regulators of Pkh1/2p. An unexpected finding was that the level of Ypk1p is greatly reduced in pkc1Δ cells, indicating that Pkc1p controls the level of Ypk1p. Homologs of Pil1p and Lsp1p are widespread in nature, and our results suggest that they may be negative regulators of PDK-like protein kinases and their downstream cellular pathways that control cell growth and survival.

Factors that regulate cell growth and survival often activate the 3-phosphoinositide-dependent protein kinase, termed PDK1 1 (1). PDK1 then activates specific members of the AGC protein kinase family that regulate diverse cellular functions necessary for growth and survival (reviewed in Refs. [2][3][4]. PDK1 homologs are found in most organisms, including two, Pkh1p and Pkh2p, in Saccharomyces cerevisiae where they have partially overlapping functions and at least one of the proteins is necessary for growth (5). One target of Pkh1p and Pkh2p is the Pkc1p-MAP kinase pathway that regulates cell wall maintenance and integrity (6). In a large-scale analysis of protein complexes, Ho et al. (7) found the predicted proteins Ygr086c and Ypl004 in a complex with either Pkh1p or Pkh2p. Here we present the results of experiments that examine the biochemical and functional roles of these two uncharacterized proteins in pathways regulated by Pkh1p and Pkh2p. Based upon our results, we assigned the name Pil1p (phosphorylation is inhibited by long chain bases) to Ygr086p and the name Lsp1p (long chain bases stimulate phosphorylation) to Ypl004p.
Pkh1p and Pkh2p show 52% amino acid identity and 69% similarity and nearly the same level of identity and similarity to human PDK1, with the similarities confined mostly to the amino-terminal protein kinase domain of the proteins. The yeast and mammalian proteins also are functionally similar, because expression of human PDK1 restores growth to inviable pkh1⌬phk2⌬ mutant cells (5). One known target of Pkh1p and Pkh2p is Pkc1p, which controls an MAP kinase pathway necessary for regulating the integrity of the cell wall (8,9) and for repolarization of the actin cytoskeleton during heat stress (10). Pkh2p was shown to phosphorylate Pkc1p in vitro and Pkc1p isolated from a pkh1 pkh2 mutant strain had reduced kinase activity, indicating that the Pkh proteins function upstream of Pkc1p (6). Additional studies showed that the Pkh proteins were necessary for preventing cell lysis due to defects in the cell wall and for repolarization of the actin cytoskeleton following a heat shock (6). Thus, the Pkh proteins are one of two pathways for regulating the Pkc1-MAP kinase cascade during heat stress. The other pathway includes membrane proteins of the WSC family that sense stress in the plasma membrane and activate the guanine nucleotide exchange factor Rom2p, which regulates the GTPase Rho1p. Active Rho1p then turns on Pkc1p-MAP kinase cascade (reviewed in Ref. 11).
Another downstream target of Pkh1p and Pkh2p is the protein kinase Ypk1p, a member of the AGC kinase family, which has been shown both in vitro and in vivo to be phosphorylated by Pkh1p (5). Ypk1p and its closely related paralog, Ypk2p (Ykr2p), perform redundant functions necessary for growth, because a ypk1 ypk2 double mutant is not viable.
The sphingolipid long chain bases (LCBs), dihydrosphingosine (DHS), and phytosphingosine (PHS) are the only regulators of Pkh1p identified to date (12). LCBs are particularly important regulators during heat stress, because their concentration transiently increases (13,14). Ypk1p and probably Ypk2p are downstream targets of this PHS-regulated signal transduction pathway that is necessary for growth of vegetative cells. Other studies have shown that the Pkh proteins and LCBs are required for endocytosis. A temperature-sensitive strain (lcb1-100) blocked in endocytosis 37°C turned out to be defective in lcb1 (15), which encodes a subunit of serine palmi-toyltransferase, the first enzyme in sphingolipid biosynthesis (16). The Ala-381 to Thr amino acid change conferred by the lcb1-100 allele results in a rapid drop in the steady-state level LCBs and greatly reduced sphingolipid synthesis when cells are shifted to 37°C (17,18). The endocytosis defect can be suppressed by multiple copies of PKH1 or PKH2 (19) and by multiple copies of PKC1 and YCK2 (20), implying that these genes perform some function necessary for endocytosis. Immunoprecipitated Pkh1p or Pkh2p were shown to phosphorylate immunoprecipitated Pkc1p, and phosphorylation was stimulated 2-to 3-fold by LCBs (19). Thus, at least at elevated temperatures, LCBs stimulate Pkh1p or Pkh2p to phosphorylate Pkc1p, which controls an uncharacterized but vital step in the internalization phase of endocytosis. A role for Ypk1p, Pkh1p, and/or Pkh2p in endocytosis is also suggested by examination of factors required for ubiquitin-dependent internalization of Ste2p, the receptor for the ␣-factor mating pheromone (21).
Roles for Ypk1p and Ypk2p in the cell wall integrity pathway and polarization of the actin cytoskeleton have been established (22). It was found that constitutively active Pkc1p or constitutively active components in the MAP kinase cascade, including Bck1p and its downstream target Mkk1p could suppress the growth defect of a ypk1⌬ypk2⌬ mutant. Specifically, ypk1⌬ypk2⌬ mutant cells were not able to properly organize the actin cytoskeleton, nor were they able to phosphorylate (activate) Slt2p, the terminal or MAP kinase in the Pkc1pcontrolled MAP kinase cascade, when cells were heat-shocked. It is unclear how Ypk1p and Ypk2p act on the Pkc1p-MAP kinase cascade. Data from another laboratory suggest that Ypk1p targets Slt2p or a pathway that acts parallel to the Pkc1p-MAP pathway to regulate cell wall synthesis (23). Ypk1p has also been implicated in the regulation of translation initiation (24).
Recently a large-scale analysis of protein complexes in S. cerevisiae revealed that Pil1p and Lsp1p were each in a complex with either Pkh1p or Pkh2p, in addition to being complexed with other proteins (7). These results suggested to us that Pil1p and Lsp1p might be substrates of Pkh1p and Pkh2p and that LCBs might control phosphorylation. Because Pil1p and Lsp1p show no sequence similarity to known proteins, possible functions cannot be inferred, therefore, we examined Pil1p and Lsp1p to gain insight into their function. We find that both Pkh1p and Pkh2p phosphorylate Pil1p and Lsp1p in vitro but that LCBs affect phosphorylation differently. Other data show that Pil1p and Lsp1p are negative regulators of heat stress resistance and of a pathway controlled by LCBs, that Pkh1p is essential to this pathway, and that downstream targets include the Pkc1p-MAP cascade and the Ypk1p signaling pathways.

EXPERIMENTAL PROCEDURES
Strains, Plasmids, and Media-Yeast strains and plasmids used in this study are listed in Tables I and II, respectively. Cells were grown in YPD (1% yeast extract, 2% peptone, 2% glucose) or complete synthetic medium (SD: 0.34% yeast nitrogen base (Difco), 1% ammonium sulfate, 2% glucose, 30 mg/liter of adenine and tyrosine, and 20 mg/liter each of histidine, leucine, lysine, methionine, uracil, and tryptophan). Synthetic medium lacking uracil (SD-Ura) was used to select cells transformed with a plasmid carrying URA3. Solid media contained 2% agar.
Catalytically inactive (kinase dead, KD (6)) versions of Pkh1p (K154R) and Pkh2p (K208R) were generated by using the GeneEditor TM in vitro site-directed mutagenesis system (Promega). Mutant constructs were verified by DNA sequence analysis. Chromosomal SLT2 was tagged at its 3Ј end with three hemagglutinin (HA) epitopes as described previously using pFA6a-3HA-HIS3MX6 as a template for generation of a PCR fragment (25). Correct insertion of the PCR fragment was verified by PCR analysis of chromosomal DNA and the tagged allele was found to be functional. Six histidine residues were added to the amino terminus of proteins by cloning genes (Table II) into pYES2/ NTA (Invitrogen, San Diego, CA) so that gene expression was driven by the galactose-inducible GAL1 promoter.
Purification of His 6 -tagged Proteins-Yeast cells, transformed with a multicopy vector expressing the desired gene under control of the GAL1 promoter (Table II), were grown overnight at 30°C to an A 600 nm of 0.4 in S-Ura medium containing 2% sucrose and 0.1% glucose. Protein overproduction was induced by adding galactose to 2.5% and incubating the culture for 5-6 h. A cell-free protein extract was prepared as described previously (6), and 10 mg was mixed with an with equal volume of wash buffer (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 20 mM imidazole). The mixture was incubated for 1 h with shaking at 4°C with 500 l of nickel-nitrilotriacetic acid-agarose (Qiagen) and then loaded into a column. The flowthrough was passed through the column again, and then 5 ml of wash buffer was passed through the column prior to elution with 2.5 ml of elution buffer (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 50 mM imidazole). His 6 -tagged proteins were also purified by immunoprecipitation from cell-free yeast extracts as described previously (19).
Immunoblotting-Protein samples were subjected to 10% SDS-PAGE followed by electroblotting onto an Immobilon-P membrane (Millipore). Membranes were blocked by incubation for 1 h at room temperature in 1% blocking solution (1% casein in Tris-buffered saline (TBS), Roche Applied Science). Membranes were then shaken with anti-His This study antibody (1:3000 dilution, Amersham Biosciences, catalogue no. 27-4710-0) overnight at 4°C. After two washes with TBS-T (0.1% Tween) and two washes with 0.5% blocking solution, membranes were incubated for 2 h with alkaline phosphatase-linked anti-mouse antibody (1:5000, Sigma). Membranes were washed three times with TBS-T, exposed to ECF substrate (Amersham Biosciences), and fluorescent signals were analyzed by using a PhosphorImager (Storm 860, Amersham Biosciences). Ypk1 was immunoblotted (12) using anti-Ypk1 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). Total protein extracts were prepared by growing cells to an A 600 nm of 0.3 at 30°C, adding myriocin as indicated in the text, switching cells to 34°C and harvesting after 6 h. Cells were centrifuged and resuspended in lysis buffer (50 mM Tris-HCl buffer, pH 8.0, containing 1 mM phenylmethylsulfonyl fluoride, 1 g/ml of leupeptin and pepstatin, 50 mM NaF, 1 mM Na 3 VO 4 , 1.5 mM MgCl 2 , 150 mM NaCl). One-half volume of acid-washed glass beads were added followed by vortexing for 3 min at 4°C. Unbroken cells and debris were removed by centrifugation at 4000 ϫ g for 10 min. A 150-g sample of the supernatant fluid was separated on a SDS-PAGE.
Slt2p Activation Assay-Quantification of phosphorylated Slt2p was done by immunoblotting of an SDS-PAGE loaded with 150 g of protein.
Stress Resistance-Heat stress resistance of log phase cells (A 600 nm of 0.3) was performed as described previously (26) as was the resistance of stationary phase cells to heat stress (27). Other stress-resistance experiments were performed as described by Huang et al. (28).
Alkaline Phosphatase Treatment of Ypk1p-Immunoprecipitates were prepared by incubating 5 l of anti-Ypk1 protein antibodies with 20 l of a 50:50 slurry of protein G-Sepharose and 300 g of cell-free extract for 1-2 h at 4°C. The beads were processed as described previously (12) and subjected to SDS-PAGE.
Ypk1 Kinase Assay-Cells grown as described for Ypk1p immunoblotting in medium containing 1 M myriocin, were centrifuged and rinsed once with ice-cold IP buffer (20 mM Tris-HCl, pH 7.5, 125 mM potassium acetate, 0.5 mM EDTA, 0.5 mM EGTA, 1 mM dithiothreitol, 1 g/ml leupeptin and pepstatin A, 0.1% Triton X-100, and 12.5% glycerol). Cells were broken with glass beads as described for Ypk1 immunoblotting. The lysate was clarified by centrifugation at 14,000 ϫ g at 4°C for 15 min and Ypk1p was immunoprecipitated from the supernatant fluid and used in a kinase assay with Cross-tide (GRPRTSSFAEG, BIOSOURCE Int.) as substrate as described previously (23). Kinase reactions were terminated by spotting a 45-l portion of the reaction mixture onto a small square of phosphocellulose paper, which were washed and counted in a liquid scintillation spectrometer (29).
Miscellaneous Assays-Protein concentrations were measured by using the Bio-Rad DC kit with bovine serum albumin as a standard. Pkh1/2p kinase activity was assayed in vitro as described previously (19). Phosphorylated Pil1p and Lsp1p was identified and quantified by using a PhosphorImager.

Pil1p Is Phosphorylated by Pkh1p and Pkh2p
-To begin to understand why Pil1p complexes with Pkh1p and Pkh2p we determined if it was a kinase substrate. The three proteins were tagged at their amino terminus with six histidines and individually overproduced in yeast by using the galactose-inducible GAL1 promoter. Proteins were partially purified by nickel affinity chromatography and used in equal amounts for in vitro phosphorylation reactions. Phosphorylated proteins were examined by SDS-PAGE and quantified by phosphorimaging analysis. In preliminary experiments we determined that phosphorylation of Pil1p by Pkh1p was linear up to 60 min (data not shown). Therefore, we used 30-min incubations for all experiments shown here.
In the absence of any LCB, Pkh1p and Pkh2p phosphorylated Pil1p (Fig. 1A, lanes 3 and 7, respectively). Contrary to expectation, phosphorylation of Pil1p by either protein kinase was not stimulated by LCBs but was actually reduced by inclusion of 50 M LCBs in the reaction (Fig. 1A, compare lane 3 with lane 4 and lane 7 with lane 8, and see below). Specifically, Pil1p phosphorylation by Pkh1p was noticeably inhibited by the biological isomer erythro-DHS and to a lesser extent by sphingosine, the major LCB found in mammals (Fig. 1A, lane 4). Pil1p phosphorylation by Pkh2p was strongly inhibited by PHS and sphingosine and inhibited to a lesser extent by erythro-DHS (Fig. 1A, lane 8). The related but chemically different lipids C18-DHS-phosphate and C 6 -ceramide were used as controls for lipid specificity and neither inhibited phosphorylation. Stearylamine, a long-chain amine also did not inhibit phosphorylation.
To demonstrate that Pkh1p and Pkh2p were responsible for phosphorylation rather than some contaminating protein kinase, we used variant Pkh1p (K154R) and Pkh2p (K208R), which lack kinase activity (KD mutants). Phosphorylation of Pil1p by either KD variant was either eliminated or greatly reduced in the presence or absence of LCB (Fig. 1A, lanes 5, 6,  9, and 10). We conclude from these data that Pil1p can be phosphorylated by Pkh1p and Pkh2p.
Phosphorylation of Pil1p by Pkh1p and Pkh2p Is Reduced by LCBs-We examined LCB inhibition of Pil1p phosphorylation in more detail by varying the concentration and type of LCB. Phosphorylation of Pil1p by Pkh1p began to be inhibited by 50 M PHS and was about 70% inhibited at the highest concentration of PHS (Fig. 1B). This concentration of PHS is about what we estimate the concentration of LCBs to be in cells grown at 25°C (13). Erythro-DHS started to inhibit at 20 M, and sphingosine started to inhibit at 50 M, whereas the non- biological isomer threo-DHS only inhibited at the highest concentration, which is most likely due to a 5% contamination with erythro-DHS as determined by HPLC (data not shown). The long-chain amine stearylamine only showed inhibition at the highest concentration used, whereas C18-DHS-phosphate and C 6 -ceramide showed no inhibition. Similar trends were seen for inhibition of Pil1p phosphorylation by Pkh2p (Fig. 1C) except that (i) the concentration of LCB needed to inhibit were slightly different, (ii) a greater level of inhibition, more that 90%, was obtained with several LCBs, and (iii) stearylamine inhibited better than in the reactions containing Pkh1p (Fig. 1B). We conclude from these data that LCBs inhibit phosphorylation of Pil1p by both Pkh1p and Pkh2p, with Pkh2p being more strongly inhibited.
Lsp1p Is Phosphorylated by Pkh1p and Pkh2p-Lsp1p, like Pil1p, was phosphorylated by Pkh1p in the absence of LCBs, and LCBs slightly stimulated phosphorylation ( Fig. 2A, lanes 3  and 4). In contrast to Pkh1p, Pkh2p only weakly phosphorylated Lsp1p in the absence of LCBs but phosphorylation was strongly stimulated by 50 M LCBs ( Fig. 2A, compare lanes 7 and 8, and see below). For example, Lsp1p phosphorylation by Pkh2 was stimulated by PHS, the biological isomer erythro-DHS and by sphingosine ( Fig. 2A, compare lanes 7 and 8). The non-biological isomer threo-DHS only stimulated phosphorylation slightly relative to the other LCBs. Stearylamine stimulated phosphorylation, but the other control lipids C18-DHSphosphate and C 6 -ceramide did not, indicating that LCBs and long-chain amines stimulate while related but chemically different sphingolipids do not. Kinase dead variants of Pkh1p (K154R) and Pkh2p (K208R) did not phosphorylate Lsp1p ( Fig.  2A, lanes 5, 6, 9, and 10). We conclude from these experiments that LCBs are not required for Pkh1p to phosphorylate Lsp1p, although they do stimulate phosphorylation. On the other hand, LCBs are required for Pkh2p to phosphorylate Lsp1p.
Phosphorylation of Lsp1p by Pkh1p and Pkh2p was examined in more detail by varying the concentration LCBs. Phosphorylation of Lsp1p by Pkh1p began to be stimulated by 20 M PHS and peak stimulation of 2.6-fold occurred at 50 M (Fig.  2B). A similar trend was seen with erythro-DHS while sphingosine only stimulated at 50 M or higher. The non-biological isomer threo-DHS showed weak stimulation at the highest concentrations used, and this stimulation is most likely due to a 5% contamination with erythro-DHS. Stimulation by stearylamine was similar to that seen erythro-DHS. Neither of the other control lipids C18-DHS-phosphate and C 6 -ceramide showed stimulation.
Deletion of PIL1 or LSP1 Enhances Heat Stress Resistance-Because the Pkh1/2 kinases have been shown to regulate the Pkc1p-MAP kinase pathway, and because this pathway controls cellular processes necessary for surviving heat stress, we determined if deletion of PIL1 or LSP1 or both genes influenced heat stress resistance in log and stationary phase cells. Log phase pil1⌬ and lsp1⌬ cells were 2-fold more resistant to heat stress than wild type cells (Fig. 3). Stationary phase pil1⌬ cells were 3-fold more resistant than wild type cells to heat stress, and lsp1⌬ cells were nearly 4-fold more resistant (Fig. 3). The double mutant cells were slightly less resistant than the single mutants, suggesting that the two proteins regulate the same or similar cellular processes that mediate heat stress. We conclude that Pil1p and Lsp1p act to down-regulate heat stress resistance.
If Pil1p and Lsp1p act to down-regulate heat stress resistance, then overproduction of either protein should produce the opposite effect and reduce heat stress resistance. Stationary phase cells with PIL1 on a multicopy vector were 7-fold less resistant to heat stress compared with cells transformed with the empty vector (Fig. 3), which is consistent with our hypothesis. On the other hand, multicopies of LSP1 slightly increased heat stress resistance, indicating that Lsp1p has a more complex mechanistic role in heat stress resistance than does Pil1p.
Pil1p and Lsp1p Down-regulate the Pkc1p-MAP Kinase Pathway-To examine in more detail the hypothesis that Pil1p and Lsp1p down-regulate the Pkc1p-MAP kinase pathway we measured phosphorylation of Slt2p, the terminal or MAP kinase in this pathway. Activation of Slt2p can be monitored with a polyclonal antibody that only recognizes Slt2p activated by dual phosphorylation of a threonine and a tyrosine residue (30,31). We found a 3-fold increase in the basal level of Slt2p phosphorylation in log phase pil1⌬ cells grown at 25°C compared with wild type cells (Fig. 4A). This difference persisted following heat shock by transfer of cells from 25°C to 39°C. We conclude that Pil1p acts to down-regulate the basal and heatinduced activity of the Pkc1-MAP kinase pathway.
The basal level of phospho-Slt2 in lsp1⌬ cells grown at 25°C was the same as in the wild type cells. But when cells were heat-shocked by transfer from 25°C to 39°C, the level of phospho-Slt2 was 2-to 3-fold higher in the mutant than it was in wild type cells (Fig. 4B). These data support the hypothesis that Lsp1p is a negative regulator of the Pkc1-MAP kinase pathway during heat stress but that it has little if any control over basal level activity of the pathway.
Pil1p and Lsp1p Work Together to Negatively Regulate Pkh1p Activity in a Pathway Controlled by Long-chain Bases-We hypothesize that Pil1p and Lsp1p regulate Pkh1p kinase activity, because both proteins form a complex with Pkh1p (7), they both are phosphorylated by Pkh1p in vitro in an LCB-sensitive reaction (Figs. 1 and 2). To examine this hypothesis we first deleted either pil1 or lsp1 or both genes in an otherwise wild type strain background and examined phenotypes that are known to be associated with loss of or reduced Pkh1/2p activity. Only very small (2-fold or less) changes were observed (data not shown) and we reasoned that this was due to redundancy in some functions of Pkh1p and Pkh2p, with one of the two kinases being less sensitive to loss of Pil1p or Lsp1p.
To circumvent the redundancy issue, we used a strain having pkh2 deleted and the wild type PKH1 allele replaced with a temperature-sensitive allele (strain INA106-3B, pkh1 D398G pkh2⌬) so that the activity of Pkh1p could be varied by changing the temperature of the incubation medium. Variants of strain INA106-3B were made with pil1, lsp1, or both genes deleted. At the permissive temperature of 30°C the lsp1⌬ cells (pkh1 D398G pkh2⌬lsp1⌬) grew the slowest of all strains, whereas the double mutant pil1⌬lsp1⌬ cells (pkh1 D398G pkh2⌬pil1⌬lsp1⌬) grew as well as the wild type (Fig. 5, left panel, compare the top and bottom rows), showing that Pil1p and Lsp1p are negatively regulating Pkh1p activity.
Because LCBs have been shown to regulate Pkh1p activity, we wanted to demonstrate that Pil1p and Lsp1p work downstream of a pathway that is regulated by LCBs. To do this we used myriocin, an inhibitor of serine palmitoyl transferase (12), the first enzyme in the pathway leading to synthesis of LCBs and sphingolipids. Our data show that Pkh1p has kinase activity, which we will refer to as basal activity, with Pil1p or Lsp1p as substrates even in the absence of an LCB (Figs. 1 and  2). LCBs enhance enzyme activity and activate the Pkh1-MAP cascade and the Ypk1/2p cascade. Thus, at a partially restrictive temperature (34°C) strain INA106-3B (pkh1 ts pkh2⌬) will contain a level of Pkh1p that is limiting for growth, and the strain should be very sensitive to myriocin because a drop in LCBs will further reduce the activity of Pkh1p and further impede growth. In contrast, in the wild type strain 15Dau the basal (not dependent upon LCBs for activity) level of Pkh1p and Pkh2p activity should remain high enough in the presence of a low dose of myriocin to promote normal growth. These predictions were verified as shown in Fig. 5. At 0.2 M myriocin wild type 15Dau cells grew as well as they did in the absence of drug, whereas growth of INA106-3B cells was nearly completely inhibited by myriocin (Fig. 5, compare the center and  right panels).
Having established conditions where growth of INA106-3B cell was dependent upon LCBs, we determined if deletion of PIL1 or LSP1 or both genes promoted growth, as predicted if they negatively regulate Pkh1p and act downstream of a pathway controlled by LCBs. The pil1⌬ and lsp1⌬ cells failed to grow just like the parental INA106-3B cells, but the double pil1⌬lsp1⌬ mutant cells grew, although not as well as the wild type 15Dau cells (Fig. 5, right panel, compare the top and  bottom rows). We conclude from the results with the double deletion mutant that Pil1p and Lsp1p work together to negatively regulated Pkh1p activity in a pathway that requires LCBs for activity.
Pil1p and Lsp1p Negatively Regulate the Ypk1p Cell Integrity Pathway-Because Pil1p and Lsp1p negatively regulate the Pkh1-MAP kinase cascade, we wanted to determine if they also regulate the parallel Ypk1/2p pathway, given that Pkh1/2p are also upstream regulators of this pathway (5,23). For these experiments the Pkc1-MAP kinase pathway was inactivated by deleting pkc1 so that growth would be dependent upon the Ypk1/2p pathway. Growth of pkc1⌬ cells with pil1, lsp1, or both genes deleted was examined. All of the mutant strains grew as well as wild type cells at 30°C, but at 34°C their growth was slightly reduced relative to wild type (Fig. 6). We reasoned that deletion of pil1 or lsp1 produced no change in growth of the pkc1⌬ cells, because the level of LCBs was sufficient to activate the Pkh1/2p-Ypk1/2p pathway and compensate for loss of Pkc1p. To test this idea a low concentration of myriocin was used to try and reduce the intracellular concentration LCBs so that growth of pkc1⌬ cells would be reduced without affecting wild type cells. These desired effects were seen with 0.5 M myriocin in the agar medium (Fig. 6). Deleting pil1, lsp1, or both genes in the pkc1⌬ cells produced a striking enhancement of growth. In the presence of 1 M myriocin only the pkc1⌬pil1⌬ or the pkc1⌬pil1⌬lsp1⌬ mutant cells grew, and they grew even better than wild type cells. We conclude that Pil1p has a more pronounced regulatory role than Lsp1p on the activity of a pathway that is necessary for growth. Evidence that the pathway involves Ypk1p is presented below.
To establish that the results shown in Fig. 6 were not an artifact of growing cells on agar medium, we performed the growth tests in liquid medium. Cells were grown to early log phase at 30°C and then switched at time zero to 34°C. Following the switch, pkc1⌬ cells divide about two times before growth stops (Fig. 7A). The same growth pattern was seen for pkc1⌬pil1⌬, pkc1⌬lsp1⌬, and pkc1⌬pil1⌬lsp1⌬ mutant cells. Thus, these results differ from those conducted with agar medium where all strains grew at 30°C (Fig. 5).
Addition of 0.5 M myriocin to liquid medium produced two changes. First, all the mutant cells went through three or four cell divisions. Second, pkc1⌬lsp1⌬ cells grew to a slightly higher density than parental pkc1⌬ cells, whereas pkc1⌬pil1⌬ and pkc1⌬pil1⌬lsp1⌬ mutant cells grew to even higher densities, with the pkc1⌬pil1⌬ cells nearly reaching the density of wild type cells (Fig. 7B). These results are similar but not identical to those observed with agar medium. before and at 30, 60, 90, and 120 min after heat shock at 39°C was measured by immunoblotting with an antibody specific for phospho-Slt2p. Another antibody that recognizes any form of Slt2p was used to measure total Slt2p. Phosphorylation was quantified by Phosphor-Imager analysis, and the values were plotted as a percentage of the wild type value at the 0-min time point. Values represent the average Ϯ S.D. of two separate experiments. The two strains in A behaved differently over the course of the experiment (p ϭ 0.027) as determined by using a generalized F-test (MIXED, SAS software package) with the degrees of freedom (1 or 2) calculated by using Satterthwaite's approximation. The two strains in B behaved differently at the 30-, 60-, and 90-min time points (p Ͻ 0.05) as determined by using the Student's t test.
Addition of 1 M myriocin to liquid medium enabled pkc1⌬pil1⌬ and pkc1⌬pil1⌬lsp1⌬ cells to grow to nearly the same density as the wild type (Fig. 7B). pkc1⌬lsp1⌬ cells did not grow much better than the parental pkc1⌬ cells. These results are very similar to those observed using agar medium, and together the two sets of data establish that Pil1p is a negative regulator of a pathway (Ypk12p, see below) that is necessary for cell growth. We conclude that in pkc1⌬ cells Pil1p works with Lsp1p to create a even stronger negative regulator of this pathway than does Pil1p by itself. Most significantly, Pil1p and the Pil1p-Lsp1p combination work downstream of a pathway regulated by LCBs.
To directly determine if deletion of pil1, lsp1, or both genes up-regulated Ypk1p activity in pkc1⌬ cells, we performed immunoblotting studies using anti-Ypk1p antibody. It has been shown previously that phosphorylated Ypk1p (the most active species) migrates slower than the non-phosphorylated, less active form and that LCB stimulation of phosphorylation is mediated by Pkh1/2. We anticipated that deletion of pil1 would generate more phospho-Ypk1p. The observed results were quite different. First, we found that only the non-phosphorylated, faster migrating species was present in cells grown at 30°C (Fig. 8, left panel). Second, following a shift to 34°C and growth for 6 h, wild type cells contained both phosphorylated and non-phosphorylated species (Fig. 8, compare the left and center panels), but unexpectedly, all of the pkc1⌬ mutant strains had a greatly reduced level of Ypk1p. Finally, addition of 1 M myriocin caused the faster migrating, non-phosphorylated species of Ypk1 to increase to a level that was slightly higher in the pkc1⌬pil1⌬ cells compared with wild type cells while in pkc1⌬pil1⌬lsp1⌬ cells the level increased to slightly less than in wild type cells (Fig. 8, right panel). As a control to demonstrate that the upper, slower migrating band of Ypk1p is phosphorylated we treated immunoprecipitated protein with calf-intestinal phosphatase (Fig. 8, CIP). These data show that deletion of pkc1 causes the level of Ypk1p to decrease when cells are given a mild heat shock, but the level can be restored if the first step in sphingolipid synthesis is partially blocked by treating cells with myriocin and if pil1 is deleted. This restoration of Ypk1 protein explains why pkc1⌬pil1⌬ and pkc1⌬pil1⌬lsp1⌬ cells are able to grow, whereas pkc1⌬ and pkc1⌬lsp1⌬ cells do not grow at 34°C in the presence of 1 M myriocin.
A prediction based upon the results shown in Fig. 8 is that Ypk1p enzyme activity should be restored in the pkc1⌬pil1⌬ and pkc1⌬pil1⌬lsp1⌬ cells but not in the pkc1⌬ and pkc1⌬lsp1⌬ cells even though the Ypk1p protein migrates like the faster, non-phosphorylated form. These predictions were verified using Ypk1p immunoprecipitated from cells grown the same way as was done for the experiments described in Fig. 8 and assayed for kinase activity using a specific peptide substrate (Cross-tide). Enzyme activity was as high in pkc1⌬pil1⌬ cells as in wild type and was about half this level in pkc1⌬pil1⌬lsp1⌬ cells, which could explain why the two mutant strains grew (Figs. 6 and 7). Enzyme activity was very low in both the strains, pkc1⌬ and pkc1⌬lsp1⌬, that failed to grow and that contained a low level of Ypk1p protein.

DISCUSSION
Our results are the first to show that Pil1p and Lsp1p are negative regulators of heat stress resistance (Fig. 3), and the PDK1-like kinase Pkh1p, along with its downstream targets, the Pkc1p-MAP kinase cascade and the Ypk1p pathway. These two pathways work in parallel to control cell wall integrity during unstressful and stressful times (23). Our conclusions are based on the observation that the basal and heat-induced level of phospho-Slt2p, the species indicative of an activated Pkc1p-MAP kinase cascade, are elevated in pil1⌬ cells (Fig. 4A) and that the heat-induced level of phospho-Slt2p is elevated in lsp1⌬ cells (Fig. 4B). In addition, we showed that deletion of pil1 or both pil1 and lsp1 enhanced growth of INA106-3B (pkh1 ts pkh2⌬) cells under moderate heat stress conditions where Pkh1p activity limits growth (Fig. 5, 34°C). Because LCBs activate Pkh1p and Pkh2p, we determined if Pil1p and Lsp1p function downstream of an LCB-regulated pathway by treating cells with low concentrations of myriocin to partially inhibit synthesis of LCBs. If Pil1p and Lsp1p are negative regulators, then abolishing them should increase Pkh1p activity and promote growth in the presence of myriocin. Deleting pil1 or deleting both pil1 and lsp1 did improve growth of INA106-3B cells (Fig. 5, 34°C panel) as predicted for negative regulators. However, myriocin did not stimulate growth (Fig. 5, compare 34°C panels with or without myriocin). Myriocin probably failed to stimulate growth in this experiment, because Pkh1p phosphorylates Pil1p and Lsp1p quite well in vitro in the absence of LCBs (Fig. 1), and, thus, the phosphorylated forms were probably present and are likely to be the ones that negatively regulate Pkh1p. Because Pkh2p is more responsive to LCBs (Figs. 1 and 2) we predict that during heat stress the transient increase in LCBs should inhibit phosphorylation of a fraction of Pil1 rendering it non-functional and allowing Pkh2p to up-regulate the Pkc1-MAP cascade and the Ypk1/2p pathways (see Fig. 10).
Other results support the idea that Pil1p and Lsp1p are negative regulators of the Ypk1p pathway and that the pathway is regulated by LCBs. Moderate heat stress was used to partially impair growth of pkc1⌬ cells so that they required the Ypk1/2p pathway(s) for growth. Deletion of pil1, lsp1, or both genes did not enhance growth of pkc1⌬ cells (Fig. 6, 34°C  panel). However, treating pkc1⌬ cells with 0.5 M myriocin strongly inhibited growth relative to wild type cells (Fig. 6, compare the two middle panels), which indicates that one or more of the Ypk1/2p pathways are dependent upon LCBs for activity, presumably via stimulation of Pkh1/2p activity. Deleting pil1, or both pil1 and lsp1, enabled pkc1⌬ cells to grow in the presence of 0.5 M myriocin (Fig. 6), a strong indication that Pil1p and Lsp1p negatively regulate Pkh1/2p activity. Doubling the concentration of myriocin to 1 M moderately inhibited growth of wild type cells but had almost no effect on pkc1⌬pil1⌬ or pkc1⌬pil1⌬lsp1⌬ cells. These results strongly support the conclusion that Pil1p is a negative regulator of the LCB 3 Pkh1/2p 3 Ypk1/2p pathway (Fig. 10). Although Lsp1p has a similar function, it is less important in this pathway than Pil1p. These conclusions were supported also by the results of cells grown in liquid cultures (Fig. 7). One interesting and unexpected effect of myriocin in the liquid cultures was that pkc1⌬ and pkc1⌬lsp1⌬ cells went through one or two more cell doublings than they did when myriocin was omitted. These results suggest that LCBs or another sphingolipid intermediate play a role in the decision to enter a new cell cycle.
To directly demonstrate the proposed role of Pil1p as a negative upstream regulator of Ypk1p activity, immunoblots were probed with anti-Ypk1p antibody. Wild type cells contain two forms of the protein with the slower migrating form being more phosphorylated than the faster migrating form (Fig. 8) (12, 23). Unexpectedly, pkc1⌬ cells had a very reduced level of Ypk1p when grown at 34°C, which was not restored to the wild type FIG. 9. Ypk1p protein kinase activity is restored in pkc1⌬ cells by deletion of PIL1 and myriocin treatment. Cells were grown as described in the legend to Fig. 7 for 6 h at 34°C in the presence of 1 M myriocin and cell-free extract were prepared. Ypk1 was precipitated from 1 mg of each extract with anti-Ypk1p antibody. Immunoprecipitated protein kinase activity was measured using Cross-tide as substrate as described under "Experimental Procedures." The average amount of radioactive phosphate incorporated into Cross-tide Ϯ S.D. for three trials is shown. level by deleting pil1 or lsp1 (Fig. 8, 34°C panel). However, treatment with myriocin restored the Ypk1 protein level (Fig.  8, right panel) and enzyme activity (Fig. 9) in pkc1⌬pil1⌬ and partially restored them in pkc1⌬pil1⌬lsp1⌬ cells. This unexpected ability of myriocin to restore Ypk1p to the wild type level explains why pkc1⌬pil1 or pkc1⌬pil1⌬lsp1⌬ cells grew well in the presence of myriocin (Figs. 6 and 7). It also supports the hypothesis that Pil1p negatively regulates an LCB-controlled pathway (Pkh1/2p) that governs Ypk1p and perhaps Ypk2p.
How myriocin restores Ypk1p in pkc1⌬ cells and how the level of Ypk1p depends upon the Pkc1-MAP kinase cascade remain to be determined. But our results could be related to an observation made by Schmelzle et al. (22). These authors found that the basal and heat-induced levels of phospho-Slt2p were reduced in ypk1⌬ cells. Together the two sets of data argue that there is a reciprocal interaction between the Pkc1-MAP cascade and the Ypk1p pathway such that each is dependent upon the other: it is not known if Ypk2p is also dependent upon the Pkc1-MAP cascade.
Our data add a new level of control and complexity to LCBregulated signal transduction pathways by showing that Pil1p and Lsp1p negatively regulate Pkh1p and its downstream effector pathways during heat stress (Fig. 10). Although we have not directly demonstrated in vivo that Pil1p and Lsp1p also regulate Pkh2p, they most likely do, because they both form a complex with Pkh2p (7) and both are substrates for phosphorylation ( Figs. 1 and 2). In our model for Pil1p and Lsp1p function (Fig. 10) we envision that under basal or non-heat stress conditions, the concentration of LCBs is low thereby allowing phosphorylation of Pil1p by Pkh1p and Pkh2p and phosphorylation of Lsp1p by Pkh1p. Our data support the idea that phosphorylated Pil1p is active (inhibitory), but experimental verification is required as are data for Lsp1p. By inhibiting Pkh1/2 p, Pil1p-P strongly down-regulates the Pkc1p-MAP cascade and the Ypk1 pathway. Our model includes the Ypk2p pathway, but this suggestion needs to be verified experimentally. Upon heat shock the concentration of LCBs transiently increases (13,14,32). Increased LCBs inhibit phosphorylation of Pil1p by Pkh1p and Pkh2p and reduce Pil1p-P (the inhibitory form) thereby allowing Pkh1/2p to phosphorylate Pkc1p and Ypk1/2p and the active Pkc1p-MAP and Ypk1/2p pathways. Within 20 -30 min after the initial heat stress, LCBs return to their basal level followed soon thereafter by a return of the Pkc1p and Ypk1p pathways to their basal state.
Our model only attempts to explain the function of Pil1p and Lsp1p during heat stress when LCB levels tend to be transiently elevated. We found no effect of pil1⌬ or lsp1⌬ on growth of cells at 25°C or 30°C in YPD medium, suggesting that their role, if any, is minor during unstressed conditions. In addition, Pil1p and Lsp1p are likely to play roles in signaling pathways and processes not depicted in Fig. 10, because both proteins were found to complex with many proteins besides Pkh1/2p (7). Furthermore, Pil1p and Lsp1p probably play roles in other stress responses, because the promoter of each gene contains three putative stress response elements (33), and their transcription is induced by several types of stress (34 -36).
Elucidating how Pil1p and Lsp1p regulate Pkh1p and Pkh2p is an important goal for future research, because their primary amino acid sequences, although highly related (72% amino acid identity and 84% similarity), are not similar to any known protein or domain and, thus, provide no clues about molecular mechanism of action. There are also homologs of each gene in Schizosaccharomyces pombe, Candida albicans, Magnaporthe grisea (rice blast fungus), Neurospora crassa, and the mouse, indicating that these proteins function in pairs and are widespread in nature. Our results suggest that at least one of their functions is to negative regulate PDK-like protein kinases and their downstream cellular processes that control cell growth and survival.