Cross-talk phosphorylations by protein kinase C and Pho85p-Pho80p protein kinase regulate Pah1p phosphatidate phosphatase abundance in Saccharomyces cerevisiae

Abstract

Yeast Pah1p is the phosphatidate phosphatase that catalyzes the penultimate step in triacylglycerol synthesis and plays a role in the transcriptional regulation of phospholipid synthesis genes. The enzyme is multiply phosphorylated, some of which is mediated by Pho85p-Pho80p, Cdc28p-cyclin B, and protein kinase A. Here, we showed that Pah1p is a bona fide substrate of protein kinase C; the phosphorylation reaction was time- and dose-dependent and dependent on the concentrations of ATP (Km = 4.5 μm) and Pah1p (Km = 0.75 μm). The stoichiometry of the reaction was 0.8 mol of phosphate/mol of Pah1p. By combining mass spectrometry, truncation analysis, site-directed mutagenesis, and phosphopeptide mapping, we identified Ser-677, Ser-769, Ser-773, and Ser-788 as major sites of phosphorylation. Analysis of Pah1p phosphorylations by different protein kinases showed that prephosphorylation with protein kinase C reduces its subsequent phosphorylation with protein kinase A and vice versa. Prephosphorylation with Pho85p-Pho80p had an inhibitory effect on its subsequent phosphorylation with protein kinase C; however, prephosphorylation with protein kinase C had no effect on the subsequent phosphorylation with Pho85p-Pho80p. Unlike its phosphorylations by Pho85p-Pho80p and protein kinase A, which cause a significant reduction in phosphatidate phosphatase activity, the phosphorylation of Pah1p by protein kinase C had a small stimulatory effect on the enzyme activity. Analysis of phosphorylation-deficient forms of Pah1p indicated that protein kinase C does not have a major effect on its location or its function in triacylglycerol synthesis, but instead, the phosphorylation favors loss of Pah1p abundance when it is not phosphorylated with Pho85p-Pho80p.

Keywords: Diacylglycerol; Lipid Metabolism; Phosphatase; Phosphatidate; Protein Kinase C (PKC); Triacylglycerol; Yeast.

FIGURE 1.

Reactions catalyzed by yeast Pah1p PAP and Dgk1p DAG kinase and the domain structure and phosphorylation sites in Pah1p. A, the reactions catalyzed by Pah1p PAP and Dgk1p DAG kinase are shown. The DAG produced in the PAP reaction is used for the synthesis of TAG (shown) and the synthesis of phosphatidylcholine and phosphatidylethanolamine (not shown). B, the domain structure of Pah1p showing the positions of the amphipathic helix (AH), NLIP domain, the haloacid dehalogenase (HAD)-like domain containing the DXDX(T/V) catalytic motif, acidic tail (AT), and the serine (S) and threonine (T) residues that are phosphorylated by PKC (this study), PKA (38), Pho85p-Pho80p (37), and Cdc28p-cyclin B (36).

FIGURE 2.

Pah1p is phosphorylated by PKC on the serine residue. Purified recombinant Pah1p (1 μg) was phosphorylated with PKC (2.5 units) and [γ-³²P]ATP (1 nmol) for 10 min. Following the reaction, Pah1p was separated from labeled ATP by SDS-PAGE. A, the polyacrylamide gel was dried and subjected to phosphorimaging analysis. After the imaging analysis, the dried gel was swollen in water and stained with Coomassie Blue. B, ³²P-labeled Pah1p in the polyacrylamide gel was transferred to a PVDF membrane followed by incubation with 6 n HCl for 90 min at 110 °C. The acid hydrolysates were separated by two-dimensional electrophoresis and subjected to phosphorimaging analysis. The positions of the standard phosphoamino acids phosphoserine (p-Ser), phosphothreonine (p-Thr) (dotted lines), and phosphotyrosine (p-Tyr) (dotted lines) are indicated in the figure. The data shown in A and B are representative of three experiments.

FIGURE 3.

Characterization of PKC activity using Pah1p as a substrate. Phosphorylation of Pah1p by PKC was measured by following the incorporation of the radiolabeled phosphate from [γ-³²P]ATP into purified recombinant Pah1p under standard reaction conditions by varying the time (A), amount of PKC (B), and concentrations of ATP (C) and Pah1p (D). Following the reactions, the samples were subjected to SDS-PAGE; the polyacrylamide gels were dried and then subjected to phosphorimaging analysis. The relative amounts of phosphate incorporated into Pah1p were quantified using ImageQuant software. The data shown in A–D are the averages of three experiments ±S.D. (error bars).

FIGURE 4.

Effect of phosphorylation on Pah1p PAP activity. Purified recombinant Pah1p (0.5 μg) was incubated with and without PKC (10 units) and ATP (2 nmol) for 5 min. The PAP activity of the phosphorylated and unphosphorylated forms of the enzyme was measured as a function of the surface concentration of PA. The molar concentration of PA was held constant at 0.2 mm, and the molar concentration of Triton X-100 was varied to obtain the indicated surface concentrations. The values indicated are the average of three experiments ±S.D. (error bars).

FIGURE 5.

Identification of Ser-677, Ser-769, Ser-773, and Ser-788 as the PKC phosphorylation sites at the C terminus of Pah1p. A, the diagrams show full-length and truncated forms of purified recombinant Pah1p used for phosphorylation and phosphopeptide mapping analysis. The positions of the phosphorylation sites are indicated in full-length Pah1p. B, samples (1 μg) of the indicated forms of Pah1p were phosphorylated with PKC (2 units) and [γ-³²P]ATP (1 nmol) for 20 min. The phosphorylated samples were resolved by SDS-PAGE, transferred to a PVDF membrane, and subjected to phosphorimaging analysis. The arrow indicates the positions of the various forms of Pah1p. C, ³²P-labeled Pah1p from the PVDF membrane was digested with l-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin. D, the indicated purified recombinant phosphorylation site mutants were phosphorylated with PKC and subjected to proteolytic digestion as described for the truncation mutants. The digested peptides were separated on cellulose thin-layer plates by electrophoresis (from left to right) in the first dimension and by chromatography (from bottom to top) in the second dimension followed by phosphorimaging analysis. The identity of the phosphorylation sites in the radioactive phosphopeptides of full-length wild type Pah1p was determined from the maps of the truncation and phosphorylation site mutant enzymes. The positions of the phosphopeptides that were absent in the mutant enzymes (indicated by the dotted line ellipse) but present in the wild type full-length enzyme are indicated in the figure. The data are representative of three independent experiments.

FIGURE 6.

Effects of the PKC phosphorylation site mutations on the phosphorylation of Pah1p. Wild type and the indicated Pah1p phosphorylation site mutants were expressed and purified from E. coli. The Pah1p enzymes were phosphorylated with PKC (2 units) and [γ-³²P]ATP (1 nmol) for 20 min (A) or for the indicated time intervals (B). Following the reaction, Pah1p was separated from labeled ATP by SDS-PAGE. The polyacrylamide gel was dried and subjected to phosphorimaging and ImageQuant analysis. To control for loading, the dried gel was swollen with water, stained with Coomassie Blue, and subjected to image analysis. The relative amount of the phosphorylated wild type enzyme was arbitrarily set at 100%. The data reported are the average of three independent experiments ±S.D. (error bars).

FIGURE 7.

Pah1p phosphorylation by PKA or Pho85p-Pho80p reduces subsequent phosphorylation by PKC. Wild type and the indicated phosphorylation site mutants were expressed and purified from E. coli. The Pah1p enzymes were phosphorylated with PKC (A), PKA (B), or Pho85p-Pho80p (C) using [γ-³²P]ATP without or with prephosphorylation by the indicated protein kinases using unlabeled ATP. Following the reactions, Pah1p was separated from labeled ATP by SDS-PAGE. The polyacrylamide gel was dried and subjected to phosphorimaging and ImageQuant analysis. To control for loading, the dried gel was swollen with water, stained with Coomassie Blue, and subjected to image analysis. The relative amount of the phosphorylated wild type enzyme was arbitrarily set at 100%. The data reported are the average of four independent experiments ±S.D. (error bars).

FIGURE 8.

Effects of the PKC and Pho85p-Pho80p phosphorylation site mutations on the abundance and location of Pah1p and TAG content. The indicated wild type and phosphorylation site mutant forms of Pah1p were expressed in pah1Δ and pah1Δ nem1Δ cells. A, cell extracts were prepared from late exponential phase cells (A_{600 nm} ∼ 0.8) and used for Western blot analysis using anti-Pah1p and anti-Pgk1p (loading control) antibodies. The levels of Pah1p and Pgk1p were quantified with ImageQuant software. The relative amount of Pah1p/Pgk1p of the wild type enzyme was arbitrarily set at 100%. B, cell extracts were fractionated into the cytosol and membrane fractions by centrifugation. The membrane fraction was resuspended in the same volume as the cytosol fraction, and equal volumes of the fractions were subjected to Western blot analysis using anti-Pah1p, anti-Pgk1p (cytosol marker), and anti-phosphatidylserine synthase (endoplasmic reticulum marker) antibodies. As described previously (100), the Western blot analysis for the marker proteins indicated highly enriched cytosol and membrane fractions. The relative amounts of cytosolic and membrane-associated Pah1p were determined for the wild type and phosphorylation site mutant forms of the enzyme by ImageQuant analysis of the data. Each data point represents the average of four experiments ±S.D. (error bars). C, cultures were grown to the stationary phase (A_{600 nm} ∼ 3) in the medium containing [2-¹⁴C]acetate (1 μCi/ml) to label lipids. The lipids were extracted and separated by one-dimensional TLC, and the phosphorimages were subjected to ImageQuant analysis. The percentages shown for TAG were normalized to the total ¹⁴C-labeled chloroform-soluble fraction. Each data point represents the average of three experiments ±S.D. (error bars).