Dependent Phosphatidate Phosphatase Research Articles

Phosphatidic Acid Mediates the Nem1-Spo7/Pah1 Phosphatase Cascade in Yeast Lipid Synthesis

In the yeast Saccharomyces cerevisiae, the PAH1-encoded Mg2+-dependent phosphatidate (PA) phosphatase Pah1 regulates the bifurcation of PA to diacylglycerol (DAG) for triacylglycerol (TAG) synthesis and to CDP-DAG for phospholipid synthesis. Pah1 function is mainly regulated via control of its cellular location by phosphorylation and dephosphorylation. Pah1 phosphorylated by multiple protein kinases is sequestered in the cytosol apart from its substrate PA in the membrane. The phosphorylated Pah1 is then recruited and dephosphorylated by the protein phosphatase complex Nem1 (catalytic subunit)-Spo7 (regulatory subunit) in the endoplasmic reticulum. The dephosphorylated Pah1 hops onto and scoots along the membrane to recognize PA for its dephosphorylation to DAG. Here, we developed a proteoliposome model system that mimics the Nem1-Spo7/Pah1 phosphatase cascade to provide a tool for studying Pah1 regulation. Purified Nem1-Spo7 was reconstituted into phospholipid vesicles prepared in accordance with the phospholipid composition of the nuclear/endoplasmic reticulum membrane. The Nem1-Spo7 phosphatase reconstituted in the proteoliposomes, which were measured 60 nm in an average diameter, was catalytically active on Pah1 phosphorylated by Pho85-Pho80, and its active site was located at the external side of the phospholipid bilayer. Moreover, we determined that PA stimulated the Nem1-Spo7 activity, and the regulatory effect was governed by the nature of the phosphate headgroup but not by the fatty acyl moiety of PA. The reconstitution system for the Nem1-Spo7/Pah1 phosphatase cascade, which starts with the phosphorylation of Pah1 by Pho85-Pho80 and ends with the production of DAG, is a significant advance to understand a regulatory cascade in yeast lipid synthesis.

UVB-dependent inhibition of lipin-1 protects against proinflammatory responses in human keratinocytes

Lipin-1 is an Mg2+-dependent phosphatidate phosphatase (PAP1) that catalyzes a critical step in the synthesis of glycerophospholipids and is also a cotranscriptional regulator. The role of lipin-1 in the regulation of inflammatory responses has been extensively studied in various cell types but not in skin cells. In the present study, the function of lipin-1 in UVB-induced proinflammatory responses was assessed in normal human epidermal keratinocytes (NHEKs). UVB radiation downregulated lipin-1 expression. Lipin-1 inhibition was mediated by UVB-dependent sterol-response element binding protein-1 (SREBP-1) inhibition. The UVB-dependent inhibition of lipin-1 and SREBP-1 was mediated by AMPK activation. UVB-induced activation of JNK was dependent on AMPK activation and mediated lipin-1 inhibition. Prevention of UVB-mediated lipin-1 repression by introducing a lipin-1 expression vector stimulated IL-6 and IL-8 production, suggesting that lipin-1 inhibition attenuates UVB-induced IL-6 and IL-8 production. The downregulation of lipin-1 ameliorated UVB-induced NF-ĸB phosphorylation, which might be attributed to the suppression of UVB-induced accumulation of free fatty acids (FFAs). Pharmacological inhibition of PAP1 with propranolol suppressed UVB-induced production of IL-6 and IL-8 in NHEKs and reconstituted human skin models. Taken together, lipin-1 is downregulated by exposure to UVB radiation, which confers protection against UVB-induced proinflammatory responses; therefore, the inhibition of lipin-1 is a potential strategy for photoaging.

Structure‐function Analysis of Yeast Pah1 Phosphatidate Phosphatase

Mg2+‐dependent phosphatidate phosphatase (PAP), encoded by PAH1 in Saccharomyces cerevisiae, catalyzes the dephosphorylation of phosphatidate to yield diacylglycerol. The diacylglycerol produced in the reaction is utilized for the synthesis of triacylglycerol or the major membrane phospholipids phosphatidylcholine and phosphatidylethanolamine. The PAP enzyme from yeast contains two conserved domains; the NLIP domain is found at the N‐terminus and the haloacid dehalogenase (HAD)‐like domain is found in the middle region of Pah1. The G80R mutation in the NLIP domain of Pah1 causes a 98% reduction in PAP activity along with other phenotypes characteristic of the pah1Δ mutation. The HAD‐like domain of Pah1 contains the DXDX (T/V) motif that is essential for catalytic activity. In this study, we carried out a structure‐function analysis of the conserved and non‐conserved regions of yeast Pah1. For constructs expressed and purified from Escherichia coli, G80R and G80A mutations, as well as deletion of the NLIP domain caused > 99% loss of PAP activity; the deletion of the non‐conserved region at the N‐terminus had little effect on PAP activity; and the deletion of the non‐conserved region at the C‐terminus caused a 3‐fold increase in PAP activity. The effects of the mutations on the in vivo function of Pah1 were examined by the ability to complement cold or warm sensitivities exhibited by the pah1Δ mutant. Cells expressing the deletion of the non‐conserved region at the N‐terminus complemented both the cold and warm sensitive phenotypes of the pah1Δ mutant, whereas the mutations in the conserved NLIP domain and the deletion of the non‐conserved region at the C‐terminus did not rescue the temperature sensitive phenotypes. The effects of the mutations on Pah1 PAP functions such as the synthesis of triacylglycerol and lipid droplet formation are being investigated.Support or Funding InformationSupported by NIH grant GM028140

Lipin-1 expression is critical for keratinocyte differentiation

Lipin-1 is an Mg(2+)-dependent phosphatidate phosphatase that facilitates the dephosphorylation of phosphatidic acid to generate diacylglycerol. Little is known about the expression and function of lipin-1 in normal human epidermal keratinocytes (NHEKs). Here, we demonstrate that lipin-1 is present in basal and spinous layers of the normal human epidermis, and lipin-1 expression is gradually downregulated during NHEK differentiation. Interestingly, lipin-1 knockdown (KD) inhibited keratinocyte differentiation and caused G1 arrest by upregulating p21 expression. Cell cycle arrest by p21 is required for commitment of keratinocytes to differentiation, but must be downregulated for the progress of keratinocyte differentiation. Therefore, reduced keratinocyte differentiation results from sustained upregulation of p21 by lipin-1 KD. Lipin-1 KD also decreased the phosphorylation/activation of protein kinase C (PKC)α, whereas lipin-1 overexpression increased PKCα phosphorylation. Treatment with PKCα inhibitors, like lipin-1 KD, stimulated p21 expression, while lipin-1 overexpression reduced p21 expression, implicating PKCα in lipin-1-induced regulation of p21 expression. Taken together, these results suggest that lipin-1-mediated downregulation of p21 is critical for the progress of keratinocyte differentiation after the initial commitment of keratinocytes to differentiation induced by p21, and that PKCα is involved in p21 expression regulation by lipin-1.

Distinct Roles of the Phosphatidate Phosphatases Lipin 1 and 2 during Adipogenesis and Lipid Droplet Biogenesis in 3T3-L1 Cells

Lipins are evolutionarily conserved Mg(2+)-dependent phosphatidate phosphatase (PAP) enzymes with essential roles in lipid biosynthesis. Mammals express three paralogues: lipins 1, 2, and 3. Loss of lipin 1 in mice inhibits adipogenesis at an early stage of differentiation and results in a lipodystrophic phenotype. The role of lipins at later stages of adipogenesis, when cells initiate the formation of lipid droplets, is less well characterized. We found that depletion of lipin 1, after the initiation of differentiation in 3T3-L1 cells but before the loading of lipid droplets with triacylglycerol, results in a reciprocal increase of lipin 2, but not lipin 3. We generated 3T3-L1 cells where total lipin protein and PAP activity levels are down-regulated by the combined depletion of lipins 1 and 2 at day 4 of differentiation. These cells still accumulated triacylglycerol but displayed a striking fragmentation of lipid droplets without significantly affecting their total volume per cell. This was due to the lack of the PAP activity of lipin 1 in adipocytes after day 4 of differentiation, whereas depletion of lipin 2 led to an increase of lipid droplet volume per cell. We propose that in addition to their roles during early adipogenesis, lipins also have a role in lipid droplet biogenesis.

Roles of proline‐rich regions of the actin patch protein App1p in its phosphatidate phosphatase activity and membrane localization

Actin patch protein App1p is a component of cortical actin patches. It has been recently identified as a Mg2+‐dependent phosphatidate phosphatase (PAP) that catalyzes the conversion of phosphatidate (PA) to diacylglycerol (DAG) and Pi. The protein contains a canonical DXDX(T/V) catalytic motif that is typical of Mg2+‐dependent phosphatase enzymes and several proline‐rich regions (PXXP motifs). These proline‐rich regions are known to interact with Src homology 3 (SH3) domains that are characteristic of actin patch proteins (e.g., Abp1p and Rvs167p) involved in endocytosis. In fact, App1p has been shown to physically interact with several actin patch proteins. The fact that App1p possesses PAP activity indicates that it may regulate the local concentrations of PA and DAG found in cortical actin patches. PA and DAG are known to facilitate membrane fission/fusion events in model systems, and they are also known to interact with and regulate enzymes that play important roles in vesicular trafficking. In this work, the proline residues in the PXXP motifs in App1p were mutated to glycine by site‐specific mutagenesis. The mutant App1p enzymes were expressed in app1Δ and app1Δ pah1Δ dpp1Δ lpp1Δ quadruple mutant cells to assess the roles of PXXP motifs in the interaction with actin patch proteins, PAP activity, and the association of App1p with membranes. Supported by NIH grant GM‐28140.

Fluorescence spectroscopy measures yeast PAH1-encoded phosphatidate phosphatase interaction with liposome membranes

Phosphatidate (PA) phosphatase, the enzyme that catalyzes the penultimate step in triacylglycerol synthesis, is a cytosolic enzyme that must associate with the membrane where its substrate PA resides. Fluorescence spectroscopy was used to measure the interaction of yeast PAH1-encoded PA phosphatase with model liposome membranes. PA phosphatase contains five tryptophan residues and exhibited inherit fluorescence that increased upon interaction with phosphatidylcholine liposomes. The interaction was enhanced by inclusion of other phospholipids and especially the substrate PA. Interaction was dependent on both the concentration of phosphatidylcholine-PA liposomes as well as the surface concentration of PA in liposomes. Mg(2+) ions, which were required for catalysis, did not affect PA phosphatase interaction with phosphatidylcholine-PA liposomes. PA phosphatase was a substrate for protein kinase A, protein kinase C, and casein kinase II, and these phosphorylations decreased PA phosphatase interaction with phosphatidylcholine-PA liposome membranes.

Nuclear Envelope Phosphatase 1-Regulatory Subunit 1 (Formerly TMEM188) Is the Metazoan Spo7p Ortholog and Functions in the Lipin Activation Pathway

Lipin-1 catalyzes the formation of diacylglycerol from phosphatidic acid. Lipin-1 mutations cause lipodystrophy in mice and acute myopathy in humans. It is heavily phosphorylated, and the yeast ortholog Pah1p becomes membrane-associated and active upon dephosphorylation by the Nem1p-Spo7p membrane complex. A mammalian ortholog of Nem1p is the C-terminal domain nuclear envelope phosphatase 1 (CTDNEP1, formerly "dullard"), but its Spo7p-like partner is unknown, and the need for its existence is debated. Here, we identify the metazoan ortholog of Spo7p, TMEM188, renamed nuclear envelope phosphatase 1-regulatory subunit 1 (NEP1-R1). CTDNEP1 and NEP1-R1 together complement a nem1Δspo7Δ strain to block endoplasmic reticulum proliferation and restore triacylglycerol levels and lipid droplet number. The two human orthologs are in a complex in cells, and the amount of CTDNEP1 is increased in the presence of NEP1-R1. In the Caenorhabditis elegans embryo, expression of nematode CTDNEP1 and NEP1-R1, as well as lipin-1, is required for normal nuclear membrane breakdown after zygote formation. The expression pattern of NEP1-R1 and CTDNEP1 in human and mouse tissues closely mirrors that of lipin-1. CTDNEP1 can dephosphorylate lipins-1a, -1b, and -2 in human cells only in the presence of NEP1-R1. The nuclear fraction of lipin-1b is increased when CTDNEP1 and NEP1-R1 are co-expressed. Therefore, NEP1-R1 is functionally conserved from yeast to humans and functions in the lipin activation pathway.

Phosphatidate Phosphatase Plays Role in Zinc-mediated Regulation of Phospholipid Synthesis in Yeast

In the yeast Saccharomyces cerevisiae, the synthesis of phospholipids is coordinately regulated by mechanisms that control the homeostasis of the essential mineral zinc (Carman, G.M., and Han, G. S. (2007) Regulation of phospholipid synthesis in Saccharomyces cerevisiae by zinc depletion. Biochim. Biophys. Acta 1771, 322-330; Eide, D. J. (2009) Homeostatic and adaptive responses to zinc deficiency in Saccharomyces cerevisiae. J. Biol. Chem. 284, 18565-18569). The synthesis of phosphatidylcholine is balanced by the repression of CDP-diacylglycerol pathway enzymes and the induction of Kennedy pathway enzymes. PAH1-encoded phosphatidate phosphatase catalyzes the penultimate step in triacylglycerol synthesis, and the diacylglycerol generated in the reaction may also be used for phosphatidylcholine synthesis via the Kennedy pathway. In this work, we showed that the expression of PAH1-encoded phosphatidate phosphatase was induced by zinc deficiency through a mechanism that involved interaction of the Zap1p zinc-responsive transcription factor with putative upstream activating sequence zinc-responsive elements in the PAH1 promoter. The pah1Δ mutation resulted in the derepression of the CHO1-encoded phosphatidylserine synthase (CDP-diacylglycerol pathway enzyme) and loss of the zinc-mediated regulation of the enzyme. Loss of phosphatidate phosphatase also resulted in the derepression of the CKI1-encoded choline kinase (Kennedy pathway enzyme) but decreased the synthesis of phosphatidylcholine when cells were deficient of zinc. This result confirmed the role phosphatidate phosphatase plays in phosphatidylcholine synthesis via the Kennedy pathway.

ChemInform Abstract: Lipin 1 in Lipid Metabolism

AbstractReview: 22 refs.

Lipin 1 in Lipid Metabolism

The gene encoding lipin 1 was identified with a positional cloning approach that localized the causative mutation in fatty liver dystrophic (fld) mice, a mouse model of lipodystrophy. The fld mouse lacks normal adipose tissue in the body, and displays metabolic dysregulation such as obesity, insulin resistance, and hypertriglyceridemia. Lipin 1 is abundantly expressed in key metabolic tissues, including adipose tissue, skeletal muscle, and liver. In the cytosol, lipin 1 acts as an Mg²⁺-dependent phosphatidate phosphatase type-1 (PAP1), catalyzing a key step in the synthesis of glycerolipids. In the nucleus, lipin 1 acts as a transcriptional coactivator through its direct interaction with peroxisome proliferator-activated receptor (PPAR) γ coactivator-1α (PGC-1α) and PPARα. Through two distinct functions in the nucleus and cytosol, lipin 1 modulates lipid metabolism and glucose homeostasis. Here we will discuss recent developments in our understanding of the role of lipin 1 in lipid metabolism.

Characterization of the human LPIN1‐encoded phosphatidate phosphatase isoforms

The mammalian LPIN1 gene encodes the Mg2+‐dependent phosphatidate (PA) phosphatase, which catalyzes the dephosphorylation of PA to produce diacylglycerol (DAG), and its expression by alternative splicing produces the α and β isoforms. Here, we found a novel splice form (γ) of human lipin 1, which differs from the α form by containing additional 26 amino acids. In addition, we characterized the enzymological properties of human LPIN1α, β, and γ‐encoded PA phosphatases purified after heterologous expression in Escherichia coli. The LPIN1‐encoded PA phosphatase enzymes followed surface dilution kinetics with respect to PA using Triton X‐100/PA mixed micelles. These enzymes had similar Km values (4.2–4.5 mol %) for PA, but their Vmax values were in the order of α (34.9 μmol/min/mg) > β (24.9 μmol/min/mg) >> γ (3.4 μmol/min/mg). Although the lipin 1 isoforms showed a significant difference in catalytic activity, they were similar with respect to reaction conditions for optimum activity, inhibitors, and effectors. Supported by NIH grant GM‐28140.

A Conserved Serine Residue Is Required for the Phosphatidate Phosphatase Activity but Not the Transcriptional Coactivator Functions of Lipin-1 and Lipin-2

Mammalian lipins (lipin-1, lipin-2, and lipin-3) are Mg2+-dependent phosphatidate phosphatase (PAP) enzymes, which catalyze a key reaction in glycerolipid biosynthesis. Lipin-1 also functions as a transcriptional coactivator in conjunction with members of the peroxisome proliferator-activated receptor family. An S734L mutation in LPIN2 causes Majeed syndrome, a human inflammatory disorder characterized by recurrent osteomyelitis, fever, dyserythropoietic anemia, and cutaneous inflammation. Here we demonstrate that mutation of the equivalent serine in mouse lipin-1 and lipin-2 to leucine or aspartate abolishes PAP activity but does not impair lipin association with microsomal membranes, the major site of glycerolipid synthesis. We also determined that lipin-2 has transcriptional coactivator activity for peroxisome proliferator-activated receptor-response elements similar to lipin-1 and that this activity is not affected by mutating the conserved serine. Therefore, our results indicate that the symptoms of the Majeed syndrome result from a loss of lipin-2 PAP activity. To characterize sites of lipin-2 action, we detected lipin-2 expression by in situ hybridization on whole mouse sections and by quantitative PCR of tissues relevant to Majeed syndrome. Lipin-2 was most prominently expressed in liver, where levels were much higher than lipin-1, and also in kidney, lung, gastrointestinal tract, and specific regions of the brain. Lipin-2 was also expressed in circulating red blood cells and sites of lymphopoiesis (bone marrow, thymus, and spleen). These results raise the possibility that the loss of lipin-2 PAP activity in erythrocytes and lymphocytes may contribute to the anemia and inflammation phenotypes observed in Majeed syndrome patients.

Regulation of phospholipid synthesis in yeast

Phospholipid synthesis in the yeast Saccharomyces cerevisiae is a complex process that involves regulation by both genetic and biochemical mechanisms. The activity levels of phospholipid synthesis enzymes are controlled by gene expression (e.g., transcription) and by factors (lipids, water-soluble phospholipid precursors and products, and covalent modification of phosphorylation) that modulate catalysis. Phosphatidic acid, whose levels are controlled by the biochemical regulation of key phospholipid synthesis enzymes, plays a central role in the regulation of phospholipid synthesis gene expression.

The Role of Lipin Proteins in Lipid Storage and Metabolism

The three members of the lipin protein family (lipin‐1, lipin‐2 and lipin‐3) are Mg2+dependent phosphatidate phosphatase (PAP) enzymes, which catalyze a key reaction in glycerolipid biosynthesis. Lipin‐1 has also been shown to act as a transcriptional coactivator to modulate gene expression in response to fasting in the liver. Each of the lipin genes exhibits a distinct tissue expression pattern suggesting unique physiological functions. Lipin‐1 is prominently expressed in adipose tissue and muscle, and mutations in the lipin‐1 gene cause lipodystrophy in the mouse and myoglobinuria in humans. In contrast, lipin‐2 is most prominently expressed in tissues such as liver, kidney, brain, and lung. Mutations in human lipin‐2 cause Majeed syndrome, an inflammatory disorder characterized by osteomyelitis, anemia, and cutaneous inflammation, but the mechanism underlying this phenotype is not understood. To address questions concerning the roles of each lipin family member in normal physiology and disease we have developed mutant mouse models for each of the lipin family members. Characterization of their phenotypes demonstrates unique, but interacting, roles for the lipin proteins in lipid storage and metabolism.

Phosphatidic acid mediates demyelination in Lpin1 mutant mice.

Lipids play crucial roles in many aspects of glial cell biology, affecting processes ranging from myelin membrane biosynthesis to axo-glial interactions. In order to study the role of lipid metabolism in myelinating glial cells, we specifically deleted in Schwann cells the Lpin1 gene, which encodes the Mg2+-dependent phosphatidate phosphatase (PAP1) enzyme necessary for normal triacylglycerol biosynthesis. The affected animals developed pronounced peripheral neuropathy characterized by myelin degradation, Schwann cell dedifferentiation and proliferation, and a reduction in nerve conduction velocity. The observed demyelination is mediated by endoneurial accumulation of the substrate of the PAP1 enzyme, phosphatidic acid (PA). In addition, we show that PA is a potent activator of the MEK-Erk pathway in Schwann cells, and that this activation is required for PA-induced demyelination. Our results therefore reveal a surprising role for PA in Schwann cell fate determination and provide evidence of a direct link between diseases affecting lipid metabolism and abnormal Schwann cell function.

Glucocorticoids and cyclic AMP selectively increase hepatic lipin-1 expression, and insulin acts antagonistically

Glucocorticoids (GCs) increase hepatic phosphatidate phosphatase (PAP1) activity. This is important in enhancing the liver's capacity for storing fatty acids as triacylglycerols (TAGs) that can be used subsequently for beta-oxidation or VLDL secretion. PAP1 catalyzes the conversion of phosphatidate to diacylglycerol, a key substrate for TAG and phospholipid biosynthesis. PAP1 enzymes in liver include lipin-1A and -1B (alternatively spliced isoforms) and two distinct gene products, lipin-2 and lipin-3. We determined the mechanisms by which the composite PAP1 activity is regulated using rat and mouse hepatocytes. Levels of lipin-1A and -1B mRNA were increased by dexamethasone (dex; a synthetic GC), and this resulted in increased lipin-1 synthesis, protein levels, and PAP1 activity. The stimulatory effect of dex on lipin-1 expression was enhanced by glucagon or cAMP and antagonized by insulin. Lipin-2 and lipin-3 mRNA were not increased by dex/cAMP, indicating that increased PAP1 activity is attributable specifically to enhanced lipin-1 expression. This work provides the first evidence for the differential regulation of lipin activities. Selective lipin-1 expression explains the GC and cAMP effects on increased hepatic PAP1 activity, which occurs in hepatic steatosis during starvation, diabetes, stress, and ethanol consumption.

Regulation of the yeast PAH1 gene encoding Mg2+‐dependent phosphatidate phosphatase

The PAH1 gene in Saccharomyces cerevisiae encodes Mg2+‐dependent phosphatidate (PA) phosphatase. PA phosphatase catalyzes the dephosphorylation of PA yielding diacylglycerol and Pi. This enzyme generates the diacylglycerol used for the synthesis of triacylglycerol, and may generate the diacylglycerol used for the synthesis of phosphatidylethanolamine and phosphatidylcholine via the Kennedy pathway. The PAH1‐encoded enzyme also controls the cellular concentration of its substrate PA, which is the precursor for phospholipids that are synthesized via the CDP‐diacylglycerol pathway. In this work, we examined the regulation of PAH1 expression in response to inositol supplementation and growth phase by utilization of a PPAH1‐lacZ reporter gene plasmid transformed into wild type cells. In the absence of inositol, the β‐galactosidase activity driven by the reporter gene was 3‐fold higher in stationary phase cells when compared with exponential phase cells. In the presence of inositol, the activity in stationary phase cells was 10‐fold higher than that found in exponential phase cells. In stationary phase cells, inositol supplementation resulted in a 2‐fold increase in β‐galactosidase activity. Inositol did not affect the expression of PAH1 in the exponential phase of growth. The transcription factors involved are being examined. Supported by NIH grant GM 28140.

The G80R mutation in the yeast lipin homolog Pah1p abolishes phosphatidate phosphatase activity

The yeast lipin homolog Pah1p is a Mg2+‐dependent phosphatidate (PA) phosphatase enzyme. Gly80 that is found in the NLIP domain of Pah1p is conserved in mammalian lipin proteins. A spontaneous G84R mutation in lipin 1 causes lipodystrophy and fatty liver in the fld2J (fatty liver dystrophy) mouse at birth. To examine the importance of Gly80 to the enzymatic function of yeast Pah1p, we constructed a mutant PAH1HA allele that contained arginine in place of the conserved glycine residue. While the G80R mutation did not affect the expression of Pah1p in yeast (as determined by immunoblot analysis using anti‐HA antibodies), the mutation had a major effect on its phosphatidate phosphatase activity. The overexpression of the G80R mutant protein did not have a significant effect on the level of PA phosphatase activity in pah1Δ mutant cells when compared with cells that overexpressed the wild type PAH1HA allele. Moreover, an immune complex containing the G80R mutant protein that was isolated from pah1Δ cells expressing the G80R PAH1HA allele had essentially no PA phosphatase activity. A His6‐tagged G80R mutant protein was expressed and purified from E. coli. The Vmax of the G80R mutant enzyme was 56‐fold lower than that of the wild type enzyme. The G80R mutation also caused a 2.5‐fold increase in the cooperativity respect to PA, but little effect on the Km value for PA. Supported by NIH grant GM 28140.

Phosphorylation of the yeast lipin homolog Pah1p by Cdc28p kinase

The yeast lipin homolog Pah1p is a Mg2+‐dependent phosphatidate phosphatase enzyme that catalyzes the dephosphorylation of phosphatidate to diacylglycerol and Pi. Pah1p is responsible for de novo lipid synthesis, its reaction being the committed step for the synthesis of the storage lipid triacylglycerol, which is formed from phosphatidate via diacylglycerol. The diacylglycerol generated in the phosphatidate phosphatase reaction is also utilized in the synthesis of the membrane phospholipids phosphatidylcholine and phosphatidylethanolamine via the Kennedy Pathway. Previous studies indicate that Pah1p is phosphorylated by cyclin‐dependent Cdc28p kinase and dephosphorylated by the Nem1p‐Spo7p phosphatase complex. A phosphorylation‐deficient Pah1p mutant exhibits elevated phosphatidate phosphatase activity suggesting that phosphorylation inhibits activity. In this work, we examined the phosphorylation of Pah1p by Cdc28p kinase in vitro. Using purified Escherichia coli‐expressed Pah1p as a substrate, Cdc28p kinase activity was dependent on the time of phosphorylation and the concentration of the kinase. In addition, Cdc28p kinase activity was dependent on the concentration of Pah1p and ATP. Serine and threonine residues were targets for phosphorylation. The phosphorylation of Pah1p resulted in the loss of its phosphatidate phosphatase activity. Supported by NIH grant GM‐28140.