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George M. Carman - One of the best experts on this subject based on the ideXlab platform.
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a review of Phosphatidate Phosphatase assays
Journal of Lipid Research, 2020Co-Authors: Prabuddha Dey, Gil-soo Han, George M. CarmanAbstract:Phosphatidate Phosphatase (PAP) catalyzes the penultimate step in the synthesis of triacylglycerol and regulates the synthesis of membrane phospholipids. There is much interest in this enzyme because it controls the cellular levels of its substrate, Phosphatidate (PA), and product, DAG; defects in the metabolism of these lipid intermediates are the basis for lipid-based diseases such as obesity, lipodystrophy, and inflammation. The measurement of PAP activity is required for studies aimed at understanding its mechanisms of action, how it is regulated, and for screening its activators and/or inhibitors. Enzyme activity is determined through the use of radioactive and nonradioactive assays that measure the product, DAG, or Pi However, sensitivity and ease of use are variable across these methods. This review summarizes approaches to synthesize radioactive PA, to analyze radioactive and nonradioactive products, DAG and Pi, and discusses the advantages and disadvantages of each PAP assay.
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A review of Phosphatidate Phosphatase assays.
Journal of lipid research, 2020Co-Authors: Prabuddha Dey, Gil-soo Han, George M. CarmanAbstract:Phosphatidate Phosphatase catalyzes the penultimate step in the synthesis of triacylglycerol and regulates the synthesis of membrane phospholipids. There is much interest in this enzyme because it controls the cellular levels of its substrate Phosphatidate and product diacylglycerol; defects in the metabolism of these lipid intermediates are the basis for lipid-based diseases such as obesity, lipodystrophy, and inflammation. The measurement of Phosphatidate Phosphatase activity is required for studies aimed at understanding its mechanisms of action, how it is regulated, and for screening its activators and/or inhibitors. Enzyme activity is determined through the use of radioactive and nonradioactive assays that measure the product diacylglycerol or Pi. However, sensitivity and ease of use are variable across these methods. This review summarizes approaches to synthesize radioactive Phosphatidate, to analyze radioactive and nonradioactive products diacylglycerol and Pi and discusses the advantages and disadvantages of each Phosphatidate Phosphatase assay.
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The Spo7 sequence LLI is required for Nem1-Spo7/Pah1 Phosphatase cascade function in yeast lipid metabolism
The Journal of biological chemistry, 2020Co-Authors: Mona Mirheydari, Gil-soo Han, Prabuddha Dey, Yeonhee Park, Geordan J. Stukey, George M. CarmanAbstract:The Nem1-Spo7 complex in the yeast Saccharomyces cerevisiae is a protein Phosphatase that catalyzes the dephosphory-lation of Pah1 Phosphatidate Phosphatase, required for its translocation to the nuclear/endoplasmic reticulum membrane. The Nem1-Spo7/Pah1 Phosphatase cascade plays a major role in triacylglycerol synthesis and in the regulation of phospholipid synthesis. In this work, we examined Spo7, a regulatory subunit required for Nem1 catalytic function, to identify residues that govern formation of the Nem1-Spo7 complex. By deletion analysis of Spo7, we identified a hydrophobic Leu-Leu-Ile (LLI) sequence comprising residues 54-56 as being required for the protein to complement the temperature-sensitive phenotype of an spo7Δ mutant strain. Mutational analysis of the LLI sequence with alanine and arginine substitutions showed that its overall hydrophobicity is crucial for the formation of the Nem1-Spo7 complex as well as for the Nem1 catalytic function on its substrate, Pah1, in vivo Consistent with the role of the Nem1-Spo7 complex in activating the function of Pah1, we found that the mutational effects of the Spo7 LLI sequence were on the Nem1-Spo7/Pah1 axis that controls lipid synthesis and related cellular processes (e.g. triacylglycerol/phospholipid synthesis, lipid droplet formation, nuclear/endoplasmic reticulum membrane morphology, vacuole fusion, and growth on glycerol medium). These findings advance the understanding of Nem1-Spo7 complex formation and its role in the Phosphatase cascade that regulates the function of Pah1 Phosphatidate Phosphatase.
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yck1 casein kinase i regulates the activity and phosphorylation of pah1 Phosphatidate Phosphatase from saccharomyces cerevisiae
Journal of Biological Chemistry, 2019Co-Authors: Azam Hassaninasab, Lu-sheng Hsieh, Wenmin Su, George M. CarmanAbstract:: The PAH1-encoded Phosphatidate Phosphatase in Saccharomyces cerevisiae plays a major role in triacylglycerol synthesis and the control of phospholipid synthesis. For its catalytic function on the nuclear/endoplasmic reticulum membrane, Pah1 translocates to the membrane through its phosphorylation/dephosphorylation. Pah1 phosphorylation on multiple serine/threonine residues is complex and catalyzed by diverse protein kinases. In this work, we demonstrate that Pah1 is phosphorylated by the YCK1-encoded casein kinase I (CKI), regulating Pah1 catalytic activity and phosphorylation. Phosphoamino acid analysis coupled with phosphopeptide mapping of the CKI-phosphorylated Pah1 indicated that it is phosphorylated mainly on multiple serine residues. Using site-directed mutagenesis and phosphorylation analysis of Pah1, we identified eight serine residues (i.e. Ser-114, Ser-475, Ser-511, Ser-602, Ser-677, Ser-705, Ser-748, and Ser-774) as the target sites of CKI. Of these residues, Ser-475 and Ser-511 were specific for CKI, whereas the others were shared by casein kinase II (Ser-705), Cdc28-cyclin B (Ser-602), Pho85-Pho80 (Ser-114, Ser-602, and Ser-748), protein kinase A (Ser-667 and Ser-774), and protein kinase C (Ser-677). CKI-mediated phosphorylation of Pah1 stimulated both its Phosphatidate Phosphatase activity and its subsequent phosphorylation by casein kinase II. However, the CKI-mediated phosphorylation of Pah1 strongly inhibited its subsequent phosphorylation by Pho85-Pho80, protein kinase A, and protein kinase C. In a reciprocal analysis, Pah1 phosphorylation by Pho85-Pho80 inhibited subsequent phosphorylation by CKI. CKI-mediated Pah1 phosphorylation was also inhibited by a peptide containing the Pah1 residues 506-517, including the kinase-specific Ser-511 residue. These findings advance our understanding of how Pah1 catalytic activity and phosphorylation are regulated by multiple protein kinases.
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Phosphatidate-mediated regulation of lipid synthesis at the nuclear/endoplasmic reticulum membrane
Biochimica et biophysica acta. Molecular and cell biology of lipids, 2019Co-Authors: Joanna M. Kwiatek, Gil-soo Han, George M. CarmanAbstract:In yeast and higher eukaryotes, phospholipids and triacylglycerol are derived from Phosphatidate at the nuclear/endoplasmic reticulum membrane. In de novo biosynthetic pathways, Phosphatidate is channeled into membrane phospholipids via its conversion to CDP-diacylglycerol. Its dephosphorylation to diacylglycerol is required for the synthesis of triacylglycerol as well as for the synthesis of phosphatidylcholine and phosphatidylethanolamine via the Kennedy pathway. In addition to the role of Phosphatidate as a precursor, it is a regulatory molecule in the transcriptional control of phospholipid synthesis genes via the Henry regulatory circuit. Pah1 Phosphatidate Phosphatase and Dgk1 diacylglycerol kinase are key players that function counteractively in the control of the Phosphatidate level at the nuclear/endoplasmic reticulum membrane. Loss of Pah1 Phosphatidate Phosphatase activity not only affects triacylglycerol synthesis but also disturbs the balance of the Phosphatidate level, resulting in the alteration of lipid synthesis and related cellular defects. The pah1Δ phenotypes requiring Dgk1 diacylglycerol kinase exemplify the importance of the Phosphatidate level in the misregulation of cellular processes. The catalytic function of Pah1 requires its translocation from the cytoplasm to the nuclear/endoplasmic reticulum membrane, which is regulated through its phosphorylation in the cytoplasm by multiple protein kinases as well as through its dephosphorylation by the membrane-associated Nem1-Spo7 protein Phosphatase complex. This article is part of a Special Issue entitled Endoplasmic reticulum platforms for lipid dynamics edited by Shamshad Cockcroft and Christopher Stefan.
Gil-soo Han - One of the best experts on this subject based on the ideXlab platform.
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A review of Phosphatidate Phosphatase assays.
Journal of lipid research, 2020Co-Authors: Prabuddha Dey, Gil-soo Han, George M. CarmanAbstract:Phosphatidate Phosphatase catalyzes the penultimate step in the synthesis of triacylglycerol and regulates the synthesis of membrane phospholipids. There is much interest in this enzyme because it controls the cellular levels of its substrate Phosphatidate and product diacylglycerol; defects in the metabolism of these lipid intermediates are the basis for lipid-based diseases such as obesity, lipodystrophy, and inflammation. The measurement of Phosphatidate Phosphatase activity is required for studies aimed at understanding its mechanisms of action, how it is regulated, and for screening its activators and/or inhibitors. Enzyme activity is determined through the use of radioactive and nonradioactive assays that measure the product diacylglycerol or Pi. However, sensitivity and ease of use are variable across these methods. This review summarizes approaches to synthesize radioactive Phosphatidate, to analyze radioactive and nonradioactive products diacylglycerol and Pi and discusses the advantages and disadvantages of each Phosphatidate Phosphatase assay.
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a review of Phosphatidate Phosphatase assays
Journal of Lipid Research, 2020Co-Authors: Prabuddha Dey, Gil-soo Han, George M. CarmanAbstract:Phosphatidate Phosphatase (PAP) catalyzes the penultimate step in the synthesis of triacylglycerol and regulates the synthesis of membrane phospholipids. There is much interest in this enzyme because it controls the cellular levels of its substrate, Phosphatidate (PA), and product, DAG; defects in the metabolism of these lipid intermediates are the basis for lipid-based diseases such as obesity, lipodystrophy, and inflammation. The measurement of PAP activity is required for studies aimed at understanding its mechanisms of action, how it is regulated, and for screening its activators and/or inhibitors. Enzyme activity is determined through the use of radioactive and nonradioactive assays that measure the product, DAG, or Pi However, sensitivity and ease of use are variable across these methods. This review summarizes approaches to synthesize radioactive PA, to analyze radioactive and nonradioactive products, DAG and Pi, and discusses the advantages and disadvantages of each PAP assay.
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The Spo7 sequence LLI is required for Nem1-Spo7/Pah1 Phosphatase cascade function in yeast lipid metabolism
The Journal of biological chemistry, 2020Co-Authors: Mona Mirheydari, Gil-soo Han, Prabuddha Dey, Yeonhee Park, Geordan J. Stukey, George M. CarmanAbstract:The Nem1-Spo7 complex in the yeast Saccharomyces cerevisiae is a protein Phosphatase that catalyzes the dephosphory-lation of Pah1 Phosphatidate Phosphatase, required for its translocation to the nuclear/endoplasmic reticulum membrane. The Nem1-Spo7/Pah1 Phosphatase cascade plays a major role in triacylglycerol synthesis and in the regulation of phospholipid synthesis. In this work, we examined Spo7, a regulatory subunit required for Nem1 catalytic function, to identify residues that govern formation of the Nem1-Spo7 complex. By deletion analysis of Spo7, we identified a hydrophobic Leu-Leu-Ile (LLI) sequence comprising residues 54-56 as being required for the protein to complement the temperature-sensitive phenotype of an spo7Δ mutant strain. Mutational analysis of the LLI sequence with alanine and arginine substitutions showed that its overall hydrophobicity is crucial for the formation of the Nem1-Spo7 complex as well as for the Nem1 catalytic function on its substrate, Pah1, in vivo Consistent with the role of the Nem1-Spo7 complex in activating the function of Pah1, we found that the mutational effects of the Spo7 LLI sequence were on the Nem1-Spo7/Pah1 axis that controls lipid synthesis and related cellular processes (e.g. triacylglycerol/phospholipid synthesis, lipid droplet formation, nuclear/endoplasmic reticulum membrane morphology, vacuole fusion, and growth on glycerol medium). These findings advance the understanding of Nem1-Spo7 complex formation and its role in the Phosphatase cascade that regulates the function of Pah1 Phosphatidate Phosphatase.
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Phosphatidate-mediated regulation of lipid synthesis at the nuclear/endoplasmic reticulum membrane
Biochimica et biophysica acta. Molecular and cell biology of lipids, 2019Co-Authors: Joanna M. Kwiatek, Gil-soo Han, George M. CarmanAbstract:In yeast and higher eukaryotes, phospholipids and triacylglycerol are derived from Phosphatidate at the nuclear/endoplasmic reticulum membrane. In de novo biosynthetic pathways, Phosphatidate is channeled into membrane phospholipids via its conversion to CDP-diacylglycerol. Its dephosphorylation to diacylglycerol is required for the synthesis of triacylglycerol as well as for the synthesis of phosphatidylcholine and phosphatidylethanolamine via the Kennedy pathway. In addition to the role of Phosphatidate as a precursor, it is a regulatory molecule in the transcriptional control of phospholipid synthesis genes via the Henry regulatory circuit. Pah1 Phosphatidate Phosphatase and Dgk1 diacylglycerol kinase are key players that function counteractively in the control of the Phosphatidate level at the nuclear/endoplasmic reticulum membrane. Loss of Pah1 Phosphatidate Phosphatase activity not only affects triacylglycerol synthesis but also disturbs the balance of the Phosphatidate level, resulting in the alteration of lipid synthesis and related cellular defects. The pah1Δ phenotypes requiring Dgk1 diacylglycerol kinase exemplify the importance of the Phosphatidate level in the misregulation of cellular processes. The catalytic function of Pah1 requires its translocation from the cytoplasm to the nuclear/endoplasmic reticulum membrane, which is regulated through its phosphorylation in the cytoplasm by multiple protein kinases as well as through its dephosphorylation by the membrane-associated Nem1-Spo7 protein Phosphatase complex. This article is part of a Special Issue entitled Endoplasmic reticulum platforms for lipid dynamics edited by Shamshad Cockcroft and Christopher Stefan.
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Casein kinase II-mediated phosphorylation of lipin 1β Phosphatidate Phosphatase at Ser-285 and Ser-287 regulates its interaction with 14-3-3β protein.
The Journal of biological chemistry, 2019Co-Authors: Meagan Hennessy, Gil-soo Han, Azam Hassaninasab, Thurl E. Harris, Mitchell E. Granade, Dana Wang, Joanna M. Kwiatek, George M. CarmanAbstract:The mammalian lipin 1 Phosphatidate Phosphatase is a key regulatory enzyme in lipid metabolism. By catalyzing Phosphatidate dephosphorylation, which produces diacylglycerol, the enzyme plays a major role in the synthesis of triacylglycerol and membrane phospholipids. The importance of lipin 1 to lipid metabolism is exemplified by cellular defects and lipid-based diseases associated with its loss or overexpression. Phosphorylation of lipin 1 governs whether it is associated with the cytoplasm apart from its substrate or with the endoplasmic reticulum membrane where its enzyme reaction occurs. Lipin 1β is phosphorylated on multiple sites, but less than 10% of them are ascribed to a specific protein kinase. Here, we demonstrate that lipin 1β is a bona fide substrate for casein kinase II (CKII), a protein kinase that is essential to viability and cell cycle progression. Phosphoamino acid analysis and phosphopeptide mapping revealed that lipin 1β is phosphorylated by CKII on multiple serine and threonine residues, with the former being major sites. Mutational analysis of lipin 1β and its peptides indicated that Ser-285 and Ser-287 are both phosphorylated by CKII. Substitutions of Ser-285 and Ser-287 with nonphosphorylatable alanine attenuated the interaction of lipin 1β with 14-3-3β protein, a regulatory hub that facilitates the cytoplasmic localization of phosphorylated lipin 1. These findings advance our understanding of how phosphorylation of lipin 1β Phosphatidate Phosphatase regulates its interaction with 14-3-3β protein and intracellular localization and uncover a mechanism by which CKII regulates cellular physiology.
David N. Brindley - One of the best experts on this subject based on the ideXlab platform.
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Conserved Residues in the N Terminus of Lipin-1 Are Required for Binding to Protein Phosphatase-1c, Nuclear Translocation, and Phosphatidate Phosphatase Activity
The Journal of biological chemistry, 2014Co-Authors: Bernard P. C. Kok, Tamara D. Skene-arnold, Ji Ling, Matthew G.k. Benesch, Jay Dewald, Thurl E. Harris, Charles F. B. Holmes, David N. BrindleyAbstract:Lipin-1 is a Phosphatidate Phosphatase in glycerolipid biosynthesis and signal transduction. It also serves as a transcriptional co-regulator to control lipid metabolism and adipogenesis. These functions are controlled partly by its subcellular distribution. Hyperphosphorylated lipin-1 remains sequestered in the cytosol, whereas hypophosphorylated lipin-1 translocates to the endoplasmic reticulum and nucleus. The serine/threonine protein Phosphatase-1 catalytic subunit (PP-1c) is a major protein dephosphorylation enzyme. Its activity is controlled by interactions with different regulatory proteins, many of which contain conserved RVXF binding motifs. We found that lipin-1 binds to PP-1cγ through a similar HVRF binding motif. This interaction depends on Mg2+ or Mn2+ and is competitively inhibited by (R/H)VXF-containing peptides. Mutating the HVRF motif in the highly conserved N terminus of lipin-1 greatly decreases PP-1cγ interaction. Moreover, mutations of other residues in the N terminus of lipin-1 also modulate PP-1cγ binding. PP-1cγ binds poorly to a phosphomimetic mutant of lipin-1 and binds well to the non-phosphorylatable lipin-1 mutant. This indicates that lipin-1 is dephosphorylated before PP-1cγ binds to its HVRF motif. Importantly, mutating the HVRF motif also abrogates the nuclear translocation and Phosphatidate Phosphatase activity of lipin-1. In conclusion, we provide novel evidence of the importance of the lipin-1 N-terminal domain for its catalytic activity, nuclear localization, and binding to PP-1cγ.
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A conserved serine residue is required for the Phosphatidate Phosphatase activity but not the transcriptional coactivator functions of lipin-1 and lipin-2.
The Journal of biological chemistry, 2009Co-Authors: Jimmy Donkor, David N. Brindley, Bernard P. C. Kok, Jay Dewald, Peixiang Zhang, Samantha Wong, Lauren O'loughlin, Karen ReueAbstract: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.
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thematic review series glycerolipids multiple roles for lipins Phosphatidate Phosphatase enzymes in lipid metabolism
Journal of Lipid Research, 2008Co-Authors: Karen Reue, David N. BrindleyAbstract:Phosphatidate Phosphatase-1 (PAP1) enzymes have a key role in glycerolipid synthesis through the conversion of Phosphatidate to diacylglycerol, the immediate precursor of triacylglycerol, phosphatidylcholine, and phosphatidylethanolamine. PAP1 activity in mammals is determined by the lipin family of proteins, lipin-1, lipin-2, and lipin-3, which each have distinct tissue expression patterns and appear to have unique physiological functions. In addition to its role in glycerolipid synthesis, lipin-1 also operates as a transcriptional coactivator, working in collaboration with known nuclear receptors and coactivators to modulate lipid metabolism gene expression. The requirement for different lipin activities in vivo is highlighted by the occurrence of lipodystrophy, insulin resistance, and neuropathy in a lipin-1-deficient mutant mouse strain. In humans, variations in lipin-1 expression levels and gene polymorphisms are associated with insulin sensitivity, metabolic rate, hypertension, and risk for the metabolic syndrome. Furthermore, critical mutations in lipin-2 result in the development of an inflammatory disorder in human patients. A key goal of future studies will be to further elucidate the specific roles and modes of regulation of each of the three lipin proteins in key metabolic processes, including triglyceride and phospholipid synthesis, fatty acid metabolism, and insulin sensitivity.
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Thematic Review Series: Glycerolipids. Multiple roles for lipins/Phosphatidate Phosphatase enzymes in lipid metabolism.
Journal of lipid research, 2008Co-Authors: Karen Reue, David N. BrindleyAbstract:Phosphatidate Phosphatase-1 (PAP1) enzymes have a key role in glycerolipid synthesis through the conversion of Phosphatidate to diacylglycerol, the immediate precursor of triacylglycerol, phosphatidylcholine, and phosphatidylethanolamine. PAP1 activity in mammals is determined by the lipin family of proteins, lipin-1, lipin-2, and lipin-3, which each have distinct tissue expression patterns and appear to have unique physiological functions. In addition to its role in glycerolipid synthesis, lipin-1 also operates as a transcriptional coactivator, working in collaboration with known nuclear receptors and coactivators to modulate lipid metabolism gene expression. The requirement for different lipin activities in vivo is highlighted by the occurrence of lipodystrophy, insulin resistance, and neuropathy in a lipin-1-deficient mutant mouse strain. In humans, variations in lipin-1 expression levels and gene polymorphisms are associated with insulin sensitivity, metabolic rate, hypertension, and risk for the metabolic syndrome. Furthermore, critical mutations in lipin-2 result in the development of an inflammatory disorder in human patients. A key goal of future studies will be to further elucidate the specific roles and modes of regulation of each of the three lipin proteins in key metabolic processes, including triglyceride and phospholipid synthesis, fatty acid metabolism, and insulin sensitivity.
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The level and compartmentalization of Phosphatidate Phosphatase-1 (lipin-1) control the assembly and secretion of hepatic VLDL.
Journal of lipid research, 2008Co-Authors: Maroun Bou Khalil, Karen Reue, Meenakshi Sundaram, Hong-yu Zhang, Philip H. Links, Jennifer F. Raven, Boripont Manmontri, Meltem Sariahmetoglu, Khai Tran, David N. BrindleyAbstract:Phosphatidate Phosphatase-1 (PAP-1) converts Phosphatidate to diacylglycerol and plays a key role in the biosynthesis of phospholipids and triacylglycerol (TAG). PAP-1 activity is encoded by members of the lipin family, including lipin-1 (1alpha and 1beta), -2, and -3. We determined the effect of lipin-1 expression on the assembly and secretion of very low density lipoproteins (VLDL) using McA-RH7777 cells. Expression of lipin-1alpha or -1beta increased the synthesis and secretion of [(3)H]glycerol-labeled lipids under either basal- or oleate-supplemented conditions. In the presence of oleate, the increased TAG secretion was mainly associated with VLDL(1) (S(f) > 100) and VLDL(2) (S(f) 20-100). Expression of lipin-1alpha or -1beta increased secretion efficiency and decreased intracellular degradation of [(35)S]apolipoprotein B-100 (apoB100). Knockdown of lipin-1 using specific short interfering RNA decreased secretion of [(3)H]glycerolipids and [(35)S]apoB100 even though total PAP-1 activity was not decreased, owing to the presence of lipin-2 and -3 in the cells. Deletion of the nuclear localization signal sequences within lipin-1alpha not only abolished nuclear localization but also resulted in impaired association with microsomal membranes. Cells expressing the cytosolic lipin-1alpha mutant failed to promote [(35)S]apoB100 synthesis or secretion, and showed compromised stimulation in [(3)H]TAG synthesis and secretion. Thus, alteration in hepatic expression of lipin-1 and its compartmentalization control VLDL assembly/secretion.
Claudio De Virgilio - One of the best experts on this subject based on the ideXlab platform.
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TORC1 regulates Pah1 Phosphatidate Phosphatase activity via the Nem1/Spo7 protein Phosphatase complex.
PloS one, 2014Co-Authors: Emmanuelle Dubots, Stéphanie Cottier, Marie-pierre Péli-gulli, Malika Jaquenoud, Séverine Bontron, Roger Schneiter, Claudio De VirgilioAbstract:The evolutionarily conserved target of rapamycin complex 1 (TORC1) controls growth-related processes such as protein, nucleotide, and lipid metabolism in response to growth hormones, energy/ATP levels, and amino acids. Its deregulation is associated with cancer, type 2 diabetes, and obesity. Among other substrates, mammalian TORC1 directly phosphorylates and inhibits the Phosphatidate Phosphatase lipin-1, a central enzyme in lipid metabolism that provides diacylglycerol for the synthesis of membrane phospholipids and/or triacylglycerol as neutral lipid reserve. Here, we show that yeast TORC1 inhibits the function of the respective lipin, Pah1, to prevent the accumulation of triacylglycerol. Surprisingly, TORC1 regulates Pah1 in part indirectly by controlling the phosphorylation status of Nem1 within the Pah1-activating, heterodimeric Nem1-Spo7 protein Phosphatase module. Our results delineate a hitherto unknown TORC1 effector branch that controls lipin function in yeast, which, given the recent discovery of Nem1-Spo7 orthologous proteins in humans, may be conserved.
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torc1 regulates pah1 Phosphatidate Phosphatase activity via the nem1 spo7 protein Phosphatase complex
PLOS ONE, 2014Co-Authors: Emmanuelle Dubots, Stéphanie Cottier, Malika Jaquenoud, Séverine Bontron, Roger Schneiter, Mariepierre Peligulli, Claudio De VirgilioAbstract:The evolutionarily conserved target of rapamycin complex 1 (TORC1) controls growth-related processes such as protein, nucleotide, and lipid metabolism in response to growth hormones, energy/ATP levels, and amino acids. Its deregulation is associated with cancer, type 2 diabetes, and obesity. Among other substrates, mammalian TORC1 directly phosphorylates and inhibits the Phosphatidate Phosphatase lipin-1, a central enzyme in lipid metabolism that provides diacylglycerol for the synthesis of membrane phospholipids and/or triacylglycerol as neutral lipid reserve. Here, we show that yeast TORC1 inhibits the function of the respective lipin, Pah1, to prevent the accumulation of triacylglycerol. Surprisingly, TORC1 regulates Pah1 in part indirectly by controlling the phosphorylation status of Nem1 within the Pah1-activating, heterodimeric Nem1-Spo7 protein Phosphatase module. Our results delineate a hitherto unknown TORC1 effector branch that controls lipin function in yeast, which, given the recent discovery of Nem1-Spo7 orthologous proteins in humans, may be conserved.
Lu-sheng Hsieh - One of the best experts on this subject based on the ideXlab platform.
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yck1 casein kinase i regulates the activity and phosphorylation of pah1 Phosphatidate Phosphatase from saccharomyces cerevisiae
Journal of Biological Chemistry, 2019Co-Authors: Azam Hassaninasab, Lu-sheng Hsieh, Wenmin Su, George M. CarmanAbstract:: The PAH1-encoded Phosphatidate Phosphatase in Saccharomyces cerevisiae plays a major role in triacylglycerol synthesis and the control of phospholipid synthesis. For its catalytic function on the nuclear/endoplasmic reticulum membrane, Pah1 translocates to the membrane through its phosphorylation/dephosphorylation. Pah1 phosphorylation on multiple serine/threonine residues is complex and catalyzed by diverse protein kinases. In this work, we demonstrate that Pah1 is phosphorylated by the YCK1-encoded casein kinase I (CKI), regulating Pah1 catalytic activity and phosphorylation. Phosphoamino acid analysis coupled with phosphopeptide mapping of the CKI-phosphorylated Pah1 indicated that it is phosphorylated mainly on multiple serine residues. Using site-directed mutagenesis and phosphorylation analysis of Pah1, we identified eight serine residues (i.e. Ser-114, Ser-475, Ser-511, Ser-602, Ser-677, Ser-705, Ser-748, and Ser-774) as the target sites of CKI. Of these residues, Ser-475 and Ser-511 were specific for CKI, whereas the others were shared by casein kinase II (Ser-705), Cdc28-cyclin B (Ser-602), Pho85-Pho80 (Ser-114, Ser-602, and Ser-748), protein kinase A (Ser-667 and Ser-774), and protein kinase C (Ser-677). CKI-mediated phosphorylation of Pah1 stimulated both its Phosphatidate Phosphatase activity and its subsequent phosphorylation by casein kinase II. However, the CKI-mediated phosphorylation of Pah1 strongly inhibited its subsequent phosphorylation by Pho85-Pho80, protein kinase A, and protein kinase C. In a reciprocal analysis, Pah1 phosphorylation by Pho85-Pho80 inhibited subsequent phosphorylation by CKI. CKI-mediated Pah1 phosphorylation was also inhibited by a peptide containing the Pah1 residues 506-517, including the kinase-specific Ser-511 residue. These findings advance our understanding of how Pah1 catalytic activity and phosphorylation are regulated by multiple protein kinases.
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phosphorylation of yeast pah1 Phosphatidate Phosphatase by casein kinase ii regulates its function in lipid metabolism
Journal of Biological Chemistry, 2016Co-Authors: Lu-sheng Hsieh, Wenmin Su, George M. CarmanAbstract:Abstract Pah1 Phosphatidate Phosphatase in Saccharomyces cerevisiae catalyzes the penultimate step in the synthesis of triacylglycerol, i.e., the production of diacylglycerol by dephosphorylation of Phosphatidate. The enzyme playing a major role in lipid metabolism is subject to phosphorylation (e.g., by Pho85-Pho80, Cdc28-cyclin B, protein kinases A and C) and dephosphorylation (e.g., by Nem1-Spo7) that regulate its cellular location, catalytic activity, and stability/degradation. In this work, we showed that Pah1 is a substrate for casein kinase II (CKII); its phosphorylation was time- and dose-dependent, and dependent on the concentrations of Pah1 (Km = 0.23 μM) and ATP (Km = 5.5 μM). By mass spectrometry, truncation analysis, site-directed mutagenesis, phosphopeptide mapping, and phosphoamino acid analysis, we identified that >90% of its phosphorylation occurs on Thr-170, Ser-250, Ser-313, Ser-705, Ser-814, and Ser-818. The CKII-phosphorylated Pah1 was a substrate for the Nem1-Spo7 protein Phosphatase, and was degraded by the 20S proteasome. The prephosphorylation of Pah1 by protein kinase A or protein kinase C reduced its subsequent phosphorylation by CKII. The prephosphorylation of Pah1 by CKII reduced its subsequent phosphorylation by protein kinase A, but not by protein kinase C. The expression of Pah1 with combined mutations of S705D and 7A, which mimic its phosphorylation by CKII and lack of phosphorylation by Pho85-Pho80, caused an increase in triacylglycerol content and lipid droplet number in cells expressing the Nem1-Spo7 Phosphatase complex.
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Phosphorylation of Yeast Pah1 Phosphatidate Phosphatase by Casein Kinase II Regulates Its Function in Lipid Metabolism.
The Journal of biological chemistry, 2016Co-Authors: Lu-sheng Hsieh, Gil-soo Han, George M. CarmanAbstract:Pah1 Phosphatidate Phosphatase in Saccharomyces cerevisiae catalyzes the penultimate step in the synthesis of triacylglycerol (i.e. the production of diacylglycerol by dephosphorylation of Phosphatidate). The enzyme playing a major role in lipid metabolism is subject to phosphorylation (e.g. by Pho85-Pho80, Cdc28-cyclin B, and protein kinases A and C) and dephosphorylation (e.g. by Nem1-Spo7) that regulate its cellular location, catalytic activity, and stability/degradation. In this work, we show that Pah1 is a substrate for casein kinase II (CKII); its phosphorylation was time- and dose-dependent and was dependent on the concentrations of Pah1 (Km = 0.23 μm) and ATP (Km = 5.5 μm). By mass spectrometry, truncation analysis, site-directed mutagenesis, phosphopeptide mapping, and phosphoamino acid analysis, we identified that >90% of its phosphorylation occurs on Thr-170, Ser-250, Ser-313, Ser-705, Ser-814, and Ser-818. The CKII-phosphorylated Pah1 was a substrate for the Nem1-Spo7 protein Phosphatase and was degraded by the 20S proteasome. The prephosphorylation of Pah1 by protein kinase A or protein kinase C reduced its subsequent phosphorylation by CKII. The prephosphorylation of Pah1 by CKII reduced its subsequent phosphorylation by protein kinase A but not by protein kinase C. The expression of Pah1 with combined mutations of S705D and 7A, which mimic its phosphorylation by CKII and lack of phosphorylation by Pho85-Pho80, caused an increase in triacylglycerol content and lipid droplet number in cells expressing the Nem1-Spo7 Phosphatase complex.
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Phosphorylation Regulates the Ubiquitin-independent Degradation of Yeast Pah1 Phosphatidate Phosphatase by the 20S Proteasome
The Journal of biological chemistry, 2015Co-Authors: Lu-sheng Hsieh, Gil-soo Han, George M. CarmanAbstract:Abstract Saccharomyces cerevisiae Pah1 Phosphatidate Phosphatase, which catalyzes the conversion of Phosphatidate to diacylglycerol for triacylglycerol synthesis and simultaneously controls Phosphatidate levels for phospholipid synthesis, is subject to the proteasome-mediated degradation in the stationary phase of growth. In this study, we examined the mechanism for its degradation using purified Pah1 and isolated proteasomes. Pah1 expressed in S. cerevisiae or Escherichia coli was not degraded by the 26S proteasome, but by its catalytic 20S core particle, indicating that its degradation is ubiquitin-independent. The degradation of Pah1 by the 20S proteasome was dependent on time and proteasome concentration at the pH optimum of 7.0. The 20S proteasomal degradation was conserved for human lipin 1 Phosphatidate Phosphatase. The degradation analysis using Pah1 truncations and its fusion with GFP indicated that proteolysis initiates at the N- and C-terminal unfolded regions. The folded region of Pah1, in particular the haloacid dehalogenase-like domain containing the DIDGT catalytic sequence, was resistant to the proteasomal degradation. The structural change of Pah1, as reflected by electrophoretic mobility shift, occurs through its phosphorylation by Pho85-Pho80, and the phosphorylation sites are located within its N- and C-terminal unfolded regions. Phosphorylation of Pah1 by Pho85-Pho80 inhibited its degradation, extending its half-life by ∼2-fold. The dephosphorylation of endogenously phosphorylated Pah1 by the Nem1-Spo7 protein Phosphatase, which is highly specific for the sites phosphorylated by Pho85-Pho80, stimulated the 20S proteasomal degradation and reduced its half-life by 2.6-fold. These results indicate that the proteolysis of Pah1 by the 20S proteasome is controlled by its phosphorylation state.
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Yeast Pah1p Phosphatidate Phosphatase is regulated by proteasome-mediated degradation.
The Journal of biological chemistry, 2014Co-Authors: Florencia Pascual, Lu-sheng Hsieh, An Iacutebal Soto-cardalda, George M. CarmanAbstract:Yeast PAH1-encoded Phosphatidate Phosphatase is the enzyme responsible for the production of the diacylglycerol used for the synthesis of triacylglycerol that accumulates in the stationary phase of growth. Paradoxically, the growth phase-mediated inductions of PAH1 and Phosphatidate Phosphatase activity do not correlate with the amount of Pah1p; enzyme abundance declined in a growth phase-dependent manner. Pah1p from exponential phase cells was a relatively stable protein, and its abundance was not affected by incubation with an extract from stationary phase cells. Recombinant Pah1p was degraded upon incubation with the 100,000 × g pellet fraction of stationary phase cells, although the enzyme was stable when incubated with the same fraction of exponential phase cells. MG132, an inhibitor of proteasome function, prevented degradation of the recombinant enzyme. Endogenously expressed and plasmid-mediated overexpressed levels of Pah1p were more abundant in the stationary phase of cells treated with MG132. Pah1p was stabilized in mutants with impaired proteasome (rpn4Δ, blm10Δ, ump1Δ, and pre1 pre2) and ubiquitination (hrd1Δ, ubc4Δ, ubc7Δ, ubc8Δ, and doa4Δ) functions. The pre1 pre2 mutations that eliminate nearly all chymotrypsin-like activity of the 20 S proteasome had the greatest stabilizing effect on enzyme levels. Taken together, these results supported the conclusion that Pah1p is subject to proteasome-mediated degradation in the stationary phase. That Pah1p abundance was stabilized in pah1Δ mutant cells expressing catalytically inactive forms of Pah1p and dgk1Δ mutant cells with induced expression of DGK1-encoded diacylglycerol kinase indicated that alteration in Phosphatidate and/or diacylglycerol levels might be the signal that triggers Pah1p degradation.