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Temporal and spatial regulation of the phosphatidate phosphatases lipin 1 and 2.

Grimsey N, Han GS, O'Hara L, Rochford JJ, Carman GM, Siniossoglou S - J. Biol. Chem. (2008)

Bottom Line: Lipins are the founding members of a novel family of Mg(2+)-dependent phosphatidate phosphatases (PAP1 enzymes) that play key roles in fat metabolism and lipid biosynthesis.Lipin 2 levels increase in lipin 1-depleted 3T3-L1 cells without rescuing the adipogenic defects, whereas depletion of lipin 2 does not inhibit adipogenesis.We propose that distinct and non-redundant functions of lipin 1 and 2 regulate lipid production during the cell cycle and adipocyte differentiation.

View Article: PubMed Central - PubMed

Affiliation: Cambridge Institute for Medical Research, University of Cambridge, Hills Road, CB2 0XY Cambridge, United Kingdom.

ABSTRACT
Lipins are the founding members of a novel family of Mg(2+)-dependent phosphatidate phosphatases (PAP1 enzymes) that play key roles in fat metabolism and lipid biosynthesis. Despite their importance, there is still little information on how their activity is regulated. Here we demonstrate that the functions of lipin 1 and 2 are evolutionarily conserved from unicellular eukaryotes to mammals. The two lipins display distinct intracellular localization in HeLa M cells, with a pool of lipin 2 exhibiting a tight membrane association. Small interfering RNA-mediated silencing of lipin 1 leads to a dramatic decrease of the cellular PAP1 activity in HeLa M cells, whereas silencing of lipin 2 leads to an increase of lipin 1 levels and PAP1 activity. Consistent with their distinct functions in HeLa M cells, lipin 1 and 2 exhibit reciprocal patterns of protein expression in differentiating 3T3-L1 adipocytes. Lipin 2 levels increase in lipin 1-depleted 3T3-L1 cells without rescuing the adipogenic defects, whereas depletion of lipin 2 does not inhibit adipogenesis. Finally, we show that the PAP1 activity of both lipins is inhibited by phosphorylation during mitosis, leading to a decrease in the cellular PAP1 activity during cell division. We propose that distinct and non-redundant functions of lipin 1 and 2 regulate lipid production during the cell cycle and adipocyte differentiation.

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The function of lipin 1 and 2 is evolutionarily conserved from yeast to mammals. A, primary structure of Pah1p, lipin 1, and lipin 2. The conserved N-terminal domain and the C-terminal PAP domain are indicated. B, lipin 2 rescues the temperature-sensitive growth defect of the pah1Δ mutant. pah1Δ cells were transformed with a 2 μ (high copy) vector containing the indicated genes, spotted on YEPD plates, and grown at 30 or 37 °C for 36 h. C, lipins can rescue the nup84Δ spo7Δ synthetic lethality. The nup84Δ spo7Δ double deletion strain carrying a URA3-containing vector expressing NUP84 was transformed with the indicated plasmids. Transformants were grown on plates containing 5-fluoroorotic acid for 3 days. No growth indicates synthetic lethality. D, lipin 2 rescues the nuclear structure defects of the pah1Δ cells. The pah1Δ mutant expressing an intranuclear reporter (PUS1-GFP) was transformed with the same plasmids as in B and visualized by confocal microscopy. Arrows point to cells containing irregularly shaped/multilobed nuclei. The percentage of cells containing a single round nucleus is given. Two different transformants per strain were analyzed and for each one the number of cells counted was 200. Bars, 5 μm. E, expression of lipin 1 and lipin 2 in yeast. Protein extracts from pah1Δ cells expressing PAH1-GFP, lipin 1-GFP or lipin 2-GFP fusions and grown at 30 °C to early logarithmic phase were analyzed by Western blot using anti-GFP antibodies.
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fig1: The function of lipin 1 and 2 is evolutionarily conserved from yeast to mammals. A, primary structure of Pah1p, lipin 1, and lipin 2. The conserved N-terminal domain and the C-terminal PAP domain are indicated. B, lipin 2 rescues the temperature-sensitive growth defect of the pah1Δ mutant. pah1Δ cells were transformed with a 2 μ (high copy) vector containing the indicated genes, spotted on YEPD plates, and grown at 30 or 37 °C for 36 h. C, lipins can rescue the nup84Δ spo7Δ synthetic lethality. The nup84Δ spo7Δ double deletion strain carrying a URA3-containing vector expressing NUP84 was transformed with the indicated plasmids. Transformants were grown on plates containing 5-fluoroorotic acid for 3 days. No growth indicates synthetic lethality. D, lipin 2 rescues the nuclear structure defects of the pah1Δ cells. The pah1Δ mutant expressing an intranuclear reporter (PUS1-GFP) was transformed with the same plasmids as in B and visualized by confocal microscopy. Arrows point to cells containing irregularly shaped/multilobed nuclei. The percentage of cells containing a single round nucleus is given. Two different transformants per strain were analyzed and for each one the number of cells counted was 200. Bars, 5 μm. E, expression of lipin 1 and lipin 2 in yeast. Protein extracts from pah1Δ cells expressing PAH1-GFP, lipin 1-GFP or lipin 2-GFP fusions and grown at 30 °C to early logarithmic phase were analyzed by Western blot using anti-GFP antibodies.

Mentions: Mammalian Lipins Are Orthologues of the Yeast Pah1p—To address whether the function of lipins is conserved throughout evolution, we asked whether the mammalian lipin 1 and 2 could function in the distantly related unicellular eukaryote Saccharomyces cerevisiae. The human lipin 1 (corresponding to the short murine α form) and mouse lipin 2 open reading frames were placed under the control of the PAH1 promoter and were expressed in a yeast pah1Δ deletion mutant that lacks the endogenous yeast lipin PAH1 gene. Lipin 2 fully rescued the temperature-sensitive growth defect of the pah1Δ strain (Fig. 1B). The pah1Δ mutant cells contain enlarged and irregularly shaped nuclei often consisting of two interconnected lobes within a single cell and also accumulate nuclear/ER membranes into the cytoplasm. These structural defects are efficiently rescued by the expression of lipin 2 in pah1Δ cells, as judged by the reappearance of round nuclei to the same extent as in pah1Δ cells expressing PAH1 (Fig. 1D). Lipin 1 could rescue the growth defect of pah1Δ at 30 °C but not at 37 °C (Fig. 1B). Consistent with this, expression of lipin 1 in pah1Δ cells resulted in a partial restoration of nuclear morphology (Fig. 1D). To compare the expression levels of the two lipins and Pah1p in the pah1Δ cells, we expressed them as C-terminal GFP fusions. The fusion proteins maintain their function because they are still able to rescue the pah1Δ growth defect as efficiently as the untagged proteins.4 The lipin 2-GFP is expressed in yeast at similar levels as the Pah1p-GFP fusion (Fig. 1E). Although an intact lipin1-GFP can be also detected, most of this fusion protein is broken down to a ∼70 kDa C-terminal product, predicted to contain the haloacid dehalogenase-like domain. No other major degradation products were detected in the lipin 1-GFP sample.4 The breakdown of the lipin 1 fusion could explain why it partially rescues the pah1Δ defects.


Temporal and spatial regulation of the phosphatidate phosphatases lipin 1 and 2.

Grimsey N, Han GS, O'Hara L, Rochford JJ, Carman GM, Siniossoglou S - J. Biol. Chem. (2008)

The function of lipin 1 and 2 is evolutionarily conserved from yeast to mammals. A, primary structure of Pah1p, lipin 1, and lipin 2. The conserved N-terminal domain and the C-terminal PAP domain are indicated. B, lipin 2 rescues the temperature-sensitive growth defect of the pah1Δ mutant. pah1Δ cells were transformed with a 2 μ (high copy) vector containing the indicated genes, spotted on YEPD plates, and grown at 30 or 37 °C for 36 h. C, lipins can rescue the nup84Δ spo7Δ synthetic lethality. The nup84Δ spo7Δ double deletion strain carrying a URA3-containing vector expressing NUP84 was transformed with the indicated plasmids. Transformants were grown on plates containing 5-fluoroorotic acid for 3 days. No growth indicates synthetic lethality. D, lipin 2 rescues the nuclear structure defects of the pah1Δ cells. The pah1Δ mutant expressing an intranuclear reporter (PUS1-GFP) was transformed with the same plasmids as in B and visualized by confocal microscopy. Arrows point to cells containing irregularly shaped/multilobed nuclei. The percentage of cells containing a single round nucleus is given. Two different transformants per strain were analyzed and for each one the number of cells counted was 200. Bars, 5 μm. E, expression of lipin 1 and lipin 2 in yeast. Protein extracts from pah1Δ cells expressing PAH1-GFP, lipin 1-GFP or lipin 2-GFP fusions and grown at 30 °C to early logarithmic phase were analyzed by Western blot using anti-GFP antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig1: The function of lipin 1 and 2 is evolutionarily conserved from yeast to mammals. A, primary structure of Pah1p, lipin 1, and lipin 2. The conserved N-terminal domain and the C-terminal PAP domain are indicated. B, lipin 2 rescues the temperature-sensitive growth defect of the pah1Δ mutant. pah1Δ cells were transformed with a 2 μ (high copy) vector containing the indicated genes, spotted on YEPD plates, and grown at 30 or 37 °C for 36 h. C, lipins can rescue the nup84Δ spo7Δ synthetic lethality. The nup84Δ spo7Δ double deletion strain carrying a URA3-containing vector expressing NUP84 was transformed with the indicated plasmids. Transformants were grown on plates containing 5-fluoroorotic acid for 3 days. No growth indicates synthetic lethality. D, lipin 2 rescues the nuclear structure defects of the pah1Δ cells. The pah1Δ mutant expressing an intranuclear reporter (PUS1-GFP) was transformed with the same plasmids as in B and visualized by confocal microscopy. Arrows point to cells containing irregularly shaped/multilobed nuclei. The percentage of cells containing a single round nucleus is given. Two different transformants per strain were analyzed and for each one the number of cells counted was 200. Bars, 5 μm. E, expression of lipin 1 and lipin 2 in yeast. Protein extracts from pah1Δ cells expressing PAH1-GFP, lipin 1-GFP or lipin 2-GFP fusions and grown at 30 °C to early logarithmic phase were analyzed by Western blot using anti-GFP antibodies.
Mentions: Mammalian Lipins Are Orthologues of the Yeast Pah1p—To address whether the function of lipins is conserved throughout evolution, we asked whether the mammalian lipin 1 and 2 could function in the distantly related unicellular eukaryote Saccharomyces cerevisiae. The human lipin 1 (corresponding to the short murine α form) and mouse lipin 2 open reading frames were placed under the control of the PAH1 promoter and were expressed in a yeast pah1Δ deletion mutant that lacks the endogenous yeast lipin PAH1 gene. Lipin 2 fully rescued the temperature-sensitive growth defect of the pah1Δ strain (Fig. 1B). The pah1Δ mutant cells contain enlarged and irregularly shaped nuclei often consisting of two interconnected lobes within a single cell and also accumulate nuclear/ER membranes into the cytoplasm. These structural defects are efficiently rescued by the expression of lipin 2 in pah1Δ cells, as judged by the reappearance of round nuclei to the same extent as in pah1Δ cells expressing PAH1 (Fig. 1D). Lipin 1 could rescue the growth defect of pah1Δ at 30 °C but not at 37 °C (Fig. 1B). Consistent with this, expression of lipin 1 in pah1Δ cells resulted in a partial restoration of nuclear morphology (Fig. 1D). To compare the expression levels of the two lipins and Pah1p in the pah1Δ cells, we expressed them as C-terminal GFP fusions. The fusion proteins maintain their function because they are still able to rescue the pah1Δ growth defect as efficiently as the untagged proteins.4 The lipin 2-GFP is expressed in yeast at similar levels as the Pah1p-GFP fusion (Fig. 1E). Although an intact lipin1-GFP can be also detected, most of this fusion protein is broken down to a ∼70 kDa C-terminal product, predicted to contain the haloacid dehalogenase-like domain. No other major degradation products were detected in the lipin 1-GFP sample.4 The breakdown of the lipin 1 fusion could explain why it partially rescues the pah1Δ defects.

Bottom Line: Lipins are the founding members of a novel family of Mg(2+)-dependent phosphatidate phosphatases (PAP1 enzymes) that play key roles in fat metabolism and lipid biosynthesis.Lipin 2 levels increase in lipin 1-depleted 3T3-L1 cells without rescuing the adipogenic defects, whereas depletion of lipin 2 does not inhibit adipogenesis.We propose that distinct and non-redundant functions of lipin 1 and 2 regulate lipid production during the cell cycle and adipocyte differentiation.

View Article: PubMed Central - PubMed

Affiliation: Cambridge Institute for Medical Research, University of Cambridge, Hills Road, CB2 0XY Cambridge, United Kingdom.

ABSTRACT
Lipins are the founding members of a novel family of Mg(2+)-dependent phosphatidate phosphatases (PAP1 enzymes) that play key roles in fat metabolism and lipid biosynthesis. Despite their importance, there is still little information on how their activity is regulated. Here we demonstrate that the functions of lipin 1 and 2 are evolutionarily conserved from unicellular eukaryotes to mammals. The two lipins display distinct intracellular localization in HeLa M cells, with a pool of lipin 2 exhibiting a tight membrane association. Small interfering RNA-mediated silencing of lipin 1 leads to a dramatic decrease of the cellular PAP1 activity in HeLa M cells, whereas silencing of lipin 2 leads to an increase of lipin 1 levels and PAP1 activity. Consistent with their distinct functions in HeLa M cells, lipin 1 and 2 exhibit reciprocal patterns of protein expression in differentiating 3T3-L1 adipocytes. Lipin 2 levels increase in lipin 1-depleted 3T3-L1 cells without rescuing the adipogenic defects, whereas depletion of lipin 2 does not inhibit adipogenesis. Finally, we show that the PAP1 activity of both lipins is inhibited by phosphorylation during mitosis, leading to a decrease in the cellular PAP1 activity during cell division. We propose that distinct and non-redundant functions of lipin 1 and 2 regulate lipid production during the cell cycle and adipocyte differentiation.

Show MeSH
Related in: MedlinePlus