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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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Subcellular localization of lipin 1 and 2. A, localization of lipin 1-HA and lipin 2-HA fusions in formaldehyde fixed HeLa M cells. Expression of lipin-HA fusions was driven by the cytomegalovirus promoter. Bars, 5 μm. B, localization of lipin 1-GFP and lipin 2-GFP fusions in live HeLa M cells. Live HeLa M cells transiently expressing a dsRed-ER reporter and lipin 1-GFP (upper panel) or lipin 2-GFP (lower panel) were visualized by confocal microscopy. Expression of the tagged lipins was driven by the cytomegalovirus promoter. Bars, 5 μm. C, fractionation of endogenous lipin 1 and 2 in HeLa M cells. The cells were lysed in either buffer alone or buffer containing 1 m NaCl, 1% Triton X-100, or both. The extracts were incubated for 20 min at 4 °C and then centrifuged at 100,000 × g for 1 h. Equal volumes from the pellet (P) and supernatant (S) were loaded on an 8% SDS-PAGE and immunoblotted using the indicated antibodies.
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fig2: Subcellular localization of lipin 1 and 2. A, localization of lipin 1-HA and lipin 2-HA fusions in formaldehyde fixed HeLa M cells. Expression of lipin-HA fusions was driven by the cytomegalovirus promoter. Bars, 5 μm. B, localization of lipin 1-GFP and lipin 2-GFP fusions in live HeLa M cells. Live HeLa M cells transiently expressing a dsRed-ER reporter and lipin 1-GFP (upper panel) or lipin 2-GFP (lower panel) were visualized by confocal microscopy. Expression of the tagged lipins was driven by the cytomegalovirus promoter. Bars, 5 μm. C, fractionation of endogenous lipin 1 and 2 in HeLa M cells. The cells were lysed in either buffer alone or buffer containing 1 m NaCl, 1% Triton X-100, or both. The extracts were incubated for 20 min at 4 °C and then centrifuged at 100,000 × g for 1 h. Equal volumes from the pellet (P) and supernatant (S) were loaded on an 8% SDS-PAGE and immunoblotted using the indicated antibodies.

Mentions: Distinct Intracellular Localizations for Lipin 1 and 2—In contrast to the PA phosphatases of the lipid phosphate phosphatase (LPP) family and other enzymes involved in phospholipid biosynthesis, lipins do not contain predicted transmembrane domains, suggesting that their interaction with membrane surfaces is more transient. Antibodies against lipin 1 and 2 failed to visualize the endogenous proteins in HeLa M cells using a variety of different fixation procedures.4 Therefore, to address their localization, we transfected HeLa M cells with lipin 1 and 2 as either HA or GFP-tagged fusions. In fixed cells, lipin 1-HA shows a predominantly cytosolic staining, whereas lipin 2-HA shows a mixed soluble and reticular staining and a nuclear envelope-associated pool that is reminiscent of ER proteins (Fig. 2A). Lipin 2-HA did not show any significant co-localization with a mitochondrial or a Golgi marker (supplemental Fig. S1). Consistent with its reticular distribution, in live cells a proportion of lipin 2-GFP fusion shows co-localization with an endoplasmic reticulum marker, whereas lipin 1-GFP shows again a diffuse cytosolic distribution (Fig. 2B).


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)

Subcellular localization of lipin 1 and 2. A, localization of lipin 1-HA and lipin 2-HA fusions in formaldehyde fixed HeLa M cells. Expression of lipin-HA fusions was driven by the cytomegalovirus promoter. Bars, 5 μm. B, localization of lipin 1-GFP and lipin 2-GFP fusions in live HeLa M cells. Live HeLa M cells transiently expressing a dsRed-ER reporter and lipin 1-GFP (upper panel) or lipin 2-GFP (lower panel) were visualized by confocal microscopy. Expression of the tagged lipins was driven by the cytomegalovirus promoter. Bars, 5 μm. C, fractionation of endogenous lipin 1 and 2 in HeLa M cells. The cells were lysed in either buffer alone or buffer containing 1 m NaCl, 1% Triton X-100, or both. The extracts were incubated for 20 min at 4 °C and then centrifuged at 100,000 × g for 1 h. Equal volumes from the pellet (P) and supernatant (S) were loaded on an 8% SDS-PAGE and immunoblotted using the indicated antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2570901&req=5

fig2: Subcellular localization of lipin 1 and 2. A, localization of lipin 1-HA and lipin 2-HA fusions in formaldehyde fixed HeLa M cells. Expression of lipin-HA fusions was driven by the cytomegalovirus promoter. Bars, 5 μm. B, localization of lipin 1-GFP and lipin 2-GFP fusions in live HeLa M cells. Live HeLa M cells transiently expressing a dsRed-ER reporter and lipin 1-GFP (upper panel) or lipin 2-GFP (lower panel) were visualized by confocal microscopy. Expression of the tagged lipins was driven by the cytomegalovirus promoter. Bars, 5 μm. C, fractionation of endogenous lipin 1 and 2 in HeLa M cells. The cells were lysed in either buffer alone or buffer containing 1 m NaCl, 1% Triton X-100, or both. The extracts were incubated for 20 min at 4 °C and then centrifuged at 100,000 × g for 1 h. Equal volumes from the pellet (P) and supernatant (S) were loaded on an 8% SDS-PAGE and immunoblotted using the indicated antibodies.
Mentions: Distinct Intracellular Localizations for Lipin 1 and 2—In contrast to the PA phosphatases of the lipid phosphate phosphatase (LPP) family and other enzymes involved in phospholipid biosynthesis, lipins do not contain predicted transmembrane domains, suggesting that their interaction with membrane surfaces is more transient. Antibodies against lipin 1 and 2 failed to visualize the endogenous proteins in HeLa M cells using a variety of different fixation procedures.4 Therefore, to address their localization, we transfected HeLa M cells with lipin 1 and 2 as either HA or GFP-tagged fusions. In fixed cells, lipin 1-HA shows a predominantly cytosolic staining, whereas lipin 2-HA shows a mixed soluble and reticular staining and a nuclear envelope-associated pool that is reminiscent of ER proteins (Fig. 2A). Lipin 2-HA did not show any significant co-localization with a mitochondrial or a Golgi marker (supplemental Fig. S1). Consistent with its reticular distribution, in live cells a proportion of lipin 2-GFP fusion shows co-localization with an endoplasmic reticulum marker, whereas lipin 1-GFP shows again a diffuse cytosolic distribution (Fig. 2B).

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