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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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Depletion of lipin 1 and 2 in HeLa M cells. A, siRNA down-regulates lipin 1 and 2 mRNA and protein expression. The cells were transfected with either a nontargeting (Control), or lipin 1, lipin 2, or lipin 1 and 2 small interfering RNA duplexes. 72 h after transfection, mRNA (left panel) or protein (right panel) levels were determined by real time PCR and immunoblotting, respectively, using the indicated antibodies. B, cell cycle profiles of cells from A were determined by flow cytometry as described under “Experimental Procedures.” The significance of the difference between control and lipin 1 siRNA-treated cells found in the G1 phase is indicated by p < 0.00005. The data are averages from eight independent experiments. C, lipin 1 is the major PAP1 enzyme in HeLa M cells. Control and lipin siRNA-treated cells were lysed 72 h after the knock-down, and PAP assays were performed as described under “Experimental Procedures.” The results shown were determined from triplicate enzyme assays ± S.D. and were reproduced in two independent experiments.
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fig3: Depletion of lipin 1 and 2 in HeLa M cells. A, siRNA down-regulates lipin 1 and 2 mRNA and protein expression. The cells were transfected with either a nontargeting (Control), or lipin 1, lipin 2, or lipin 1 and 2 small interfering RNA duplexes. 72 h after transfection, mRNA (left panel) or protein (right panel) levels were determined by real time PCR and immunoblotting, respectively, using the indicated antibodies. B, cell cycle profiles of cells from A were determined by flow cytometry as described under “Experimental Procedures.” The significance of the difference between control and lipin 1 siRNA-treated cells found in the G1 phase is indicated by p < 0.00005. The data are averages from eight independent experiments. C, lipin 1 is the major PAP1 enzyme in HeLa M cells. Control and lipin siRNA-treated cells were lysed 72 h after the knock-down, and PAP assays were performed as described under “Experimental Procedures.” The results shown were determined from triplicate enzyme assays ± S.D. and were reproduced in two independent experiments.

Mentions: Reciprocal Regulation of Lipin 1 and 2 Controls Cellular PAP1 Activity in HeLa M Cells—To investigate their cellular functions, we depleted lipin 1 and lipin 2 in HeLa M cells using siRNA. To this end we used two different siRNA oligonucleotides for each lipin that could, independently, deplete efficiently the respective proteins 72 h after their transfection (Fig. 3A and data not shown). Notably, depletion of either lipin resulted in an increase of both the mRNA and protein levels of the other lipin (Fig. 3A), suggesting that the knock-down cells activate a compensatory mechanism. Cell cycle analysis shows that depletion of lipin 1 for 72 h causes a 6.3% increase in the relative number of cells in G1 phase, whereas depletion of lipin 2 has no effect (Fig. 3B).


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)

Depletion of lipin 1 and 2 in HeLa M cells. A, siRNA down-regulates lipin 1 and 2 mRNA and protein expression. The cells were transfected with either a nontargeting (Control), or lipin 1, lipin 2, or lipin 1 and 2 small interfering RNA duplexes. 72 h after transfection, mRNA (left panel) or protein (right panel) levels were determined by real time PCR and immunoblotting, respectively, using the indicated antibodies. B, cell cycle profiles of cells from A were determined by flow cytometry as described under “Experimental Procedures.” The significance of the difference between control and lipin 1 siRNA-treated cells found in the G1 phase is indicated by p < 0.00005. The data are averages from eight independent experiments. C, lipin 1 is the major PAP1 enzyme in HeLa M cells. Control and lipin siRNA-treated cells were lysed 72 h after the knock-down, and PAP assays were performed as described under “Experimental Procedures.” The results shown were determined from triplicate enzyme assays ± S.D. and were reproduced in two independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Depletion of lipin 1 and 2 in HeLa M cells. A, siRNA down-regulates lipin 1 and 2 mRNA and protein expression. The cells were transfected with either a nontargeting (Control), or lipin 1, lipin 2, or lipin 1 and 2 small interfering RNA duplexes. 72 h after transfection, mRNA (left panel) or protein (right panel) levels were determined by real time PCR and immunoblotting, respectively, using the indicated antibodies. B, cell cycle profiles of cells from A were determined by flow cytometry as described under “Experimental Procedures.” The significance of the difference between control and lipin 1 siRNA-treated cells found in the G1 phase is indicated by p < 0.00005. The data are averages from eight independent experiments. C, lipin 1 is the major PAP1 enzyme in HeLa M cells. Control and lipin siRNA-treated cells were lysed 72 h after the knock-down, and PAP assays were performed as described under “Experimental Procedures.” The results shown were determined from triplicate enzyme assays ± S.D. and were reproduced in two independent experiments.
Mentions: Reciprocal Regulation of Lipin 1 and 2 Controls Cellular PAP1 Activity in HeLa M Cells—To investigate their cellular functions, we depleted lipin 1 and lipin 2 in HeLa M cells using siRNA. To this end we used two different siRNA oligonucleotides for each lipin that could, independently, deplete efficiently the respective proteins 72 h after their transfection (Fig. 3A and data not shown). Notably, depletion of either lipin resulted in an increase of both the mRNA and protein levels of the other lipin (Fig. 3A), suggesting that the knock-down cells activate a compensatory mechanism. Cell cycle analysis shows that depletion of lipin 1 for 72 h causes a 6.3% increase in the relative number of cells in G1 phase, whereas depletion of lipin 2 has no effect (Fig. 3B).

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