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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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Mitotic phosphorylation of lipin 1 and 2 regulates their PAP1 activity. A, lipins are phosphorylated on Cdk1 motifs during mitosis. Left panel, Endogenous lipin 1 and lipin 2 were immunoprecipitated (IP) from extracts of asynchronous (Asy) or mitotic HeLa M cells synchronized either by nocodazole arrest (Noc) or double thymidine arrest at the G1/S boundary followed by release and collection during mitosis (8–10 h post-release) (D. Thy). Immune pellets were analyzed by Western blotting using anti-lipin 1, anti-lipin 2, and anti-phospho-Ser/Thr-Pro (MPM2) antibodies. Right panel, flow cytometry of the cells used for the lipin immunoprecipitations. B, mitotic phosphorylation of lipins inhibits their PAP1 activity. Upper panel, immunoprecipitated lipin 1 and 2 from asynchronous or nocodazole-treated HeLa M cells with or without incubation with λ-phosphatase (λPPase) were analyzed by Western blotting as indicated. Mock immunoprecipitations were performed using the preimmune lipin 1 and 2 sera. Lower panel, immune pellets from the above immunoprecipitations were assayed for PAP1 activity as described under “Experimental Procedures.” The results shown were determined from triplicate enzyme assays ± S.D. The data for the asynchronous and nocodazole samples are the averages from two independent experiments. C, total PAP1 activity decreases during mitosis. Lysates from asynchronous or mitotic cells were assayed for PAP1 and PAP2 activity as described under “Experimental Procedures.” The results shown were determined from triplicate enzyme assays ± S.D. D, mitotic phosphorylation of lipins regulates their membrane recruitment. Lysates from asynchronous or mitotic cells were centrifuged at 100,000 × g. Equal volumes from the pellet (P) and supernatant (S) were immunoblotted using the indicated antibodies.
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fig7: Mitotic phosphorylation of lipin 1 and 2 regulates their PAP1 activity. A, lipins are phosphorylated on Cdk1 motifs during mitosis. Left panel, Endogenous lipin 1 and lipin 2 were immunoprecipitated (IP) from extracts of asynchronous (Asy) or mitotic HeLa M cells synchronized either by nocodazole arrest (Noc) or double thymidine arrest at the G1/S boundary followed by release and collection during mitosis (8–10 h post-release) (D. Thy). Immune pellets were analyzed by Western blotting using anti-lipin 1, anti-lipin 2, and anti-phospho-Ser/Thr-Pro (MPM2) antibodies. Right panel, flow cytometry of the cells used for the lipin immunoprecipitations. B, mitotic phosphorylation of lipins inhibits their PAP1 activity. Upper panel, immunoprecipitated lipin 1 and 2 from asynchronous or nocodazole-treated HeLa M cells with or without incubation with λ-phosphatase (λPPase) were analyzed by Western blotting as indicated. Mock immunoprecipitations were performed using the preimmune lipin 1 and 2 sera. Lower panel, immune pellets from the above immunoprecipitations were assayed for PAP1 activity as described under “Experimental Procedures.” The results shown were determined from triplicate enzyme assays ± S.D. The data for the asynchronous and nocodazole samples are the averages from two independent experiments. C, total PAP1 activity decreases during mitosis. Lysates from asynchronous or mitotic cells were assayed for PAP1 and PAP2 activity as described under “Experimental Procedures.” The results shown were determined from triplicate enzyme assays ± S.D. D, mitotic phosphorylation of lipins regulates their membrane recruitment. Lysates from asynchronous or mitotic cells were centrifuged at 100,000 × g. Equal volumes from the pellet (P) and supernatant (S) were immunoblotted using the indicated antibodies.

Mentions: Mitotic Phosphorylation of Lipin 1 and 2 Inhibits Their PA Phosphatase Activity—We next asked whether the function of the two lipins could also be regulated by post-translational modifications. The fact that the yeast lipin Pah1p is phosphorylated during mitosis (10) and that the mammalian lipins can functionally replace the yeast enzyme (Fig. 1) prompted us to test whether lipin 1 and 2 are also mitotically phosphorylated. To address this possibility, endogenous lipin 1 and 2 were immunoprecipitated from asynchronous or mitotic HeLa M cells, generated by nocodazole arrest, and analyzed by Western blotting. As seen in Fig. 7A, both lipins undergo mobility shifts that reduce their electrophoretic mobility in the nocodazole-arrested samples. This mobility shift is due to phosphorylation because it can be reversed in a concentration-dependent manner after incubation of the immunoprecipitated proteins with λ-phosphatase (Fig. 7B and data not shown). Importantly, the hyperphosphorylated lipin 1 and 2 forms are recognized by MPM2, a monoclonal antibody that is specific for phospho-Ser/Thr-Pro sites, the minimal Cdk1 phosphorylation motif (20) (Fig. 7A).


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)

Mitotic phosphorylation of lipin 1 and 2 regulates their PAP1 activity. A, lipins are phosphorylated on Cdk1 motifs during mitosis. Left panel, Endogenous lipin 1 and lipin 2 were immunoprecipitated (IP) from extracts of asynchronous (Asy) or mitotic HeLa M cells synchronized either by nocodazole arrest (Noc) or double thymidine arrest at the G1/S boundary followed by release and collection during mitosis (8–10 h post-release) (D. Thy). Immune pellets were analyzed by Western blotting using anti-lipin 1, anti-lipin 2, and anti-phospho-Ser/Thr-Pro (MPM2) antibodies. Right panel, flow cytometry of the cells used for the lipin immunoprecipitations. B, mitotic phosphorylation of lipins inhibits their PAP1 activity. Upper panel, immunoprecipitated lipin 1 and 2 from asynchronous or nocodazole-treated HeLa M cells with or without incubation with λ-phosphatase (λPPase) were analyzed by Western blotting as indicated. Mock immunoprecipitations were performed using the preimmune lipin 1 and 2 sera. Lower panel, immune pellets from the above immunoprecipitations were assayed for PAP1 activity as described under “Experimental Procedures.” The results shown were determined from triplicate enzyme assays ± S.D. The data for the asynchronous and nocodazole samples are the averages from two independent experiments. C, total PAP1 activity decreases during mitosis. Lysates from asynchronous or mitotic cells were assayed for PAP1 and PAP2 activity as described under “Experimental Procedures.” The results shown were determined from triplicate enzyme assays ± S.D. D, mitotic phosphorylation of lipins regulates their membrane recruitment. Lysates from asynchronous or mitotic cells were centrifuged at 100,000 × g. Equal volumes from the pellet (P) and supernatant (S) were immunoblotted using the indicated antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig7: Mitotic phosphorylation of lipin 1 and 2 regulates their PAP1 activity. A, lipins are phosphorylated on Cdk1 motifs during mitosis. Left panel, Endogenous lipin 1 and lipin 2 were immunoprecipitated (IP) from extracts of asynchronous (Asy) or mitotic HeLa M cells synchronized either by nocodazole arrest (Noc) or double thymidine arrest at the G1/S boundary followed by release and collection during mitosis (8–10 h post-release) (D. Thy). Immune pellets were analyzed by Western blotting using anti-lipin 1, anti-lipin 2, and anti-phospho-Ser/Thr-Pro (MPM2) antibodies. Right panel, flow cytometry of the cells used for the lipin immunoprecipitations. B, mitotic phosphorylation of lipins inhibits their PAP1 activity. Upper panel, immunoprecipitated lipin 1 and 2 from asynchronous or nocodazole-treated HeLa M cells with or without incubation with λ-phosphatase (λPPase) were analyzed by Western blotting as indicated. Mock immunoprecipitations were performed using the preimmune lipin 1 and 2 sera. Lower panel, immune pellets from the above immunoprecipitations were assayed for PAP1 activity as described under “Experimental Procedures.” The results shown were determined from triplicate enzyme assays ± S.D. The data for the asynchronous and nocodazole samples are the averages from two independent experiments. C, total PAP1 activity decreases during mitosis. Lysates from asynchronous or mitotic cells were assayed for PAP1 and PAP2 activity as described under “Experimental Procedures.” The results shown were determined from triplicate enzyme assays ± S.D. D, mitotic phosphorylation of lipins regulates their membrane recruitment. Lysates from asynchronous or mitotic cells were centrifuged at 100,000 × g. Equal volumes from the pellet (P) and supernatant (S) were immunoblotted using the indicated antibodies.
Mentions: Mitotic Phosphorylation of Lipin 1 and 2 Inhibits Their PA Phosphatase Activity—We next asked whether the function of the two lipins could also be regulated by post-translational modifications. The fact that the yeast lipin Pah1p is phosphorylated during mitosis (10) and that the mammalian lipins can functionally replace the yeast enzyme (Fig. 1) prompted us to test whether lipin 1 and 2 are also mitotically phosphorylated. To address this possibility, endogenous lipin 1 and 2 were immunoprecipitated from asynchronous or mitotic HeLa M cells, generated by nocodazole arrest, and analyzed by Western blotting. As seen in Fig. 7A, both lipins undergo mobility shifts that reduce their electrophoretic mobility in the nocodazole-arrested samples. This mobility shift is due to phosphorylation because it can be reversed in a concentration-dependent manner after incubation of the immunoprecipitated proteins with λ-phosphatase (Fig. 7B and data not shown). Importantly, the hyperphosphorylated lipin 1 and 2 forms are recognized by MPM2, a monoclonal antibody that is specific for phospho-Ser/Thr-Pro sites, the minimal Cdk1 phosphorylation motif (20) (Fig. 7A).

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