Abstract
Lipins are the founding members of a novel family of Mg2+-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.
Highlights
The phospholipid composition of biological membranes is crucial for many aspects of cell physiology, including growth, differentiation, and transport [1, 2]
We provide the first evidence that lipin 1 and 2 exhibit distinct intracellular localization in HeLa M cells and show that lipin 1 is the major PAP1 enzyme in HeLa M cells. siRNA-mediated depletion of the two lipins shows that their expression is reciprocally regulated both in HeLa M cells and differentiating adipocytes
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
Summary
Plasmids, and Yeast Methods—Unless otherwise stated, all of the reagents were supplied by Sigma-Aldrich. Cell Cycle Synchronization—For the double thymidine block, HeLa M cells were arrested with 2.5 mM thymidine for 19 h and released by washing two times in medium and allowing them to rest for 9 h, followed by a second 16 h arrest with 2.5 mM thymidine. Mitotic cells were collected by shake-off every 30 min, between 8 and 11 h after release from the second cell cycle arrest. The cells were released from the block by washing two times in medium and allowing them to recover for 4 h. This was followed by a second 12-h arrest using 50 ng/ml of nocodazole. The remaining primers were used for the construction of the shRNAs targeting lipin 1 and 2, as described under “Experimental Procedures.”
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