Abstract
Lipins 1, 2, and 3 are Mg2+-dependent phosphatidic acid phosphatases and catalyze the penultimate step of triacylglycerol synthesis. We have previously investigated the biochemistry of lipins 1 and 2 and shown that di-anionic phosphatidic acid (PA) augments their activity and lipid binding and that lipin 1 activity is negatively regulated by phosphorylation. In the present study, we show that phosphorylation does not affect the catalytic activity of lipin 3 or its ability to associate with PA in vitro The lipin proteins each contain a conserved polybasic domain (PBD) composed of nine lysine and arginine residues located between the conserved N- and C-terminal domains. In lipin 1, the PBD is the site of PA binding and sensing of the PA electrostatic charge. The specific arrangement and number of the lysines and arginines of the PBD vary among the lipins. We show that the different PBDs of lipins 1 and 3 are responsible for the presence of phosphoregulation on the former but not the latter enzyme. To do so, we generated lipin 1 that contained the PBD of lipin 3 and vice versa. The lipin 1 enzyme with the lipin 3 PBD lost its ability to be regulated by phosphorylation but remained downstream of phosphorylation by mammalian target of rapamycin. Conversely, the presence of the lipin 1 PBD in lipin 3 subjected the enzyme to negative intramolecular control by phosphorylation. These results indicate a mechanism for the observed differences in lipin phosphoregulation in vitro.
Highlights
Lipins 1, 2, and 3 are Mg2؉-dependent phosphatidic acid phosphatases and catalyze the penultimate step of triacylglycerol synthesis
The lipin family of Mg2ϩ-dependent phosphatidic acid (PA)3 phosphatases consists of three members, which form diacylglycerol from PA in neutral and phospholipid synthesis [1,2,3,4,5,6]
The polybasic domain (PBD) is conserved among PAP enzymes, but the specific arrangement and number of lysines and arginines vary between the mammalian lipins 1–3
Summary
The phosphatidic acid–binding, polybasic domain is responsible for the differences in the phosphoregulation of lipins 1 and 3. They hydrogen-bond with the PA headgroup and cause a dissociation of the final proton This causes a switch in the charge from mono- to di-anionic and locks the protein onto its lipid-ligand through strengthened electrostatic interactions. This model was termed the hydrogen bond switch mechanism and has been demonstrated experimentally by an induction in lipin activity in the presence of di-anionic PA [14, 15, 21]. Each lipin contains a cluster of nine lysines and arginines known as the polybasic domain (PBD) This region on lipin 1 was identified as the primary PA-binding site and a membrane/ nuclear localization sequence [13, 22, 23]. The PBD is conserved among PAP enzymes, but the specific arrangement and number of lysines and arginines vary between the mammalian lipins 1–3
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