The phosphatidic acid–binding, polybasic domain is responsible for the differences in the phosphoregulation of lipins 1 and 3

Abstract

Lipins 1, 2, and 3 are Mg-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 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.

Crystal structure of a lipin/Pah phosphatidic acid phosphatase

“…Membrane association. In vitro mammalian lipins and Sc Pah1 interact strongly with liposomes containing the anionic lipid substrate, PA 24,[35][36][37] . Tt Pah2 also preferentially interacted with PA-containing liposomes, in comparison with neutral or anionic PS-enriched liposomes (Fig.…”

Mechanism of sphingosine 1-phosphate clearance from blood

“…Human SphK1, SphK2, and catalytically inactive SphK2(D211A) (numbering refers to isoform c, NCBI accession # NP_001191089) cDNAs containing a carboxyl-terminal V5 epitope tag were sub-cloned into the pAdTRACK-CMV shuttle vector and adenoviruses (subclass C, serotype 5) were generated using the pAdEASY system as described previously [26,27]. A lack of kinase activity of the mutant SphK2(D211A) construct was verified using our yeast-based sphingolipid kinase assay [28].…”

Polybasic RKKR motif in the linker region of lipid droplet (LD)–associated protein CIDEC inhibits LD fusion activity by interacting with acidic phospholipids

“…Well-known examples include some small GTPases (Ras, Rab, Arf, and Rho proteins) with polybasic clusters being targeted to the plasma membrane through interaction with phospholipids such as phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 4,5-bisphosphate (52). The polybasic domain was also reported to regulate the phosphorylation of Lipin1 (53). Separately, a polybasic KKR motif in NiV-F protein modulates cell-cell fusion (54).…”