A Phosphatidic Acid Binding/Nuclear Localization Motif Determines Lipin1 Function in Lipid Metabolism and Adipogenesis

Abstract

Lipins are phosphatidic acid phosphatases with a pivotal role in regulation of triglyceride and glycerophospholipid metabolism. Lipin1 is also an amplifier of PGC-1␣, a nuclear coactivator of PPAR-␣ responsive gene transcription. Lipins do not contain recognized membrane-association domains, but interaction of these enzymes with cellular membranes is necessary for access to their phospholipid substrate. We identified a role for a conserved polybasic amino acid motif in an N-terminal domain previously implicated as a determinant of nuclear localization in selective binding of lipin1␤ to phosphatidic acid, using blot overlay assays and model bilayer membranes. Studies using lipin1␤ polybasic motif variants establish that this region is also critical for nuclear import and raise the possibility that nuclear/cytoplasmic shuttling of lipin1␤ is regulated by PA. We used pharmacological agents and lipin1␤ polybasic motif mutants to explore the role of PA-mediated membrane association and nuclear localization on lipin1␤ function in phospholipid metabolism and adipogenic differentiation. We identify a role for the lipin1 polybasic motif as both a lipid binding motif and a primary nuclear localization sequence. These two functions are necessary for full expression of the biological activity of the protein in intracellular lipid metabolism and transcriptional control of adipogenesis.

Rhabdomyolysis-Associated Mutations in Human LPIN1 Lead to Loss of Phosphatidic Acid Phosphohydrolase Activity

“…Twentyfour hours later, cells were fixed and incubated with DAPI, anti-V5, and anti-calnexin (cytoplasmic marker). Cells were stained with appropriate secondary antibodies and visualized by fluorescence for subcellular protein distribution analysis as described previously (Ren et al 2010). …”

The Catalytic Efficiency of Lipin 1β Increases by Physically Interacting with the Proto-oncoprotein c-Fos

“…The lipin 1 PBD is also its primary nuclear localization signal. It has been proposed that the dual role for PBM is regulated through PA levels; elevated PA content on cytoplasmic membranes antagonizes nuclear localization of lipin 1 (10). There is evidence that the lipins in the nucleus play a role in directly regulating transcription of genes involved in fatty acid oxidation (11).…”

Conserved Residues in the N Terminus of Lipin-1 Are Required for Binding to Protein Phosphatase-1c, Nuclear Translocation, and Phosphatidate Phosphatase Activity

“…Our group demonstrated the ability of Mg 2ϩ -dependent PAP activity (now attributed to the lipins) to translocate from the cytosol onto the membranes of the endoplasmic reticulum when stimulated with unsaturated fatty acids due to the increase in negative charge on the membrane surface (15)(16)(17)37). This was essentially corroborated after the lipins were discovered to be responsible for PAP activity (4,5,9). Since 2007, several groups have shown that the association of lipins with the endoplasmic reticulum and nucleus is decreased by the introduction of negative charges caused by hyperphosphorylation (5-7, 9, 11, 14).…”

“…10). Previous work demonstrated that PAP catalytic activity does not dictate nuclear lipin-1 localization (9,11). Therefore, exclusion of the lipin-1 HARA mutant from the nucleus cannot be explained by the loss of catalytic PAP activity.…”

“…Lipins are predominantly cytosolic proteins that translocate to their sites of action in the endoplasmic reticulum and nucleus (5)(6)(7)(8)(9)(10)(11)(12)(13). These changes are dictated by a polybasic nuclear localization motif (6,9,14), which also promotes an electrostatic interaction with negatively charged phosphatidate, fatty acids, and acyl-CoA esters on the membrane surface (9, 14 -17). Conversely, positively charged amphiphilic compounds, such as chlorpromazine and sphingosine, reverse this translocation (16,18).…”