PostDoc Fellowship: Hongmei Ren Regulation of Nuclear Cytoplasmic Shuttling of the PA Phosphatase Lipin

Phosphatidic acid (PA) is an intracellular lipid signal and a metabolic intermediate in the synthesis of glycero- and glycerophospho- lipids. Lipins are Mg2+-dependent “Type 1” phosphatidic acid phosphatases. Studies with lipin 1 deficient mice identify this lipin isoform as a key regulator of triglyceride and phospholipid metabolism linked to adiposity, fat storage and lipoprotein synthesis. Mammalian lipin1 is a direct transcriptional co-activator of PPAR alpha-responsive genes suggesting that the protein functions both within the nucleus and at intracellular membrane sites. While the catalytic activity of lipin1 appears dispensable for this transcriptional co-activator function, the precise contributions of the lipid phosphatase and transcriptional activities to the integrated role of lipin1 in metabolic regulation are presently largely undefined. Lipins are soluble proteins without recognized membrane-association domains. Interaction of lipins with cellular membranes is clearly necessary for proper access to their phospholipid substrate. In the past 18 months we investigated the binding of purified lipin1 to different lipids using blot overlay assays and model bilayer membranes and identified a role for a conserved polybasic amino acid motif previously suggested to contain a nuclear localization sequence selective binding of lipin 1 to PA. Studies using these lipin 1 polybasic motif variants clearly establish that this region is also critical for nuclear import and raise the interesting possibility that nuclear/cytoplasmic shuttling of Lipin 1 is regulated by PA. By expressing Lipin1 polybasic motif mutants in fibroblasts from lipin 1 deficient fld mice we identified a critical role for PA-mediated membrane association and nuclear localization on lipin 1 function in phospholipid metabolism and adpiogenic differentiation. This application requests funds for a third year of support. I will complete and extend my studies of nuclear cytoplasmic transport of lipin 1 to facilitate publication of my research. I have also made the surprising observation that a catalytically inactive mutant of lipin 1 is unable to induce expression of adipogenic genes in fld MEFs suggesting that a substrate or product of the enzyme is necessary for these effects. I will therefore obtain additional training in the use of state of the art mass spectrometry approaches to identify and test candidate lipid mediators of lipin 1 function as a transcriptional activator.