Molecular mechanism of the Lipin1 activation by c-Fos

CARDOZO GIZZI AM; CAPUTTO, BL

Buenos Aires

Congreso; XLVIII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2013

Sociedad Argentina de Investigación en Bioquímica

The oncoprotein c-Fos activates phospholipid synthesis through a mechanism independent of its genomic AP-1 activity. To accomplish an overall activation of this synthesis, key metabolic steps are positively affected, including Phosphatidate Phosphohydrolase (PAP1). PAP1 catalyzes Phosphatidic Acid conversion to diacylglicerol. The mammalian enzymes responsible for PAP1 activity are the Lipin family, which are emerging as critical regulators of lipid metabolism. Lipins may govern the pathways by which phospholipids are synthesized and control the cellular content of signaling lipids. Herein, we studied in vitro the activity of mammalian Lipin 1 (responsible of most PAP1 activity in mammals) purified to homogeneity. Lipin1 activity was measured with or without recombinant c-Fos in PA/Triton X-100 mixed micelles varying the assay conditions in order to clarify the activation mechanism. The kcat of the enzyme is doubled upon c-Fos addition, while the Km remains unaltered. Confocal microscopy revealed a co-localization pattern in the perinuclear region of the cell, so we decided to go further into the characterization. In contrast to Co-localization, FRET (Förster Resonance Energy Transfer) can establish a direct protein-protein interaccion. A robust method of detecting FRET occurrence is FLIM (Fluorescence Lifetime Imaging). We found that Lipin1b and c-Fos directly interact at the perinuclear region. Results support our hypothesis that c-Fos physically associates with the phospholipid synthesis enzymes that it activates and reinforce the concept of a protein capable of increasing pivotal enzymatic activities per se.