Interaction of the adipocyte fatty acid-binding protein with the hormone-sensitive lipase: regulation by fatty acids and phosphorylation.
Adipocyte fatty acid-binding protein (AFABP/aP2) forms a physical complex with the hormone-sensitive lipase (HSL) and AFABP/aP2-null mice exhibit reduced basal and hormone-stimulated lipolysis. To identify the determinants affecting the interaction fluorescence resonance energy transfer (FRET) imaging was used in conjunction with a mutagenesis strategy to evaluate the roles AFABP/aP2 fatty ... acid binding and HSL phosphorylation have in complex formation as well as determine the HSL binding site on AFABP/aP2. The nonfatty acid binding mutant of AFABP/aP2 (R126Q) failed to form a FRET-competent complex with HSL either under basal or forskolin-stimulated conditions, indicating that lipid binding is required for association. Once bound to HSL and on the surface of the lipid droplet, YFP-AFABP/aP2 (but not YFP-HSL) exhibited energy transfer between the fusion protein and BODIPY-C12-labeled triacylglycerol. Serine to alanine mutations at the two PKA phosphorylation sites of HSL (659 and 660), or at the AMPK phosphorylation sites (565), blocked FRET between HSL and AFABP/aP2. Substitution of isoleucine for lysine at position 21 of AFABP/aP2 (K21I), but not 31 (K31I), resulted in a non-HSL-binding protein indicating that residues on helix alphaI of AFABP/aP2 define a component of the HSL binding site. These results indicate that the ligand-bound form of AFABP/aP2.interacts with the activated, phosphorylated HSL and that the association is likely to be regulatory; either delivering FA to inhibit HSL (facilitating feedback inhibition) or affecting multicomponent complex formation on the droplet surface.
Mesh Terms:
Adipocytes, Amino Acid Substitution, Animals, Cell Line, Cloning, Molecular, Fatty Acid-Binding Proteins, Fatty Acids, Fluorescence Resonance Energy Transfer, Humans, Mice, Models, Molecular, Mutagenesis, Site-Directed, Phosphorylation, Recombinant Fusion Proteins, Sterol Esterase
Adipocytes, Amino Acid Substitution, Animals, Cell Line, Cloning, Molecular, Fatty Acid-Binding Proteins, Fatty Acids, Fluorescence Resonance Energy Transfer, Humans, Mice, Models, Molecular, Mutagenesis, Site-Directed, Phosphorylation, Recombinant Fusion Proteins, Sterol Esterase
J. Biol. Chem.
Date: Nov. 02, 2007
PubMed ID: 17785468
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