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.
Smith
Anne J
AJ
Department of Biochemistry, Molecular Biology, and Biophysics, Imaging Center, The University of Minnesota, Minneapolis 55455, USA.
Thompson
Brian R
BR
Sanders
Mark A
MA
Bernlohr
David A
DA
D017667:Adipocytes / Q000378:metabolism*; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D050556:Fatty Acid-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005227:Fatty Acids / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002787:Sterol Esterase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*