Mutational analysis of the hormone-sensitive lipase translocation reaction in adipocytes.
Lipolysis in adipocytes governs the release of fatty acids for the supply of energy to various tissues of the body. This reaction is mediated by hormone-sensitive lipase (HSL), a cytosolic enzyme, and perilipin, which coats the lipid droplet surface in adipocytes. Both HSL and perilipin are substrates for polyphosphorylation by protein kinase A (PKA), and phosphorylation of perilipin is required to induce HSL to translocate from the cytosol to the surface of the lipid droplet, a critical step in the lipolytic reaction (Sztalryd C., Xu, G., Dorward, H., Tansey, J. T., Contreras, J.A, Kimmel, A. R., and Londos, C. (2003) J. Cell Biol. 161, 1093-1103). In the present paper we demonstrate that phosphorylation at one of the two more recently discovered PKA sites within HSL, serines 659 and 660, is also required to effect the translocation reaction. Translocation does not occur when these serines residues are mutated simultaneously to alanines. Also, mutation of the catalytic Ser-423 eliminates HSL translocation, showing that the inactive enzyme does not migrate to the lipid droplet upon PKA activation. Thus, HSL translocation requires the phosphorylation of both HSL and perilipin.
Su
Chun-Li
CL
Laboratory of Cellular and Developmental Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-8028, USA.
Sztalryd
Carole
C
Contreras
Juan Antonio
JA
Holm
Cecilia
C
Kimmel
Alan R
AR
Londos
Constantine
C
D041721:3T3-L1 Cells; D017667:Adipocytes / Q000378:metabolism*; D000818:Animals; D015152:Blotting, Northern; D002352:Carrier Proteins; D002384:Catalysis; D020134:Catalytic Domain; D002454:Cell Differentiation; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D004252:DNA Mutational Analysis; D005990:Glycerol / Q000378:metabolism; D015151:Immunoblotting; D050356:Lipid Metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D009154:Mutation; D000070778:Perilipin-1; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D021381:Protein Transport; D012694:Serine / Q000737:chemistry; D002787:Sterol Esterase / Q000378:metabolism*
D002352:Carrier Proteins; D000070778:Perilipin-1; D010750:Phosphoproteins; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases; D002787:Sterol Esterase; D005990:Glycerol