title: Differential phosphorylation of perilipin 1A at the initiation of lipolysis revealed by novel monoclonal antibodies and high content analysis.
abstract: Lipolysis in adipocytes is regulated by phosphorylation of lipid droplet-associated proteins, including perilipin 1A and hormone-sensitive lipase (HSL). Perilipin 1A is potentially phosphorylated by cAMP(adenosine 3',5'-cyclic monophosphate)-dependent protein kinase (PKA) on several sites, including conserved C-terminal residues, serine 497 (PKA-site 5) and serine 522 (PKA-site 6). To characterize perilipin 1A phosphorylation, novel monoclonal antibodies were developed, which selectively recognize perilipin 1A phosphorylation at PKA-site 5 and PKA-site 6. Utilizing these novel antibodies, as well as antibodies selectively recognizing HSL phosphorylation at serine 563 or serine 660, we used high content analysis to examine the phosphorylation of perilipin 1A and HSL in adipocytes exposed to lipolytic agents. We found that perilipin PKA-site 5 and HSL-serine 660 were phosphorylated to a similar extent in response to forskolin (FSK) and L-γ-melanocyte stimulating hormone (L-γ-MSH). In contrast, perilipin PKA-site 6 and HSL-serine 563 were phosphorylated more slowly and L-γ-MSH was a stronger agonist for these sites compared to FSK. When a panel of lipolytic agents was tested, including multiple concentrations of isoproterenol, FSK, and L-γ-MSH, the pattern of results was virtually identical for perilipin PKA-site 5 and HSL-serine 660, whereas a distinct pattern was observed for perilipin PKA-site 6 and HSL-serine 563. Notably, perilipin PKA-site 5 and HSL-serine 660 feature two arginine residues upstream from the phospho-acceptor site, which confers high affinity for PKA, whereas perilipin PKA-site 6 and HSL-serine 563 feature only a single arginine. Thus, we suggest perilipin 1A and HSL are differentially phosphorylated in a similar manner at the initiation of lipolysis and arginine residues near the target serines may influence this process.
authors: Vala Sciences Inc, San Diego, California, United States of America. pmcdonough@valasciences.com
authors: Patrick M
authors: PM
authors: McDonough
authors: Dominique
authors: D
authors: Maciejewski-Lenoir
authors: Sean M
authors: SM
authors: Hartig
authors: Rita A
authors: RA
authors: Hanna
authors: Ross
authors: R
authors: Whittaker
authors: Andrew
authors: A
authors: Heisel
authors: James B
authors: JB
authors: Nicoll
authors: Benjamin M
authors: BM
authors: Buehrer
authors: Kurt
authors: K
authors: Christensen
authors: Maureen G
authors: MG
authors: Mancini
authors: Michael A
authors: MA
authors: Mancini
authors: Dean P
authors: DP
authors: Edwards
authors: Jeffrey H
authors: JH
authors: Price
mesh_terms: D041721:3T3-L1 Cells; D017667:Adipocytes / Q000166:cytology / Q000378:metabolism*; D000328:Adult; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D000918:Antibody Specificity; D001120:Arginine / Q000737:chemistry / Q000378:metabolism*; D015153:Blotting, Western; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D050356:Lipid Metabolism / Q000187:drug effects / Q000502:physiology*; D008066:Lipolysis / Q000502:physiology*; D051379:Mice; D000070778:Perilipin-1; D010750:Phosphoproteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D034741:RNA, Small Interfering / Q000235:genetics; D012694:Serine / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D000070778:Perilipin-1; D010750:Phosphoproteins; D034741:RNA, Small Interfering; D012694:Serine; D001120:Arginine; D017868:Cyclic AMP-Dependent Protein Kinases