Differential Phosphorylation of Perilipin 1A at the Initiation of Lipolysis Revealed by Novel Monoclonal Antibodies and High Content Analysis |
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Authors: | Patrick M McDonough Dominique Maciejewski-Lenoir Sean M Hartig Rita A Hanna Ross Whittaker Andrew Heisel James B Nicoll Benjamin M Buehrer Kurt Christensen Maureen G Mancini Michael A Mancini Dean P Edwards Jeffrey H Price |
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Institution: | 1. Vala Sciences Inc, San Diego, California, United States of America.; 2. Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, United States of America.; 3. Zen-Bio, Inc, Research Triangle Park, North Carolina, United States of America.; 4. Signal Transduction Program, The Sanford-Burnham Institute for Medical Research, La Jolla, California, United States of America.; The University of Queensland, Australia, |
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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. |
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