Differential phosphorylation of perilipin 1A at the initiation of lipolysis revealed by novel monoclonal antibodies and high content analysis.
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,...
| Main Authors: | , , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2013-01-01
|
| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3566132?pdf=render |
| _version_ | 1811231030288318464 |
|---|---|
| author | 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 |
| author_facet | 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 |
| author_sort | Patrick M McDonough |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-12T10:38:07Z |
| format | Article |
| id | doaj.art-64a1d093fe334737ba6d03f6e2eee559 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-04-12T10:38:07Z |
| publishDate | 2013-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-64a1d093fe334737ba6d03f6e2eee5592022-12-22T03:36:40ZengPublic Library of Science (PLoS)PLoS ONE1932-62032013-01-0182e5551110.1371/journal.pone.0055511Differential phosphorylation of perilipin 1A at the initiation of lipolysis revealed by novel monoclonal antibodies and high content analysis.Patrick M McDonoughDominique Maciejewski-LenoirSean M HartigRita A HannaRoss WhittakerAndrew HeiselJames B NicollBenjamin M BuehrerKurt ChristensenMaureen G ManciniMichael A ManciniDean P EdwardsJeffrey H PriceLipolysis 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.http://europepmc.org/articles/PMC3566132?pdf=render |
| spellingShingle | 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 Differential phosphorylation of perilipin 1A at the initiation of lipolysis revealed by novel monoclonal antibodies and high content analysis. PLoS ONE |
| title | Differential phosphorylation of perilipin 1A at the initiation of lipolysis revealed by novel monoclonal antibodies and high content analysis. |
| title_full | Differential phosphorylation of perilipin 1A at the initiation of lipolysis revealed by novel monoclonal antibodies and high content analysis. |
| title_fullStr | Differential phosphorylation of perilipin 1A at the initiation of lipolysis revealed by novel monoclonal antibodies and high content analysis. |
| title_full_unstemmed | Differential phosphorylation of perilipin 1A at the initiation of lipolysis revealed by novel monoclonal antibodies and high content analysis. |
| title_short | Differential phosphorylation of perilipin 1A at the initiation of lipolysis revealed by novel monoclonal antibodies and high content analysis. |
| title_sort | differential phosphorylation of perilipin 1a at the initiation of lipolysis revealed by novel monoclonal antibodies and high content analysis |
| url | http://europepmc.org/articles/PMC3566132?pdf=render |
| work_keys_str_mv | AT patrickmmcdonough differentialphosphorylationofperilipin1aattheinitiationoflipolysisrevealedbynovelmonoclonalantibodiesandhighcontentanalysis AT dominiquemaciejewskilenoir differentialphosphorylationofperilipin1aattheinitiationoflipolysisrevealedbynovelmonoclonalantibodiesandhighcontentanalysis AT seanmhartig differentialphosphorylationofperilipin1aattheinitiationoflipolysisrevealedbynovelmonoclonalantibodiesandhighcontentanalysis AT ritaahanna differentialphosphorylationofperilipin1aattheinitiationoflipolysisrevealedbynovelmonoclonalantibodiesandhighcontentanalysis AT rosswhittaker differentialphosphorylationofperilipin1aattheinitiationoflipolysisrevealedbynovelmonoclonalantibodiesandhighcontentanalysis AT andrewheisel differentialphosphorylationofperilipin1aattheinitiationoflipolysisrevealedbynovelmonoclonalantibodiesandhighcontentanalysis AT jamesbnicoll differentialphosphorylationofperilipin1aattheinitiationoflipolysisrevealedbynovelmonoclonalantibodiesandhighcontentanalysis AT benjaminmbuehrer differentialphosphorylationofperilipin1aattheinitiationoflipolysisrevealedbynovelmonoclonalantibodiesandhighcontentanalysis AT kurtchristensen differentialphosphorylationofperilipin1aattheinitiationoflipolysisrevealedbynovelmonoclonalantibodiesandhighcontentanalysis AT maureengmancini differentialphosphorylationofperilipin1aattheinitiationoflipolysisrevealedbynovelmonoclonalantibodiesandhighcontentanalysis AT michaelamancini differentialphosphorylationofperilipin1aattheinitiationoflipolysisrevealedbynovelmonoclonalantibodiesandhighcontentanalysis AT deanpedwards differentialphosphorylationofperilipin1aattheinitiationoflipolysisrevealedbynovelmonoclonalantibodiesandhighcontentanalysis AT jeffreyhprice differentialphosphorylationofperilipin1aattheinitiationoflipolysisrevealedbynovelmonoclonalantibodiesandhighcontentanalysis |