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.

Adipose tissue development and the molecular regulation of lipid metabolism

“…The PKA phosphorylation sites of both murine and human PLIN1 have previously been described [50]. These key phosphorylation sites maximize the activation of ATGL-dependent lipolysis, and the fragmentation and dispersion of lipid droplets to increase the surface area for lipase binding [75,76] in mice, and are conserved in humans [77], suggesting similar regulation. More recent data suggests another level of lipolytic control, with CGI-58-PLIN1 interactions being regulated by endogenous ligands, including long-chain acyl-CoA in vitro [78].…”

Exercise and the Regulation of Adipose Tissue Metabolism

“…61 Murine PLIN1 has six PKA phosphorylation sites, 62 and Ser-492 and Ser-517 are critical for the regulation of lipolysis. 63 Phosphorylation of Ser-517 maximizes activation of ATGL-dependent lipolysis, 64 and PKA-mediated phosphorylation of Ser-492 drives fragmentation and dispersion of lipid droplets, which increases the surface area for lipase binding. 65 Although these results pertain to murine PLIN1, these sites are conserved in human PLIN1 at Ser-81, Ser-436, Ser-497, and Ser-522, 63 suggesting a similar physiological function in humans (Fig.…”

The effects of dietary macronutrient composition on lipid metabolism-associated factor gene expression in the adipose tissue of chickens are influenced by fasting and refeeding

“…In the activated state, phosphorylation of PLN1 results in the release of CGI-58, which can then activate ATGL [40]. Therefore, further research is needed to evaluate how fasting affects phosphorylated PLN1 levels in the WAT of chickens.…”