The Phosphorylation of Serine 492 of Perilipin A Directs Lipid Droplet Fragmentation and Dispersion

Department of Nutritional Sciences, Rutgers, The State University of New Jersey, New Brunswick, New Jersey 08901, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 05/2006; 281(17):11901-9. DOI: 10.1074/jbc.M600171200
Source: PubMed


Perilipin A is a key regulator of triacylglycerol storage and hydrolysis in adipocytes; phosphorylation of perilipin A by protein kinase A facilitates maximal lipolysis. Chronic stimulation of lipolysis in 3T3-L1 adipocytes causes large perinuclear lipid droplets to fragment into myriad dispersed perilipin A-covered microlipid droplets. In cultured fibroblasts stably expressing ectopic perilipin A, clustered lipid droplets disperse throughout the cytoplasm upon incubation of the cells with forskolin and isobutylmethylxanthine (IBMX) to elevate levels of cAMP and activate protein kinase A, mirroring events observed in adipocytes. Furthermore, diethylum-belliferyl phosphate inhibits stimulated lipolysis but not the dispersion of lipid droplets, suggesting that products of lipolysis are not required for this remodeling process. We hypothesized that protein kinase A-mediated phosphorylation of perilipin A triggers the remodeling of lipid droplets. The mutation of serine 492 of perilipin A to alanine prevented the dispersion of clustered lipid droplets in fibroblasts stably expressing the mutated perilipin upon incubation with forskolin and IBMX. In contrast, the substitution of serines 81, 222, 276, or 433 with alanine, either singly or in combinations, did not affect the protein kinase A-mediated remodeling of lipid droplets. Interestingly, substitution of serines 433, 492, and 517 of perilipin A with glutamic acid residues blocked the dispersion of clustered lipid droplets in cells incubated with forskolin and IBMX, indicating that the addition of a negative charge does not mimic a phosphate group. We conclude that protein kinase A-mediated phosphorylation of serine 492 of perilipin A drives the fragmentation and dispersion of lipid droplets.

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    • "Although the extent varies with the cell type, clustering of LDs is a common phenotype when cells are incubated with fatty acids (Boström et al., 2005; Garcia et al., 2003; Kuerschner et al., 2008; Murphy et al., 2010; Spandl et al., 2011; Turró et al., 2006; Wang et al., 2011a). In adipocytes, it is regulated by phosphorylation of PLIN1 (Marcinkiewicz et al., 2006; Orlicky et al., 2013). However, most non-adipocyte cells do not express PLIN1. "
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    ABSTRACT: Lipid droplets are found in all cell types. Normally present at low levels in the brain, they accumulate in tumours and are associated with neurodegenerative diseases. However, little is known about the mechanisms controlling their homeostasis in the brain. We have found that the longest GRAF1 isoform, GRAF1a, is enriched in the brains of neonates. Endogenous GRAF1a is found on lipid droplets in oleic-acid fed primary glial cells. Exclusive localization requires a GRAF1a-specific hydrophobic segment and two membrane-binding regions, a BAR and a PH domain. Overexpression of GRAF1a promotes lipid droplet clustering, inhibits droplet mobility and severely perturbs lipolysis following the chase of fatty acid-overloaded cells. Under these conditions, GRAF1a concentrates at the interface between lipid droplets. Although GRAF1 knockout mice do not show any gross abnormal phenotype, the total lipid droplet volume that accumulates in GRAF1(-/-) primary glia upon incubation with fatty acids is reduced compared to GRAF1(+/+) cells. These results provide additional insights into the mechanisms contributing to lipid droplet growth in non-adipocyte cells, and suggest that proteins with membrane sculpting BAR domains play a role in droplet homeostasis.
    Full-text · Article · Sep 2014 · Journal of Cell Science
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    • "The amount of perilipin A detected in the floating fat-cake fraction was similar between WT and PRIP-DKO mice under fed and fasting conditions (Fig. 2B, D). It was reported that phosphorylation of perilipin A at Ser492 is required for maximal lipolysis and triggers a massive remodeling of lipid droplets that increases the surface area of lipid droplets available to lipases [39], [40]. Therefore, phosphorylation of perilipin A Ser492 was examined using a phosphospecific antibody; the results showed that PRIP-DKO mice exhibited higher phosphorylation of perilipin A Ser492 in adipose tissues under both fed and fasting conditions (Fig. 2B, D), indicating that more phosphorylated (active) perilipin A was present on lipid droplets in PRIP-DKO adipocytes. "
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    ABSTRACT: Phosphorylation of hormone-sensitive lipase (HSL) and perilipin by protein kinase A (PKA) promotes the hydrolysis of lipids in adipocytes. Although activation of lipolysis by PKA has been well studied, inactivation via protein phosphatases is poorly understood. Here, we investigated whether phospholipase C-related catalytically inactive protein (PRIP), a binding partner for protein phosphatase 1 and protein phosphatase 2A (PP2A), is involved in lipolysis by regulating phosphatase activity. PRIP knockout (PRIP-KO) mice displayed reduced body-fat mass as compared with wild-type mice fed with standard chow ad libitum. Most other organs appeared normal, suggesting that mutant mice had aberrant fat metabolism in adipocytes. HSL in PRIP-KO adipose tissue was highly phosphorylated compared to that in wild-type mice. Starvation of wild-type mice or stimulation of adipose tissue explants with the catabolic hormone, adrenaline, translocated both PRIP and PP2A from the cytosol to lipid droplets, but the translocation of PP2A was significantly reduced in PRIP-KO adipocytes. Consistently, the phosphatase activity associated with lipid droplet fraction in PRIP-KO adipocytes was significantly reduced and was independent of adrenaline stimulation. Lipolysis activity, as assessed by measurement of non-esterified fatty acids and glycerol, was higher in PRIP-KO adipocytes. When wild-type adipocytes were treated with a phosphatase inhibitor, they showed a high lipolysis activity at the similar level to PRIP-KO adipocytes. Collectively, these results suggest that PRIP promotes the translocation of phosphatases to lipid droplets to trigger the dephosphorylation of HSL and perilipin A, thus reducing PKA-mediated lipolysis.
    Full-text · Article · Jun 2014 · PLoS ONE
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    • "This process leads to the formation of the single LD in mature adipocytes.68 Conversely, TG hydrolysis induces LD fragmentation and shrinking69,70 with a rapid formation of small and micro LDs (<1 μm) in adipocytes.71–73 Although, both small and micro LD originate during lipolysis, small LDs would be generated from the fission of large LDs, whereas micro LDs would be formed with TG produced by the re-esterification of FAs.72 Micro LDs and small LDs are considered physiologically relevant in the response to energy demands.72,73 "
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    ABSTRACT: The main cells of the adipose tissue of animals, adipocytes, are characterized by the presence of large cytosolic lipid droplets (LDs), which store triglyceride (TG) and cholesterol. However, most cells have LDs and the ability to store lipids. LDs have a well-known central role in storage and provision of fatty acids and cholesterol. However, the complexity of the regulation of lipid metabolism on the surface of the LDs is still a matter of intense study. Beyond this role, a number of recent studies have suggested that LDs have major functions in other cellular processes, such as protein storage and degradation, and infection and immunity. Thus, our perception of LDs, from simple globules of fat to highly dynamic organelles of unexpected complexity, has been radically transformed. Here we compiled some recent evidence supporting the emerging view that LDs act as platforms connecting a number of relevant metabolic and cellular functions.
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