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

Abstract

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, diethylumbelliferyl 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.Adipose tissue contains the largest energy reserve in the body, stored as triacylglycerol in the intracellular lipid droplets of adipocytes. Triacylglycerol stores are hydrolyzed to mobilize energy during fasting and extended exercise. Catecholamines bind to ␤-adrenergic receptors on the plasma membranes of adipocytes to initiate a G-protein-mediated signaling cascade that activates adenylyl cyclase, thus increasing intracellular cAMP levels. Protein kinase A (or cAMP-dependent protein kinase) is then activated and phosphorylates several proteins required for the hydrolysis of triacylglycerol and consequent mobilization of fatty acids. Following phosphorylation by protein kinase A, hormone-sensitive lipase translocates from the cytosol onto lipid droplets (1-5), where it hydrolyzes triacylglycerol and diacylglycerol (6 -8). Perilipin A is another major substrate for protein kinase A (9, 10).Perilipin A localizes to lipid droplets in adipocytes and plays roles in facilitating both the storage and hydrolysis of triacylglycerol. In adipocytes, two isoforms of perilipin are derived from the alternate splicing of mRNA transcribed from a single gene (10, 11); perilipin A is the predominant protein isoform, whereas perilipin B is a minor isoform. Perilipins A and B share a common sequ...

Perilipin Promotes Hormone-sensitive Lipase-mediated Adipocyte Lipolysis via Phosphorylation-dependent and -independent Mechanisms

“…This interaction may facilitate access of LD-associated HSL to stored neutral lipid, thereby initiating lipolysis. While the present study was in review, Brasaemle and co-workers (43) reported that chronic PKA-dependent phosphorylation of Peri A induced LD remodeling (i.e. fragmentation and dispersion) independently of lipase action and suggested that this remodeling promoted lipolysis.…”

Effect of dermcidin, an antimicrobial peptide, on body fat mobilization in normal mice

“…In the basal state, perilipin acts as a barrier to lipases, thereby maintaining a low rate of basal lipolysis. However, upon phosphorylation by PKA, perilipin facilitates the accessibility of lipases to lipid stores, thereby promoting lipolysis (Brasaemle et al 2000, Martinez-Botas et al 2000, Tansey et al 2001, Souza et al 2002, Zhang et al 2003, Marcinkiewicz et al 2006). In the current study, the mRNA level of HSL was increased, whereas the protein level of perilipin was decreased in Ad-DCD-injected mice.…”

Not Just Fat: The Structure and Function of the Lipid Droplet

“…Another phenomenon that is ascribed to perilipin A is the appearance of microsized LDs after lipolytic stimulation (Marcinkiewicz et al 2006). Disruption of a sphere into many small spheres may be a mechanism to facilitate lipolysis by increasing the surface-to-volume ratio, but microsized LD formation has not been shown to correlate closely to release of lipolytic products (Bickel et al 2009), and seem to be generated in a region distant from the original LD (Yamaguchi et al 2007;Nagayama et al 2010).…”