Uniform TitleThe PKA-mediated phosphorylation of serine 492 of perilipin A promotes lipolysis by triggering lipid droplet dispersion
NameLiang, Xiaofang (author), Brasaemle, Dawn (chair), Carman, George (internal member), Cohick, Wendie (internal member), Rutgers University, Graduate School - New Brunswick,
DescriptionThe lipid droplet protein perilipin A plays a key role in regulating triglyceride storage and hydrolysis. The phosphorylation of perilipin A by PKA facilitates lipolysis mediated by hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) in adipocytes. Previous studies show that the phosphorylation of serine 492 of perilipin A drives lipid droplet fragmentation and dispersion following PKA-activation. These studies address the hypothesis that lipid droplet dispersion triggered by the phosphorylation of serine 492 promotes PKA-stimulated lipolysis. Adenoviral constructs encoding ectopic perilipin A (PeriA) and three mutated forms were generated, including 1) Not5, with all PKA site serines mutated to alanine or glutamic acid except PKA site #5; 2) S492A, with a single mutation of the fifth PKA site serine to alanine; and 3) All5, with all PKA sites serines substituted with alanine or glutamic acid. All forms of perilipin A and β-galactosidase were expressed in NIH 3T3 CarΔ fibroblasts, which express ATGL but lack endogenous perilipin A and HSL. Cells expressing unmodified perilipin A and mutated forms had similar levels of basal lipolysis. Six hours treatment with forskolin/IBMX in PeriA cells increases lipolysis by 2.2-fold, to levels similar to those of control cells expressing β-galactosidase. When compared to lipolysis in PeriA cells, phosphorylation of serine 492 alone (Not5) contributes to 77.5% of maximal lipolysis, whereas phosphorylation of all five other PKA sites (S492A) yields only 66.6% of maximal lipolysis. Stimulated lipolysis in cells expressing perilipin lacking all PKA sites (All5) increased only 16% over basal levels. Immunofluorescence microscopy shows that phosphorylation of serine 492 is necessary and sufficient to drive the full dispersion of lipid droplets following PKA-stimulation. In conclusion, PKA-mediated phosphorylation of serine 492 of perilipin A facilitates lipolysis at least partially through the stimulation of lipid droplet dispersion.
NoteIncludes bibliographical references (p. 53-58).
CollectionGraduate School - New Brunswick Electronic Theses and Dissertations
Organization NameRutgers, The State University of New Jersey
RightsThe author owns the copyright to this work.