Analysis of lipolytic protein trafficking and interactions in adipocytes.
This work examined the colocalization, trafficking, and interactions of key proteins involved in lipolysis during brief cAMP-dependent protein kinase A (PKA) activation. Double label immunofluorescence analysis of 3T3-L1 adipocytes indicated that PKA activation increases the translocation of hormonesensitive lipase (HSL) to perilipin A (Plin)-containing droplets and increases the colocalization of ... adipose tissue triglyceride lipase (Atgl) with its coactivator, Abhd5. Imaging of live 3T3-L1 preadipocytes transfected with Aquorea victoria-based fluorescent reporters demonstrated that HSL rapidly and specifically translocates to lipid droplets (LDs) containing Plin, and that this translocation is partially dependent on Plin phosphorylation. HSL closely, if not directly, interacts with Plin, as indicated by fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) experiments. In contrast, tagged Atgl did not support FRET or BiFC with Plin, although it did modestly translocate to LDs upon stimulation. Abhd5 strongly interacted with Plin in the basal state, as indicated by FRET and BiFC. PKA activation rapidly (within minutes) decreased FRET between Abhd5 and Plin, and this decrease depended upon Plin phosphorylation. Together, these results indicate that Plin mediates hormone-stimulated lipolysis via direct and indirect mechanisms. Plin indirectly controls Atgl activity by regulating accessibility to its coactivator, Abhd5. In contrast, Plin directly regulates the access of HSL to substrate via close, if not direct, interactions. The differential interactions of HSL and Atgl with Plin and Abhd5 also explain the findings that following stimulation, HSL and Atgl are differentially enriched at specific LDs.
Mesh Terms:
1-Acylglycerol-3-Phosphate O-Acyltransferase, 3T3 Cells, Adipocytes, Animals, Carboxylic Ester Hydrolases, Carrier Proteins, Cyclic AMP, Cyclic AMP-Dependent Protein Kinases, Cytoplasmic Structures, Enzyme Activation, Esterases, Humans, Lipase, Mice, Phospholipases A, Phosphoproteins, Protein Transport
1-Acylglycerol-3-Phosphate O-Acyltransferase, 3T3 Cells, Adipocytes, Animals, Carboxylic Ester Hydrolases, Carrier Proteins, Cyclic AMP, Cyclic AMP-Dependent Protein Kinases, Cytoplasmic Structures, Enzyme Activation, Esterases, Humans, Lipase, Mice, Phospholipases A, Phosphoproteins, Protein Transport
J. Biol. Chem.
Date: Feb. 23, 2007
PubMed ID: 17189257
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