Dynamic Palmitoylation Links Cytosol-Membrane Shuttling of Acyl-protein Thioesterase-1 and Acyl-protein Thioesterase-2 with That of Proto-oncogene H-Ras Product and Growth-associated Protein-43

• Published in: The Journal of biological chemistry 2013-03, Vol.288 (13), p.9112-9125
• Main Authors: Kong, Eryan, Peng, Shiyong, Chandra, Goutam, Sarkar, Chinmoy, Zhang, Zhongjian, Bagh, Maria B., Mukherjee, Anil B.
• Format: Article
• Language: English
• Subjects: Acyl-protein Thioesterases; Animals; Astrocytes - cytology; Catalytic Domain; Cell Biology; Cell Membrane - metabolism; Cells, Cultured; Cytosol - metabolism; Cytosol-Membrane Trafficking; Dynamic Palmitoylation; Enzyme Mechanisms; Enzymes; GAP-43; GAP-43 Protein - metabolism; H-Ras; Humans; Membrane Trafficking; Mice; Microscopy, Confocal; Mutagenesis; Mutation; Neurons - metabolism; NIH 3T3 Cells; Oncogene; Palmitic Acid - chemistry; Palmitic Acid - metabolism; Protein Binding; Ras; ras Proteins - metabolism; Subcellular Fractions - metabolism; Thiolester Hydrolases - metabolism; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.421073

Acyl-protein thioesterase-1 (APT1) and APT2 are cytosolic enzymes that catalyze depalmitoylation of membrane-anchored, palmitoylated H-Ras and growth-associated protein-43 (GAP-43), respectively. However, the mechanism(s) of cytosol-membrane shuttling of APT1 and APT2, required for depalmitoylating their substrates H-Ras and GAP-43, respectively, remained largely unknown. Here, we report that both APT1 and APT2 undergo palmitoylation on Cys-2. Moreover, blocking palmitoylation adversely affects membrane localization of both APT1 and APT2 and that of their substrates. We also demonstrate that APT1 not only catalyzes its own depalmitoylation but also that of APT2 promoting dynamic palmitoylation (palmitoylation-depalmitoylation) of both thioesterases. Furthermore, shRNA suppression of APT1 expression or inhibition of its thioesterase activity by palmostatin B markedly increased membrane localization of APT2, and shRNA suppression of APT2 had virtually no effect on membrane localization of APT1. In addition, mutagenesis of the active site Ser residue to Ala (S119A), which renders catalytic inactivation of APT1, also increased its membrane localization. Taken together, our findings provide insight into a novel mechanism by which dynamic palmitoylation links cytosol-membrane trafficking of APT1 and APT2 with that of their substrates, facilitating steady-state membrane localization and function of both. Background: How cytosolic thioesterases (APT1 and APT2) depalmitoylate their membrane-anchored substrates remains unclear. Results: APT1 and APT2 require palmitoylation for membrane localization. Although APT1 depalmitoylates itself, H-Ras, and APT2, APT2 depalmitoylates GAP-43. Conclusion: Dynamic palmitoylation regulates steady-state membrane localization and function of cytosolic thioesterases and their substrates. Significance: The findings may provide new insight into the regulation and function of cytosolic thioesterases and their substrates.