title: DHHC5 protein palmitoylates flotillin-2 and is rapidly degraded on induction of neuronal differentiation in cultured cells.
abstract: Post-translational palmitoylation of intracellular proteins is mediated by protein palmitoyltransferases belonging to the DHHC family, which share a common catalytic Asp-His-His-Cys (DHHC) motif. Several members have been implicated in neuronal development, neurotransmission, and synaptic plasticity. We previously observed that mice homozygous for a hypomorphic allele of the ZDHHC5 gene are impaired in context-dependent learning and memory. To identify potentially relevant protein substrates of DHHC5, we performed a quantitative proteomic analysis of stable isotope-labeled neuronal stem cell cultures from forebrains of normal and DHHC5-GT (gene-trapped) mice using the bioorthogonal palmitate analog 17-octadecynoic acid. We identified ∼300 17-octadecynoic acid-modified and hydroxylamine-sensitive proteins, of which a subset was decreased in abundance in DHHC5-GT cells. Palmitoylation and oligomerization of one of these proteins (flotillin-2) was abolished in DHHC5-GT neuronal stem cells. In COS-1 cells, overexpression of DHHC5 markedly stimulated the palmitoylation of flotillin-2, strongly suggesting a direct enzyme-substrate relationship. Serendipitously, we found that down-regulation of DHHC5 was triggered within minutes following growth factor withdrawal from normal neural stem cells, a maneuver that is used to induce neural differentiation in culture. The effect was reversible for up to 4 h, and degradation was partially prevented by inhibitors of ubiquitin-mediated proteolysis. These findings suggest that protein palmitoylation can be regulated through changes in DHHC PAT levels in response to differentiation signals.
authors.affiliation: Hamon Center for Therapeutic Oncology Research and Department of Internal Medicine, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-8593.
authors.forename: Yi
authors.initials: Y
authors.lastname: Li
authors.affiliation: Skaggs Institute of Chemical Biology and Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California 92037.
authors.forename: Brent R
authors.initials: BR
authors.lastname: Martin
authors.affiliation: Skaggs Institute of Chemical Biology and Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California 92037.
authors.forename: Benjamin F
authors.initials: BF
authors.lastname: Cravatt
authors.affiliation: Hamon Center for Therapeutic Oncology Research and Department of Internal Medicine, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-8593. Electronic address: sandra.hofmann@utsouthwestern.edu.
authors.forename: Sandra L
authors.initials: SL
authors.lastname: Hofmann
mesh_terms: D000217:Acyltransferases; D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D006801:Humans; D054878:Lipoylation; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D058953:Neural Stem Cells / Q000166:cytology / Q000378:metabolism; D009474:Neurons / Q000166:cytology* / Q000378:metabolism*; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D059748:Proteolysis
chemical_list: D008565:Membrane Proteins; C106354:flotillins; D000217:Acyltransferases; C549503:DHHC5 protein, mouse
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