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.
Highlights
The substrates and regulation of DHHC protein palmitoyl acyltransferases (PATs) are largely unknown
Quantitative Profiling of Protein Palmitoylation in Mutant Neuronal Stem Cells—To study the cellular role of DHHC5, we dissected the forebrains of post-natal day 1 DHHC5-GT mice and littermate controls and cultured them under conditions that select for cells maintaining neural stem cell pluripotency [20]
100% strongly expressed Sox2, a marker of NSCs. These cultures have been continually passaged for 1 year and maintain elevated Sox2 expression with greatly reduced levels of DHHC5 (ϳ5% of normal, the residual caused by splicing around the gene trap cassette) [16]
Summary
The substrates and regulation of DHHC protein palmitoyl acyltransferases (PATs) are largely unknown. Results: Flotillin-2 palmitoylation is abolished in DHHC5 gene-targeted neural stem cells, and neuronal differentiation induces DHHC5 turnover. 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. The mechanisms are incompletely understood and limited to only a few examples, in particular, the palmitoylation of G protein ␣ subunit G␣s [12] and the neuronal scaffolding protein PSD-95 [13] In both instances, palmitate turnover is accelerated in response to extracellular signals by receptor activation, contributing to down-regulation. We identify the abundant lipid raft protein flotillin-2 as a substrate for DHHC5 and show that it is under-palmitoylated in DHHC5-GT neural stem cells and brain. We find that DHHC5 declines rapidly (within minutes) on induction of neural differentiation, demonstrating a previously unsuspected mode of DHHC PAT regulation
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