DHHC5 Protein Palmitoylates Flotillin-2 and Is Rapidly Degraded on Induction of Neuronal Differentiation in Cultured Cells
DHHC5 Protein Palmitoylates Flotillin-2 and Is Rapidly Degraded on Induction of Neuronal Differentiation in Cultured Cells
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DOI：
10.1074/jbc.m111.306183
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发表时间：
2012-01-02
影响因子：
4.8
通讯作者：
Hofmann, Sandra L.
中科院分区：
文献类型：
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作者：
Li, Yi;Martin, Brent R.;Hofmann, Sandra L.
细胞内蛋白质的翻译后棕榈酰化由属于DHHC家族的蛋白质棕榈酰转移酶介导，其共享共同的催化Asp-His-His-Cys（DHHC）基序。一些成员与神经元发育、神经传递和突触可塑性有关。我们先前观察到，ZDHHC 5基因的亚型等位基因纯合子小鼠在情境依赖性学习和记忆中受损。为了鉴定DHHC 5的潜在相关蛋白质底物，我们使用生物正交棕榈酸酯类似物17-十八炔酸对来自正常和DHHC 5-GT（基因捕获）小鼠前脑的稳定同位素标记的神经元干细胞培养物进行了定量蛋白质组学分析。我们鉴定了类似于300个17-十八炔酸修饰的和羟胺敏感的蛋白质，其中一个子集在DHHC 5-GT细胞中丰度降低。在DHHC 5-GT神经干细胞中，这些蛋白质之一（flotillin-2）的棕榈酰化和寡聚化被废除。在COS-1细胞中，DHHC 5的过表达显著刺激了flotillin-2的棕榈酰化，强烈表明了直接的酶-底物关系。偶然地，我们发现DHHC 5的下调在从正常神经干细胞中撤出生长因子后几分钟内被触发，这是一种用于诱导培养中神经分化的策略。这种作用在4小时内是可逆的，并且通过泛素介导的蛋白水解抑制剂部分阻止了降解。这些发现表明，蛋白质棕榈酰化可以通过响应分化信号的DHHC PAT水平的变化来调节。
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 similar to 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.