Lievens Patricia Marie-Jeanne, Kuznetsova Tatiana, Kochlamazashvili Gaga, Cesca Fabrizia, Gorinski Natalya, Galil Dalia Abdel, Cherkas Volodimir, Ronkina Natalia, Lafera Juri, Gaestel Matthias, Ponimaskin Evgeni, Dityatev Alexander
Department of Neuroscience and Brain Technologies, Istituto Italiano di Tecnologia, Genoa, Italy Department of Neurosciences, Biomedicine and Movement Sciences, Section of Biology and Genetics, University of Verona, Verona, Italy.
Department of Neuroscience and Brain Technologies, Istituto Italiano di Tecnologia, Genoa, Italy St. Petersburg State University, St. Petersburg, Russia.
Mol Cell Biol. 2016 Aug 12;36(17):2208-25. doi: 10.1128/MCB.00144-16. Print 2016 Sep 1.
The neural cell adhesion molecule (NCAM) mediates cell-cell and cell-matrix adhesion. It is broadly expressed in the nervous system and regulates neurite outgrowth, synaptogenesis, and synaptic plasticity. Previous in vitro studies revealed that palmitoylation of NCAM is required for fibroblast growth factor 2 (FGF2)-stimulated neurite outgrowth and identified the zinc finger DHHC (Asp-His-His-Cys)-containing proteins ZDHHC3 and ZDHHC7 as specific NCAM-palmitoylating enzymes. Here, we verified that FGF2 controlled NCAM palmitoylation in vivo and investigated molecular mechanisms regulating NCAM palmitoylation by ZDHHC3. Experiments with overexpression and pharmacological inhibition of FGF receptor (FGFR) and Src revealed that these kinases control tyrosine phosphorylation of ZDHHC3 and that ZDHHC3 is phosphorylated by endogenously expressed FGFR and Src proteins. By site-directed mutagenesis, we found that Tyr18 is an FGFR1-specific ZDHHC3 phosphorylation site, while Tyr295 and Tyr297 are specifically phosphorylated by Src kinase in cell-based and cell-free assays. Abrogation of tyrosine phosphorylation increased ZDHHC3 autopalmitoylation, enhanced interaction with NCAM, and upregulated NCAM palmitoylation. Expression of ZDHHC3 with tyrosine mutated in cultured hippocampal neurons promoted neurite outgrowth. Our findings for the first time highlight that FGFR- and Src-mediated tyrosine phosphorylation of ZDHHC3 modulates ZDHHC3 enzymatic activity and plays a role in neuronal morphogenesis.
神经细胞黏附分子（NCAM）介导细胞间和细胞与基质间的黏附。它在神经系统中广泛表达，并调节神经突生长、突触形成和突触可塑性。先前的体外研究表明，成纤维细胞生长因子2（FGF2）刺激的神经突生长需要NCAM的棕榈酰化，并确定含锌指DHHC（天冬氨酸-组氨酸-组氨酸-半胱氨酸）的蛋白ZDHHC3和ZDHHC7为特定的NCAM棕榈酰化酶。在此，我们证实FGF2在体内控制NCAM的棕榈酰化，并研究ZDHHC3调节NCAM棕榈酰化的分子机制。对FGF受体（FGFR）和Src进行过表达及药理学抑制的实验表明，这些激酶控制ZDHHC3的酪氨酸磷酸化，且ZDHHC3被内源性表达的FGFR和Src蛋白磷酸化。通过定点诱变，我们发现Tyr18是FGFR1特异性的ZDHHC3磷酸化位点，而在基于细胞和无细胞的实验中，Tyr295和Tyr297被Src激酶特异性磷酸化。酪氨酸磷酸化的缺失增加了ZDHHC3的自身棕榈酰化，增强了与NCAM的相互作用，并上调了NCAM的棕榈酰化。在培养的海马神经元中表达酪氨酸突变的ZDHHC3促进了神经突生长。我们的研究结果首次表明，FGFR和Src介导的ZDHHC3酪氨酸磷酸化调节ZDHHC3的酶活性，并在神经元形态发生中起作用。
