Regulation of NCAM palmitoylation through DHHC3 phosphorylation by specific tyrosine kinases.

The neural cell adhesion molecule NCAM is broadly expressed on the surface of neural cells. Recent studies have shown that activation of FGF receptor (FGFR) by FGF2 leads to S-palmitoylation of the two major transmembrane isoforms, NCAM140 and 180, translocation of NCAM to lipid rafts, and stimulation of neurite outgrowth of hippocampal neurons. S-palmitoylation of NCAM is catalyzed by two closely related members of the DHHC (Asp-His-His-Cys)-containing protein family - DHHC3 and DHHC7. The aim of our study was to understand the mechanism, by which FGFR signaling regulates DHHC3-mediated NCAM palmitoylation. First, by means of selective inhibitors and co-expression experiments in neuroblastoma N2a cells, we found that FGFR and Src kinases regulate tyrosine phosphorylation of DHHC3. Co-immunoprecitation (co-IP) experiments have shown that DHHC3 directly interacted with Src. Moreover, in mutagenesis study we determined specific tyrosines responsible for basal and for Src- or FGFR1-stimulated tyrosine phosphorylation of DHHC3. Next, we verified the functional role of tyrosine phosphorylation of DHHC3. Metabolic labeling with radioactive palmitate revealed that tyrosine-deficient mutated form of DHHC3 has higher palmitoylation activity towards its substrate NCAM180. Furthermore, co-IP analysis demonstrated that the mutated form interacts stronger with NCAM, and Click-iT method illustrated that it is more autopalmitoylated. Finally, analyzing neurite outgrowth in cultured hippocampal neurons, we demonstrated the physiological role of DHHC3 tyrosine phosphorylation in neuronal development. Altogether, our results reveal that FGFR and Src mediate DHHC3 tyrosine phosphorylation and highlight the role of DHHC3 phosphorylation in regulation of DHHC3 catalytic activity towards NCAM and in neuronal morphogenesis.