S-palmitoylation is the postranslational addition of palmitate to cysteine residues of target proteins, which plays a role in their trafficking, subcellular localization, stability or function. Recently, zinc finger DHHC (Asp-His-His-Cys)-containing proteins have emerged as a large family of acyl transferases (PAT). Palmitoylation of specific neuro-substrates was shown to be important for synaptic function, plasticity, neuronal migration and maturation. In this context, a recent by study revealed that activation of fibroblast growth factor receptors (FGFRs) by FGF2 leads to palmitoylation of the neural cell adhesion molecule (NCAM) by DHHC3 and stimulation of neurite outgrowth. FGF:FGFR interaction induces the activation of signaling cascades operating through the sequential activation of tyrosine (Y) phosphorylation sites on target proteins. We thus questioned whether any role was played by tyrosine phosphorylation in the regulation of DHHC3 activity. We show that wild-type (wt) DHHC3 becomes highly tyrosine phosphorylated when co-transfected with Src or FGFR1 in neuroblastoma cells. Application of the selective Src inhibitor PP2 reduces the phosphorylation of DHHC3, which is further decreased by the FGFR inhibitor PD173074. DHHC3 has 5 tyrosines located in its cytoplasmic domains. By generating single and triple Y to F (phenylalanine) mutants we were able to dissect the ones responsible for src (Y295, Y297) or FGFR1 (Y18) mediated phosphorylation. Interestingly, the DHHC3 mutant lacking all 5 tyrosines is significantly more autopalmitoylated, as assed by the Click-IT protocol and shows higher palmitoylation activity towards NCAM compared to the wt. In sum, we describe for the first time a role for tyrosine phosphorylation in the regulation of the levels of DHHC3 autopalmitoylation, a postranslational event required for NCAM interaction and palmitoylation. Ongoing experiments aim to study how DHHC3 tyrosine phosphorylation modulates neuronal plasticity.
Role of DHHC3 tyrosine phosphorylation in regulating NCAM palmitoylation
LIEVENS, Patricia;ROMANELLI, Maria;
2013-01-01
Abstract
S-palmitoylation is the postranslational addition of palmitate to cysteine residues of target proteins, which plays a role in their trafficking, subcellular localization, stability or function. Recently, zinc finger DHHC (Asp-His-His-Cys)-containing proteins have emerged as a large family of acyl transferases (PAT). Palmitoylation of specific neuro-substrates was shown to be important for synaptic function, plasticity, neuronal migration and maturation. In this context, a recent by study revealed that activation of fibroblast growth factor receptors (FGFRs) by FGF2 leads to palmitoylation of the neural cell adhesion molecule (NCAM) by DHHC3 and stimulation of neurite outgrowth. FGF:FGFR interaction induces the activation of signaling cascades operating through the sequential activation of tyrosine (Y) phosphorylation sites on target proteins. We thus questioned whether any role was played by tyrosine phosphorylation in the regulation of DHHC3 activity. We show that wild-type (wt) DHHC3 becomes highly tyrosine phosphorylated when co-transfected with Src or FGFR1 in neuroblastoma cells. Application of the selective Src inhibitor PP2 reduces the phosphorylation of DHHC3, which is further decreased by the FGFR inhibitor PD173074. DHHC3 has 5 tyrosines located in its cytoplasmic domains. By generating single and triple Y to F (phenylalanine) mutants we were able to dissect the ones responsible for src (Y295, Y297) or FGFR1 (Y18) mediated phosphorylation. Interestingly, the DHHC3 mutant lacking all 5 tyrosines is significantly more autopalmitoylated, as assed by the Click-IT protocol and shows higher palmitoylation activity towards NCAM compared to the wt. In sum, we describe for the first time a role for tyrosine phosphorylation in the regulation of the levels of DHHC3 autopalmitoylation, a postranslational event required for NCAM interaction and palmitoylation. Ongoing experiments aim to study how DHHC3 tyrosine phosphorylation modulates neuronal plasticity.
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