Protein palmitoylation, the common posttranslational modification with the lipid palmitate, plays a pivotal role in protein trafficking and function. Our previous studies revealed that activation of fibroblast growth factor (FGF) receptor(s) by FGF2 leads to palmitoylation of the neural cell adhesion molecule (NCAM), its translocation to GM1 ganglioside-enriched lipid rafts, and stimulation of neurite outgrowth of cultured hippocampal neurons (1). Now, we used the Acyl-Biotin-Exchange (ABE) method and show a 1.3-fold increase in NCAM palmitoylation in mice injected with FGF2 in vivo. Furthermore, we analyzed the mechanisms of this regulation. Two members of the DHHC family, DHHC3 and DHHC7, were identified to mediate palmitoylation of NCAM in heterologous N2a cells (1). Because FGF:FGFR interaction leads to the activation of the canonical Ras/MAPK pathway and to the recruitment and activation of PLCγ and Src family tyrosine kinases (2), we investigated the tyrosine phosphorylation of DHHCs. Here, we present data showing that DHHC3 is highly tyrosine phosphorylated by activated FGFR1, suggesting a potential role of DHHC3 phosphorylation for regulation of its palmitoylating activity. We also report that treatment with the Src inhibitor PP2 significantly reduces DHHC3 tyrosine phosphorylation. Based on this evidence, we performed co-immunoprecipitation assays and found that Src but not FGFR1 is pulled-down by DHHC3, suggesting a possible direct interaction between the two molecules. Furthermore, DHHC3 isolated from brain homogenates appeared tyrosine phosphorylated, thus confirming the data obtained in the cell lines. Ongoing mutagenesis experiments aim to identify DHHC3 regulatory tyrosines. The generated DHHC3 mutants are assayed with the non-radioactive Click-IT palmitoylation assay that we specifically developed for NCAM. In summary, we demonstrated the FGFR-dependent phosphorylation of DHHC proteins, which might regulate palmitoylation activities of these enzymes towards to their substrates, including NCAM.
FGF receptor-mediated tyrosine phosphorylation of DHHC palmitoyltransferases: a mechanism to regulate the levels of NCAM palmitoylation?
LIEVENS, Patricia;
2012-01-01
Abstract
Protein palmitoylation, the common posttranslational modification with the lipid palmitate, plays a pivotal role in protein trafficking and function. Our previous studies revealed that activation of fibroblast growth factor (FGF) receptor(s) by FGF2 leads to palmitoylation of the neural cell adhesion molecule (NCAM), its translocation to GM1 ganglioside-enriched lipid rafts, and stimulation of neurite outgrowth of cultured hippocampal neurons (1). Now, we used the Acyl-Biotin-Exchange (ABE) method and show a 1.3-fold increase in NCAM palmitoylation in mice injected with FGF2 in vivo. Furthermore, we analyzed the mechanisms of this regulation. Two members of the DHHC family, DHHC3 and DHHC7, were identified to mediate palmitoylation of NCAM in heterologous N2a cells (1). Because FGF:FGFR interaction leads to the activation of the canonical Ras/MAPK pathway and to the recruitment and activation of PLCγ and Src family tyrosine kinases (2), we investigated the tyrosine phosphorylation of DHHCs. Here, we present data showing that DHHC3 is highly tyrosine phosphorylated by activated FGFR1, suggesting a potential role of DHHC3 phosphorylation for regulation of its palmitoylating activity. We also report that treatment with the Src inhibitor PP2 significantly reduces DHHC3 tyrosine phosphorylation. Based on this evidence, we performed co-immunoprecipitation assays and found that Src but not FGFR1 is pulled-down by DHHC3, suggesting a possible direct interaction between the two molecules. Furthermore, DHHC3 isolated from brain homogenates appeared tyrosine phosphorylated, thus confirming the data obtained in the cell lines. Ongoing mutagenesis experiments aim to identify DHHC3 regulatory tyrosines. The generated DHHC3 mutants are assayed with the non-radioactive Click-IT palmitoylation assay that we specifically developed for NCAM. In summary, we demonstrated the FGFR-dependent phosphorylation of DHHC proteins, which might regulate palmitoylation activities of these enzymes towards to their substrates, including NCAM.
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