An early step in signaling from activated receptor tyrosine kinases (RTKs) is the recruitment of cytosolic adaptor proteins to autophosphorylated tyrosines in the receptor cytoplasmic domains. Fibroblast growth factor receptor substrate 2α (FRS2α) associates via its phosphotyrosine-binding domain (PTB) to FGF receptors (FGFRs). Upon FGFR activation, FRS2α undergoes phosphorylation on multiple tyrosines, triggering recruitment of the adaptor Grb2 and the tyrosine phosphatase Shp2, resulting in stimulation of PI3K/AKT and MAPK signaling pathways. FRS2α also undergoes N-myristoylation, which was shown to be important for its localization to membranes and its ability to stimulate downstream signaling events (Kouhara et al., 1997). Here we show that FRS2α is also palmitoylated in cells and that cysteines 4 and 5 account for the entire modification. We further show that mutation of those two cysteines interferes with FRS2α localization to the plasma membrane (PM), and we quantify this observation using fluorescence fluctuation spectroscopy approaches. Importantly, prevention of myristoylation by introduction of a G2A mutation also abrogates palmitoylation, raising the possibility that signaling defects previously ascribed to the G2A mutant may actually be due to a failure of that mutant to undergo palmitoylation. Our results demonstrate that FRS2α undergoes coupled myristoylation and palmitoylation. Unlike stable cotranslational modifications, such as myristoylation and prenylation, palmitoylation is reversible due to the relative lability of the thioester linkage. Therefore, palmitoylation may provide a mechanism, in addition to phosphorylation, for dynamic regulation of FRS2 and its downstream signaling pathways.
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*E-mail: email@example.com. Tel: 214-645-6119. Fax: 214-645-6124. ORCID Yan Chen: 0000-0002-0900-8338 Joseph P. Albanesi: 0000-0002-6864-8140 Author Contributions J.P.A., B.B., Y.C., H.L., J.L., and J.D.M. were responsible for experimental design. B.B. and C.A.T. performed palmitoylation analysis experiments. Y.J.C. and H.Q.S. performed fluorescence light microscopy localization experiments. I.A., J.H., and Y.C. performed fluorescence fluctuation spectroscopy experiments. Funding This research was supported by National Institutes of Health (NIH) grants [GM121536 (J.P.A., H.Y., and J.D.M.) and GM064589 (Y.C., J.H., and J.D.M.)] and a Basic Science Pilot Grant from the Simmons Comprehensive Cancer Center, UTSW (J.P.A.). I.A. was supported by an NIH grant (T32 AI083196). Notes The authors declare no competing financial interest.