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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.

We previously reported that recruitment of the Type IIA phosphatidylinositol 4-kinase (PI4K2A) to autophagosomes by GABARAP, a member of the Atg8 family of autophagy-related proteins, is important for autophagosome-lysosome fusion. Because both PI4K2A and GABARAP have also been implicated in the intracellular trafficking of plasma membrane receptors in the secretory/endocytic pathway, we characterized their interaction in cells under non-autophagic conditions. Fluorescence fluctuation spectroscopy measurements revealed that GABARAP exists predominantly as a cytosolic monomer in live cells, but is recruited to small cytoplasmic vesicles upon overexpression of PI4K2A. C-terminal lipidation of GABARAP, which is essential for its autophagic activities, is not necessary for its recruitment to these PI4K2A-containing transport vesicles. However, a GABARAP truncation mutant lacking C-terminal residues 103–117 fails to bind to PI4K2A, is not recruited to cytoplasmic vesicles, and does not co-distribute with PI4K2A on subcellular organelles. These observations suggest that that the PI4K2A-GABARAP interaction plays a role in membrane trafficking both under autophagic and non-autophagic conditions.

The dimeric Crotalus atrox venom PLA2 is part of the secreted phospholipase A2 (PLA2) enzyme family that interacts at the lipid-solution interface to hydrolyze the sn-2 acyl ester bond of phospholipids. We have employed fluorescence correlation spectroscopy (FCS) to study the monomer-dimer equilibrium of the C. atrox venom PLA2 in solution, in the presence of urea, and in the presence of monomeric and micellar n-dodecylphosphocholine (C12-PN), a phosphatidylcholine analogue. Dilution experiments show that PLA2 is an extremely tight dimer, Kd < or = 0.01 nM, in solution. Urea was introduced to weaken the subunit's association, and an estimate for the PLA(2) dimer dissociation constant in buffer was obtained by linear extrapolation. The derived dissociation constant was at least several orders of magnitude greater than that suggested from the dilution experiments, indicating a complex interaction between urea and the PLA2 dimer. FCS data indicate that the PLA2 dimer begins to dissociate at 10 mM C12-PN in 10 mM Ca2+ and at 5 mM C12-PN in 1 mM EDTA. The PLA2 tryptophan fluorescence displayed spectral shifts and intensity changes upon interacting with C12-PN. On the basis of the FCS and tryptophan fluorescence results, we postulate an intermediate state where the two monomers are in loose interaction within a protein-lipid comicelle. As the concentration of C12-PN was increased, complete dissociation of the dimer was observed, inferred from the doubling of the particle number, and the average diffusion constant decreased to approximately 60 microm2/s, consistent with PLA2 associated with a C12-PN micelle. The presence of Ca2+ makes the comicelle intermediate more stable, retarding the separation of the monomers in the micellar suspension. Our data clearly indicate that PLA2, though a strong dimer in the absence of lipids, is dissociated by micellar C12-PN and supports the monomer hypothesis for PLA2 action.

Fourier transform infrared (FTIR) spectroscopy of cytochrome c oxidase from Rhodobacter sphaeroides reveals multiple CO stretch bands that are associated with different conformational substates of the enzyme. Here we report the temperature dependence of the infrared bands for the CO bound to the Fea3 heme iron and to CuB. We have also studied the kinetics of ligand return from Fea3 to CuB using temperature derivative spectroscopy (TDS). Two classes of substates (alpha/beta) can be distinguished from their different properties with regard to the width of the IR band, the temperature dependence of the peak position, and the peak of the enthalpy distribution. The pronounced temperature dependence of the stretch frequencies in the beta conformation and the lack thereof in the alpha conformation implies very different dynamic behavior in the active site and reflects structural differences between the two conformations, most likely a shift of the position of CuB in response to a change in its stereochemical environment. Similar conformational changes will be necessary during the catalytic cycle of the enzyme when dioxygen is bound in the active site.

The problem of protein stability is addressed with spectroscopic studies of equilibrium and kinetic properties of the binding of fluorescein to high-affinity monoclonal anti-fluorescyl antibodies (Mab 4-4-20), Fab fragments, and single-chain antibodies (SCA). SCA molecules contain only the variable domains of the antibody and an amino acid linker. The influence of glycerol on the antigen binding reaction is studied by circular dichroism, fluorescence, and absorption spectroscopy. The presence of glycerol in the solvent lowers the affinity of SCA for the ligand drastically, and the affinity even decreases toward lower temperatures. This effect is not observed in Fab and Mab. Analysis of the temperature jump kinetics shows that the dissociation reaction can be modeled as a two-state transition. The CD spectra indicate that the domain structure of the SCA remains unaltered in the presence of glycerol. Therefore, it is concluded that glycerol promotes the dissociation of the two variable domains of SCA. In Fab and Mab, the constant domains provide additional stabilization of the molecular structure at the antigen binding site.