Syk/Zap70 family kinases are essential for signaling via multichain immune-recognition receptors such as tetrameric (αβγ2) FcεRI. Syk activation is generally attributed to cis binding of its tandem SH2 domains to dual phosphotyrosines within FcεRIγ-ITAMs (immunoreceptor tyrosine-based activation motifs). However, the mechanistic details of Syk docking on γ homodimers are unresolved. Here, we estimate that multivalent interactions for WT Syk improve cis-oriented binding by three orders of magnitude. We applied molecular dynamics (MD), hybrid MD/worm-like chain polymer modeling, and live cell imaging to evaluate relative binding and signaling output for all possible cis and trans Syk–FcεRIγ configurations. Syk binding is likely modulated during signaling by autophosphorylation on Y130 in interdomain A, since a Y130E phosphomimetic form of Syk is predicted to lead to reduced helicity of interdomain A and alter Syk’s bias for cis binding. Experiments in reconstituted γ-KO cells, whose γ subunits are linked by disulfide bonds, as well as in cells expressing monomeric ITAM or hemITAM γ-chimeras, support model predictions that short distances between γ ITAM pairs are required for trans docking. We propose that the full range of docking configurations improves signaling efficiency by expanding the combinatorial possibilities for Syk recruitment, particularly under conditions of incomplete ITAM phosphorylation.

Crosstalk between different receptor tyrosine kinases (RTKs) is thought to drive oncogenic signaling and allow therapeutic escape. EGFR and RON are two such RTKs from different subfamilies, which engage in crosstalk through unknown mechanisms. We combined high-resolution imaging with biochemical and mutational studies to ask how EGFR and RON communicate. EGF stimulation promotes EGFR-dependent phosphorylation of RON, but ligand stimulation of RON does not trigger EGFR phosphorylation – arguing that crosstalk is unidirectional. Nanoscale imaging reveals association of EGFR and RON in common plasma membrane microdomains. Two-color single particle tracking captured formation of complexes between RON and EGF-bound EGFR. Our results further show that RON is a substrate for EGFR kinase, and that transactivation of RON requires formation of a signaling competent EGFR dimer. These results support a role for direct EGFR/RON interactions in propagating crosstalk, such that EGF-stimulated EGFR phosphorylates RON to activate RON-directed signaling.

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The physical properties of amorphous biomolecules play a critical role in stability of food, pharmaceutical, and biotechnological products. Consequently, they have an impact on formulation design and quality control of amorphous products. Molecular mobility, heterogeneity, and air permeability have been monitored in amorphous materials using Generally Recognized as Safe (GRAS) luminescent probes, i.e., tryptophan and erythrosine B. Given its GRAS status and widespread availability, we hypothesize that riboflavin can be used as a convenient and safe intrinsic luminescent probe to study the physical properties of amorphous films. Riboflavin phosphorescence has only been reported at temperatures below 77K and in solid matrices. The objectives of this study were: 1)to investigate riboflavin phosphorescence sensitivity towards molecular mobility in a disaccharide model system, 2)to characterize the temperature dependence of riboflavin phosphorescence in amorphous films. Sucrose was used as a model system to assess riboflavin's applicability as a phosphorescent probe of the physical state of edible films. Steady-state and time-resolved phosphorescence of riboflavin in amorphous sucrose films were collected over the temperature range from À30 C to 60 C. Emission spectra were fitted with a log-normal function. The performance of a stretched exponential and a multi-exponential function were evaluated to characterize riboflavin lifetimes. The rate constant of non-radiative decay (kTS0), a measure of molecular mobility, was calculated from riboflavin's average lifetime. Measures of the distribution of energetically distinct environments were obtained from bandwidth (G), stretching exponential factor (b), and fractional amplitudes (ai) parameters. The analysis of fitted parameters as a function of temperature revealed two temperature regimes below Tg in sucrose films with a transition at 10 C. Implications of these findings on riboflavin's potential applications as a luminescent probe of physical properties of amorphous biomolecules and edible films are discussed.