ja7b09387_si_001.pdf (1.13 MB)
Allosteric Modulation of Grb2 Recruitment to the Intrinsically Disordered Scaffold Protein, LAT, by Remote Site Phosphorylation
journal contribution
posted on 2017-11-28, 15:34 authored by William
Y. C. Huang, Jonathon A. Ditlev, Han-Kuei Chiang, Michael K. Rosen, Jay T. GrovesTyrosine phosphorylation of membrane
receptors and scaffold proteins
followed by recruitment of SH2 domain-containing adaptor proteins
constitutes a central mechanism of intracellular signal transduction.
During early T-cell receptor (TCR) activation, phosphorylation of
linker for activation of T cells (LAT) leading to recruitment of adaptor
proteins, including Grb2, is one prototypical example. LAT contains
multiple modifiable sites, and this multivalency may provide additional
layers of regulation, although this is not well understood. Here,
we quantitatively analyze the effects of multivalent phosphorylation
of LAT by reconstituting the initial reactions of the TCR signaling
pathway on supported membranes. Results from a series of LAT constructs
with combinatorial mutations of tyrosine residues reveal a previously
unidentified allosteric mechanism in which the binding affinity of
LAT:Grb2 depends on the phosphorylation at remote tyrosine sites.
Additionally, we find that LAT:Grb2 binding affinity is altered by
membrane localization. This allostery mainly regulates the kinetic
on-rate, not off-rate, of LAT:Grb2 interactions. LAT is an intrinsically
disordered protein, and these data suggest that phosphorylation changes
the overall ensemble of configurations to modulate the accessibility
of other phosphorylated sites to Grb2. Using Grb2 as a phosphorylation
reporter, we further monitored LAT phosphorylation by TCR ζ
chain-recruited ZAP-70, which suggests a weakly processive catalysis
on membranes. Taken together, these results suggest that signal transmission
through LAT is strongly gated and requires multiple phosphorylation
events before efficient signal transmission is achieved.