Allosteric Modulation of Grb2 Recruitment to the Intrinsically Disordered Scaffold Protein, LAT, by Remote Site Phosphorylation

William Y.C. Huang, Jonathon A. Ditlev, Han Kuei Chiang, Michael K. Rosen, Jay T. Groves

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

Tyrosine 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.

Original languageEnglish (US)
Pages (from-to)18009-18015
Number of pages7
JournalJournal of the American Chemical Society
Volume139
Issue number49
DOIs
StatePublished - Dec 13 2017

Fingerprint

Intrinsically Disordered Proteins
Phosphorylation
T-cells
Scaffolds (biology)
Scaffolds
Chemical activation
Modulation
Proteins
T-Lymphocytes
T-Cell Antigen Receptor
Tyrosine
Membranes
src Homology Domains
Signal transduction
Catalysis
Cell Communication
Signal Transduction

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

Allosteric Modulation of Grb2 Recruitment to the Intrinsically Disordered Scaffold Protein, LAT, by Remote Site Phosphorylation. / Huang, William Y.C.; Ditlev, Jonathon A.; Chiang, Han Kuei; Rosen, Michael K.; Groves, Jay T.

In: Journal of the American Chemical Society, Vol. 139, No. 49, 13.12.2017, p. 18009-18015.

Research output: Contribution to journalArticle

Huang, William Y.C. ; Ditlev, Jonathon A. ; Chiang, Han Kuei ; Rosen, Michael K. ; Groves, Jay T. / Allosteric Modulation of Grb2 Recruitment to the Intrinsically Disordered Scaffold Protein, LAT, by Remote Site Phosphorylation. In: Journal of the American Chemical Society. 2017 ; Vol. 139, No. 49. pp. 18009-18015.
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