Article Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
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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,*,†,§ and Jay T. Groves*,†,‡ †
The Howard Hughes Medical Institute Summer Institute, Marine Biological Laboratory, Woods Hole, Massachusetts 02543, United States ‡ Department of Chemistry, University of California, Berkeley, Berkeley, California 94720, United States § Department of Biophysics and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75390, United States S Supporting Information *
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.
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INTRODUCTION
receptor signaling scaffolds including EGFR, nephrin, and PD-1, has multiple phosphorylatable tyrosine sites that recruit downstream SH2-domain adaptor proteins such as Grb2, Gads, and PLCγ.1−4,8−11 Transmission of signals depends on the phosphorylation and recruitment events at these sites. Furthermore, multivalency creates opportunities for additional regulatory controls, which are only now beginning to be appreciated. For instance, recent observations in LAT:Grb2:SOS12−15 and nephrin:Nck:N-WASP16,17 reveal the formation of extended multimolecular assemblies in response to multivalent phosphorylation. These assemblies can even exhibit gelation phase transitions driven by tyrosine phosphorylation, which may govern downstream signal transmission, amplification, and noise suppression.
Tyrosine phosphorylation plays a central role in intracellular signal transduction.1−4 There are numerous examples of signaling molecules in which phosphorylation of specific tyrosine residues (Y) leads to functional alterations (e.g., activating or deactivating) by modulating the structure of the folded proteins.5 Alternatively, phosphorylated tyrosine residues can function as recruitment sites for other signaling proteins that contain SH2 domains. In the case of T-cell receptor (TCR) signaling (Figure 1A), engagement of the TCR with agonist ligand leads to phosphorylation of multiple tyrosine residues on the cytoplasmic immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR. The kinase ZAP-70 is then recruited to these phosphotyrosine (pY) sites where it is phosphorylated at sites Y315, Y319 and Y493 by the Src family kinase Lck.6,7 Phosphorylation of ZAP-70 at these residues releases autoinhibition, enabling ZAP-70 to phosphorylate tyrosine sites on LAT.1,4 LAT, like other © XXXX American Chemical Society
Received: September 2, 2017
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DOI: 10.1021/jacs.7b09387 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
Article
Journal of the American Chemical Society
binding at one pY site is favorably altered by the phosphorylation of remote tyrosine sites. Although this effect can be described as allosteric modulation (i.e., action at a distance), it fundamentally differs from classical allostery (in which remote effects on binding are achieved through ligandbinding induced changes in the folded protein structure)18,19 since LAT is an intrinsically disordered protein. Measurements described here indicate that allosteric regulation is primarily manifested in the kinetic on-rates of LAT:Grb2 interactions. This suggests that phosphorylation of LAT at remote sites changes the overall ensemble of LAT configurations, rendering other pY sites more accessible to Grb2 binding. We also found that LAT phosphorylation by membrane-recruited ZAP-70 is weakly processive using Grb2 as the phosphotyrosine reporter. Collectively, these observations suggest that signal transmission through LAT is suppressed for weak signals, but amplified for a strong input that successfully leads to multiple phosphorylation of LAT. Furthermore, quantitative values of the molecular parameters measured here depend on membrane localization, underscoring the importance of reconstitution in a membrane format.
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RESULTS In Vitro Membrane Reconstitution of LAT Phosphorylation by TCR ζ Chain-Recruited ZAP-70. Here we introduce a two-dimensional enzymatic assay on a supported membrane platform to quantitatively characterize the phosphorylation of LAT by TCR ζ chain-recruited ZAP-70 and Grb2 recruitment in real time (Figure 1A). Prephosphorylated cytoplasmic domain of TCR ζ chain (pTCR) and fluorescently labeled LAT-Alexa Fluor 568 (referred to as LAT) were chelated onto DOPC bilayers containing 4% NiNTA-modified lipids via N-terminal His8-tags (Figure 1A).20 LAT contains nine tyrosine residues and Grb2 binds to the three most distal tyrosine residues (Y171, Y191, and Y226), while a fourth tyrosine residue (Y132) is bound by PLCγ1.8,21 In these studies, we utilize an engineered LAT construct (residue 48− 233) containing only these four tyrosine residues (LAT4Y) to systematically quantify the effects of multivalent tyrosine phosphorylation on Grb2 recruitment; other tyrosine residues were mutated to phenylalanine (F) (Figure 1). This LAT construct and the mutation strategy follows prior work on LAT.8,15 The binding affinities of LAT4Y to Grb2 are later compared to those of LAT mutants containing single or triple tyrosine residues in systematic permutations (Table S1, S2). The densities of laterally fluid LAT can be controlled from 50 to 3000 molecule/μm2 and can be accurately measured using fluorescence correlation spectroscopy (FCS) (Figure S1). For comparison, physiological densities of LAT are 100−1000 molecule/μm2.22 Although not addressed in the experiments described here, this system is amenable to further elaboration of membrane lipid and protein context to explore these additional dimensions of the system. The reconstituted assay was optimized for the following kinetic considerations to resolve LAT phosphorylation: (i) Grb2 binds the pY rapidly (
