Phosphotyrosine-mediated LAT assembly on membranes drives kinetic bifurcation in recruitment dynamics of the Ras activator SOS

Abstract

The assembly of cell surface receptors with downstream signaling molecules is a commonly occurring theme in multiple signaling systems. However, little is known about how these assemblies modulate reaction kinetics and the ultimate propagation of signals. Here, we reconstitute phosphotyrosine-mediated assembly of extended linker for the activation of T cells (LAT):growth factor receptor-bound protein 2 (Grb2):Son of Sevenless (SOS) networks, derived from the T-cell receptor signaling system, on supported membranes. Single-molecule dwell time distributions reveal two, well-differentiated kinetic species for both Grb2 and SOS on the LAT assemblies. The majority fraction of membrane-recruited Grb2 and SOS both exhibit fast kinetics and single exponential dwell time distributions, with average dwell times of hundreds of milliseconds. The minor fraction exhibits much slower kinetics, extending the dwell times to tens of seconds. Considering this result in the context of the multistep process by which the Ras GEF (guanine nucleotide exchange factor) activity of SOS is activated indicates that kinetic stabilization from the LAT assembly may be important. This kinetic proofreading effect would additionally serve as a stochastic noise filter by reducing the relative probability of spontaneous SOS activation in the absence of receptor triggering. The generality of receptor-mediated assembly suggests that such effects may play a role in multiple receptor proximal signaling processes.

Keywords: kinetic proofreading; membrane dwell time; protein assembly; signal transduction; single molecule.

Fig. 1.

Quantification of the input-response function of LAT:Grb2:SOS assembly in TCR signaling. Schematic of TCR signaling pathway (Top) and the in vitro-reconstituted system (Bottom). Engagement of TCR with pMHC results in phosphorylation of LAT, which triggers assembly reaction on the cytoplasmic side of the plasma membrane. LAT assembly promotes SOS membrane recruitment and activation, which propagates downstream signals. To quantitatively assess the input-response function at the LAT signaling node, the signaling geometry of LAT is reconstituted on supported membranes. Phosphorylation of LAT is triggered by membrane-bound tyrosine kinase Hck, which results in Grb2 recruitment. The assembly reaction is initiated by the addition of SOS proline-rich domain and can be reversed by tyrosine phosphatase, YopH.

Fig. S1.

Characterization of LAT on supported membranes. (A) FRAP measurements of Alexa Fluor 555-labeled His₆-LAT on supported membranes. After exposure of high intensity of light for 10 s, the fluorescent images are taken every 20 s to monitor the recovery process. Recovery fraction of intensity at 3 min are used as a benchmark to estimate the mobile fraction of the membrane proteins. Typically, membrane-tethered LAT has a mobile fraction of >90% in our experiments. The low percentage of immobile LAT is not functional in terms of Grb2 recruitment, supported by the observation that all membrane-bound Grb2 are mobile (Movie S3). Note that the long-dwelling species are mobile at a longer time scale, with motion distinctively different from immobile particles. The bottom plot shows the line scan of the intensity profile along the diameter of the bleaching spot. (B) Step-size distribution from single-particle tracking experiments. The data are well fitted to a single diffusive population with a diffusion coefficient of 1.8 μm²/s (dashed line), suggesting that LAT is monomeric on supported membranes.

Fig. S2.

Calibration curve for LAT density. LAT density is calibrated by establishing a linear calibration curve between the average epifluorescence intensity and the density determined by FCS. The light source for epifluorescence is found to be stable in our optical setup and has low variations between different days (±2% power). Fluorescence intensities are background-subtracted.

Fig. S3.

Phosphorylation of LAT. Kinetics of LAT phosphorylation monitored by visualizing the time dependent recruitment of Alexa647-Grb2 (10 nM solution concentration) in the presence of Hck. In the absence of ATP, a low level of nonspecific interaction is quantified as the noise of the assay. Addition of 1 mM ATP leads to a drastic increase in Grb2 recruitment. The rise of the intensity is primarily attributed to the phosphorylation reaction, whereas the plateau at the later stage indicates the dynamical equilibrium of pLAT:Grb2 binding kinetics. Compared with the bleaching curve of immobilized Grb2 (gray) on a different sample, the existence of a plateau suggests that pLAT:Grb2 binding kinetics occurs at a time scale faster than 5 s, consistent with the single-molecule analysis.

Fig. S4.

LAT phosphorylation by Zap70^KD in solution. The Grb2 dwell time histogram with LAT phosphorylated by Hck on the membrane (purple) or Zap70^KD in solution (brown). Differences between Hck- and Zap70-dependent LAT phosphorylation are not observed.

Fig. 2.

LAT:Grb2:SOS is sufficient to drive an assembly network on membrane surfaces. (A) Epifluorescence images of LAT undergoing an assembly reaction. After injection of 5.8 µM Grb2 and 1.45 µM SOS, small puncta of densely assembled proteins (red arrow) appear within a few minutes. The emergence of a macroscopic protein-dense phase was observed after 30 min. (B) Reversibility of the assembly. The phase boundary disintegrated abruptly into a homogenous phase after incubation of 10 µM phosphatase YopH. (C) Different geometries and sizes of LAT assemblies by manipulating the surface densities of LAT ranging from 600 to 4,000 molecules per square micron in the presence of 5.8 µM Grb2 and 1.45 µM SOS.

Fig. S5.

Phase diagram of the LAT:Grb2:SOS assembly. Phase diagram generated by titration of LAT membrane density and Grb2-SOS solution concentration. Within 1 h of observation time, higher density/concentration of protein leads to a LAT:Grb2:SOS assembly structure more readily. The y axis denotes Grb2 concentration, whereas SOS concentration is 25% of Grb2.

Fig. S6.

FRAP of LAT in protein assembly. (A) FRAP images of His₆-LAT-Alexa Fluor 555 in LAT:Grb2:SOS assemblies. The recovery of intensity suggests that the assembly structure is dynamic. The restoration of the spatial pattern suggests that the LAT assemblies have a stable geometry, with only local fluctuations of boundaries, and that the system has reached a quasistationary state suitable for single-molecule experiments. (B) The plot of the recovery process in FRAP experiments. Dashed lines are fitting to an exponential curve: τ_D for LAT only, LAT after YopH treatment, and LAT assembly are 113, 115, 704 s, respectively.

Fig. S7.

The role of multivalency in the formation of protein assembly. (A) Single phosphorylation site LAT mutants (LAT Y9, LAT Y8, and LAT Y7) fail to self-organize into protein assemblies in the presence of 5.8 μM Grb2, 1.45 μM SOS, and 1 mM ATP after 1 h. The densities of LAT mutants were estimated to be 5,400 molecules per square micron. For wild-type LAT, an assembly forms around 20–30 min of incubation under the same condition. LATY7 is LATY110A,Y127A,Y132A,Y191A, and Y226A; LATY8 is LATY110A,Y127A,Y132A,Y171A, and Y226A; LATY9 is LATY110A,Y127A,Y132A,Y171A, and Y191A. Y36, Y45, and Y64 of LAT have not been observed to be phosphorylated by tyrosine kinases (31). (B) Absence of a kinase to phosphorylate LAT fails to form assemblies. (C) Images of Grb2 before and after injection of 10 μM tyrosine phosphatase (YopH) in the solution. Grb2 recruitments are abolished after dephosphorylation.

Fig. S8.

Binding kinetics are independent of the geometry or size of LAT assembly. The dwell time distributions of Grb2 are used to show that the intensive properties of the assembly, such as the binding kinetics, are independent of the size or geometry of the assembly. Dwell time histograms acquired at 21 and 2 Hz are plotted in A and B, respectively. By titrating the density of LAT from 600 to 8,000 molecules per square micron, the dwell time distributions from different sizes of assembly are similar.

Fig. 3.

Input-response function parameterized by single-molecule dwell time analysis. (A) Single-molecule images showing the gradual increase in Grb2 recruitment following LAT phosphorylation. (B) Diffusion and dissociation kinetics for membrane recruited Grb2 measured from single-particle tracking. Single-step photobleaching confirmed that the tracked Grb2 is a single molecule. The existence of a change point was detected using a Bayesian algorithm. (C) Dwell time histogram of Grb2 (purple histogram). After correction of photobleaching (black dots), the dwell time of LAT:Grb2 is 0.65 ± 0.10 s, setting the baseline of the input-response function before any assembly structure. Dashed line is fitting to a single kinetic population (Eq. S2).

Fig. 4.

Protein assembly creates kinetic bifurcation in the recruitment dynamics of Grb2 and SOS. (A) The kinetic species of interest are evaluated at the two different frame rates of 2 and 21 Hz. The top lane shows the time-lapse imaging of Grb2 and SOS at the framerate of 2 Hz. The assembly structure is marked by labeled LAT (red). Membrane-bound Grb2 (yellow) exhibits two kinetic populations: short dwell times (white arrow) and long dwell times (white tracks). The statistics are summarized in the dwell time histograms: Grb2 before assembly structure (black), Grb2 in protein assembly (purple), and SOS in protein assembly (blue). The bottom row shows dwell time histogram of Grb2 and SOS in protein assembly acquired at the framerate of 21 Hz. The fast kinetic species of Grb2 in protein assembly (purple) exhibits identical rate as the binding kinetics of pLAT:Grb2 before assembly (black). The fast kinetic species of SOS (blue) is also well described by a single kinetic population, with a fitted k_app,SOS of 3.4 s⁻¹. (B) Grb2 and SOS are recruited to the LAT assembly. Strong correlations are observed between epifluorescence images of LAT (Left) and the reconstructed images of Grb2 (Top Right) and SOS (Lower Right), obtained by compiling all single-molecule recruitment events within 400 s. (C) By mapping trajectories with apparent dwell time greater than 10 s onto the LAT patterns, it is evident that the long-dwelling species localize to the assembly structure.

Fig. S9.

LAT assembly leads to increase in Grb2 recruitment. By counting the recruitment events, the recruitment rate is obtained by a linear curve fitting (dashed line). In the absence of the LAT:Grb2:SOS assembly, the recruitment rate of Grb2 is 2,100 molecules/(s·μm²) (purple curve). The presence of protein assemblies leads to an increased recruitment rate of 10,000 molecule/(s·μm²) (blue curve). The recruitment events are estimated by mixing dilute fluorescent proteins and unlabeled proteins. In these experiments, the dilution factor is roughly 5 × 10⁻⁶. Both experiments contain the same Grb2 concentration and LAT density.

Fig. 5.

Mechanistic requirements for kinetic proofreading of SOS activation on membranes. (A) Kinetic model for SOS activation. Following membrane recruitment of SOS, conformational transitions are required to release auto-inhibition. The subscript for SOS denotes the kinetic intermediates, where N is the total number of kinetic intermediates preceding activation. k₁, k_2,…,k_N are the rate constants for the corresponding transitions of kinetic intermediates, and k_A is the activation rate constant. (B) The activation time distribution with different numbers of kinetic intermediates. The time dependence of activation for n > 0 result in the inequality of activation rates in C. (C) The rate of SOS activation as a function of the ratio between the mean dwell time and mean transition time. k_A = 1 s⁻¹ in this numerical example. Short dwelling species has a lower activation rate than long-dwelling species when n > 0, indicating that kinetic proofreading is in play.

Fig. 6.

Assembly-dependent membrane recruitments can achieve kinetic proofreading. Modulation of membrane dwell times can control the activation rate of cytosolic enzyme such as SOS. Kinetic proofreading of SOS ensures that receptor-dependent triggering events are distinguishable from spontaneous membrane localizations. This accuracy is especially important in TCR triggering, where detection of low agonist densities is crucial (the top right image shows that low density of TCR:pMHC complex can lead to activation). Regulation of the recruitment dynamics of biochemical networks through protein assembly thus provides a mechanism for controlling the amplification and noise filtration in signal transduction. Live cell image courtesy of Jenny J. Lin.