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

Huang, William; Yan, Qingrong; Lin, Wan‐Chen; Chung, Jean K.; Hansen, Scott D.; Christensen, Sune M.; Tu, Hsiung‐Lin; Kuriyan, John; Groves, Jay T.

doi:10.1073/pnas.1602602113

Cited by 116 publications

(186 citation statements)

References 49 publications

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“…Similar weak, multivalent interactions are prevalent among CME adaptors 45 . It has been proposed that such interactions facilitate enhanced recruitment of downstream adaptors and promote stability of the adaptor network through avidity 43,46,47 . To probe the requirement for multivalent interactions in cells, we replaced the coiled-coil domain of Eps15 with the Cry2 PHR domain, which provided light-activated control over assembly of the initiator network ( Figure 3).…”

Section: Importantly Binding Between Eps15 and Fcho1 Is Mediated By mentioning

confidence: 99%

Liquid-like protein interactions catalyze assembly of endocytic vesicles

Day

Kago

Wang

et al. 2019

Preprint

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During clathrin-mediated endocytosis, dozens of proteins assemble into an interconnected network at the plasma membrane. As early initiators of endocytosis, Eps15 and Fcho1 are responsible for locally concentrating downstream components on the membrane surface. However, they must also permit dynamic rearrangement of proteins within the budding vesicle. How do initiator proteins meet these competing demands? Here we show that Eps15 and Fcho1 rely on weak, liquid-like interactions to efficiently catalyze endocytosis. In reconstitution experiments, these weak interactions promote the assembly of protein droplets with liquid-like properties, including rapid coalescence and dynamic exchange of protein components. To probe the physiological role of liquid-like interactions among initiator proteins, we tuned the strength of initiator protein assembly in real time using light-inducible oligomerization of Eps15. Low light levels drove initiator proteins into liquid-like assemblies, restoring normal rates of endocytosis in mammalian Eps15 knockout cells. In contrast, initiator proteins formed solid-like assemblies upon exposure to higher light levels.Assembly of these structures stalled vesicle budding, likely owing to insufficient molecular rearrangement. These findings suggest that liquid-like assembly of early initiator proteins provides an optimal catalytic platform for endocytosis.Clathrin-mediated endocytosis (CME) is driven by the assembly of a highly interconnected network of adaptor proteins that participate in cargo selection, clathrin recruitment, and membrane bending. A small subset of adaptor proteins is the first to arrive at endocytic sites 1,2 . These initiators include the Fcho proteins (Fcho1/2), Eps15/Eps15R, and intersectin, which associate together in a complex thought to serve as a membrane-associated scaffold for recruitment of downstream adaptors 3 .Specifically, Fcho and Eps15 work together to recruit AP-2, which promotes maturation of the nascent endocytic structure 4-6 . Fcho also binds the adaptors HRB and Dab2 7,8 .Eps15 interacts with numerous other adaptors, including Epsin and AP180/CALM 9,10 .Meanwhile, the minimal initiator complex itself is highly interconnected. Fcho binds Eps15 through interactions between its C-terminal µHD and motifs in Eps15's intrinsically disordered C-terminus 6,11 . Fcho exists as a homodimer, mediated by its F-BAR domain 12 . Eps15 not only dimerizes via its coiled-coil domain, but can likely also take on an antiparallel tetrameric conformation mediated by interactions between its Nterminal EH domains and C-terminal disordered region 11,13 . Therefore, each Fcho dimer can interact with multiple Eps15 oligomers, suggesting an interconnected initiator protein network. Loss of both Fcho1 and Eps15 results in less productive CME 3,6 .As initiators of endocytosis, the network formed by Fcho and Eps15 must meet the competing demands of (i) locally concentrating downstream adaptors, while (ii) permitting them to rapidly rearrange as the endocytic structure grows and cha...

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Section: Importantly Binding Between Eps15 and Fcho1 Is Mediated By mentioning

confidence: 99%

Liquid-like protein interactions catalyze assembly of endocytic vesicles

Day

Kago

Wang

et al. 2019

Preprint

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“…In addition, the ability to gate noise can be important to LAT signaling because multiple mechanisms of signal amplification occur downstream of LAT phosphorylation. Other than the observed enhancement of on rates by LAT allostery, the dwell times of Grb2 and downstream proteins are extended by LAT:Grb2:SOS assemblies when LAT is substantially phosphorylated 14,15 . These events eventually lead to further downstream amplification steps such as positive feedback of SOS:Ras interactions 29,30 .…”

Section: Discussionmentioning

confidence: 87%

“…S2). The SH2 domain of Grb2 bound to pY residues on LAT with fast (~100 ms) off-rates and established a steady state during LAT phosphorylation 14 . A typical phosphorylation trace of Grb2 fluorescent intensity consisted of two phases: i) the increase in Grb2 fluorescent intensity reflecting LAT phosphorylation by membrane-associated ZAP-70 and, ii) the plateau of Grb2 fluorescent intensity reflecting the affinities between Grb2 and fully phosphorylated LAT(Fig.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2017

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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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“…This is a parameter that can change dramatically, and locally, in response to receptor signaling activity, for one example. Recent observations of two-dimensional phase separating protein condensates at the cell membrane (Banjade and Rosen, 2014;Su et al, 2016;Huang et al, 2016) present another mechanism likely to substantially alter the membrane mechanics. A general point, which is further underscored by this work, is that the membrane and the cortical cytoskeleton should be considered as a composite material; it may not be realistic to think of them as two separate structures.…”

Section: Previewsmentioning

confidence: 99%