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Positive feedback between the T cell kinase Zap70 and its substrate LAT acts as a clustering-dependent signaling switch.

ABSTRACT: Protein clustering is pervasive in cell signaling, yet how signaling from higher-order assemblies differs from simpler forms of molecular organization is still poorly understood. We present an optogenetic approach to switch between oligomers and heterodimers with a single point mutation. We apply this system to study signaling from the kinase Zap70 and its substrate linker for activation of T cells (LAT), proteins that normally form membrane-localized condensates during T cell activation. We find that fibroblasts expressing synthetic Zap70:LAT clusters activate downstream signaling, whereas one-to-one heterodimers do not. We provide evidence that clusters harbor a positive feedback loop among Zap70, LAT, and Src-family kinases that binds phosphorylated LAT and further activates Zap70. Finally, we extend our optogenetic approach to the native T cell signaling context, where light-induced LAT clustering is sufficient to drive a calcium response. Our study reveals a specific signaling function for protein clusters and identifies a biochemical circuit that robustly senses protein oligomerization state.

SUBMITTER: Dine E

PROVIDER: S-EPMC8292983 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Lck promotes Zap70-dependent LAT phosphorylation by bridging Zap70 to LAT.

Project description:T cell-antigen receptor (TCR) signaling requires the sequential activities of the kinases Lck and Zap70. Upon TCR stimulation, Lck phosphorylates the TCR, thus leading to the recruitment, phosphorylation, and activation of Zap70. Lck binds and stabilizes phosho-Zap70 by using its SH2 domain, and Zap70 phosphorylates the critical adaptors LAT and SLP76, which coordinate downstream signaling. It is unclear whether phosphorylation of these adaptors occurs through passive diffusion or active recruitment. We report the discovery of a conserved proline-rich motif in LAT that mediates efficient LAT phosphorylation. Lck associates with this motif via its SH3 domain, and with phospho-Zap70 via its SH2 domain, thereby acting as a molecular bridge that facilitates the colocalization of Zap70 and LAT. Elimination of this proline-rich motif compromises TCR signaling and T cell development. These results demonstrate the remarkable multifunctionality of Lck, wherein each of its domains has evolved to orchestrate a distinct step in TCR signaling.

| S-EPMC6202249 | biostudies-literature

Lck promotes Zap70-dependent LAT phosphorylation by bridging Zap70 to LAT

Project description:T cell antigen receptor (TCR) signaling depends upon the kinases Lck and Zap70. Lck phosphorylates the TCR, facilitating Zap70 recruitment to the stimulated TCR. Lck also phosphorylates Zap70, relieving its auto-inhibition and activating its catalytic domain. Zap70 then phosphorylates the critical adaptors LAT and SLP76 which serve to nucleate key effector molecules required for downstream responses. However, mechanisms facilitating the interaction of Zap70 with its substrates have not been described. We report an evolutionarily conserved proline-rich motif in LAT is important for Zap70-induced phosphorylation of LAT and downstream signaling. This LAT proline-rich motif associated with the Lck SH3 domain, thereby facilitating Zap70-mediated phosphorylation of LAT and downstream functions. Our results suggest Lck orchestrates multiple steps in TCR signaling including the newly described facilitation of the interaction of Zap70 with its substrate LAT. This previously unrecognized feature of TCR proximal signaling may contribute to the development of more immunomodulatory therapies.

2019-11-12 | PXD008258 | Pride

mDia1/3-dependent actin polymerization spatiotemporally controls LAT phosphorylation by Zap70 at the immune synapse.

Project description:The mechanism by which the cytosolic protein Zap70 physically interacts with and phosphorylates its substrate, the transmembrane protein LAT, upon T cell receptor (TCR) stimulation remains largely obscure. In this study, we found that the pharmacological inhibition of formins, a major class of actin nucleators, suppressed LAT phosphorylation by Zap70, despite TCR stimulation-dependent phosphorylation of Zap70 remaining intact. High-resolution imaging and three-dimensional image reconstruction revealed that localization of phosphorylated Zap70 to the immune synapse (IS) and subsequent LAT phosphorylation are critically dependent on formin-mediated actin polymerization. Using knockout mice, we identify mDia1 and mDia3, which are highly expressed in T cells and which localize to the IS upon TCR activation, as the critical formins mediating this process. Our findings therefore describe previously unsuspected roles for mDia1 and mDia3 in the spatiotemporal control of Zap70-dependent LAT phosphorylation at the IS through regulation of filamentous actin, and underscore their physiological importance in TCR signaling.

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Substrate rigidity-dependent positive feedback regulation between YAP and ROCK2.

Project description:Extracellular matrix (ECM) stiffness influences gene expression, leading to modulation of various cellular functions. While ROCK2 regulates actomyosin activity as well as cell migration and proliferation, expression of ROCK2 is increased in response to stiffening ECM. However, the mechanism underlying rigidity-dependent ROCK2 expression remains elusive. Here, we show that YAP, a mechanically regulated transcription coactivator, upregulates ROCK2 expression in an ECM rigidity-dependent manner. YAP interacted with the ROCK2 promoter region in an actomyosin activity-dependent manner. Knockdown of YAP decreased ROCK2 expression while activity of the ROCK2 promoter was upregulated by expressing constitutively active YAP. Furthermore, we found that ROCK2 expression promotes transcriptional activation by YAP. Our results reveal a novel positive feedback loop between YAP and ROCK2, which is modulated by ECM stiffness.

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Positive feedback sharpens the anaphase switch.

Project description:At the onset of anaphase, sister-chromatid cohesion is dissolved abruptly and irreversibly, ensuring that all chromosome pairs disjoin almost simultaneously. The regulatory mechanisms that generate this switch-like behaviour are unclear. Anaphase is initiated when a ubiquitin ligase, the anaphase-promoting complex (APC), triggers the destruction of securin, thereby allowing separase, a protease, to disrupt sister-chromatid cohesion. Here we demonstrate that the cyclin-dependent kinase 1 (Cdk1)-dependent phosphorylation of securin near its destruction-box motif inhibits securin ubiquitination by the APC. The phosphatase Cdc14 reverses securin phosphorylation, thereby increasing the rate of securin ubiquitination. Because separase is known to activate Cdc14 (refs 5 and 6), our results support the existence of a positive feedback loop that increases the abruptness of anaphase. Consistent with this model, we show that mutations that disrupt securin phosphoregulation decrease the synchrony of chromosome segregation. Our results also suggest that coupling securin degradation with changes in Cdk1 and Cdc14 activities helps coordinate the initiation of sister-chromatid separation with changes in spindle dynamics.

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Project description:The ability of activation-induced cytidine deaminase (AID) to efficiently mediate class-switch recombination (CSR) is dependent on its phosphorylation at Ser38; however, the trigger that induces AID phosphorylation and the mechanism by which phosphorylated AID drives CSR have not been elucidated. Here we found that phosphorylation of AID at Ser38 was induced by DNA breaks. Conversely, in the absence of AID phosphorylation, DNA breaks were not efficiently generated at switch (S) regions in the immunoglobulin heavy-chain locus (Igh), consistent with a failure of AID to interact with the endonuclease APE1. Additionally, deficiency in the DNA-damage sensor ATM impaired the phosphorylation of AID at Ser38 and the interaction of AID with APE1. Our results identify a positive feedback loop for the amplification of DNA breaks at S regions through the phosphorylation- and ATM-dependent interaction of AID with APE1.

| S-EPMC4005274 | biostudies-literature

Hunger states switch a flip-flop memory circuit via a synaptic AMPK-dependent positive feedback loop.

Project description:Synaptic plasticity in response to changes in physiologic state is coordinated by hormonal signals across multiple neuronal cell types. Here, we combine cell-type-specific electrophysiological, pharmacological, and optogenetic techniques to dissect neural circuits and molecular pathways controlling synaptic plasticity onto AGRP neurons, a population that regulates feeding. We find that food deprivation elevates excitatory synaptic input, which is mediated by a presynaptic positive feedback loop involving AMP-activated protein kinase. Potentiation of glutamate release was triggered by the orexigenic hormone ghrelin and exhibited hysteresis, persisting for hours after ghrelin removal. Persistent activity was reversed by the anorexigenic hormone leptin, and optogenetic photostimulation demonstrated involvement of opioid release from POMC neurons. Based on these experiments, we propose a memory storage device for physiological state constructed from bistable synapses that are flipped between two sustained activity states by transient exposure to hormones signaling energy levels.

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Tyrosine phosphorylation acts as a molecular switch to full-scale activation of the eIF2alpha RNA-dependent protein kinase.

Project description:Phosphorylation of the alpha-subunit of translation eukaryotic initiation factor-2 (eIF2) leads to the inhibition of protein synthesis in response to diverse conditions of stress. Serine/threonine RNA-dependent protein kinase (PKR) is an eIF2alpha kinase family member induced by type I IFN and activated in response to dsRNA or virus infection. Herein, we demonstrate that human PKR is a dual specificity kinase phosphorylated at Y101, Y162 and Y293 in vitro and in vivo. Site-specific tyrosine phosphorylation is essential for efficient dsRNA-binding, dimerization, kinase activation and eIF2alpha phosphorylation of PKR. Biologically, tyrosine phosphorylation of PKR mediates the antiviral and antiproliferative properties of the kinase through its ability to control translation. Our data demonstrate an important role of tyrosine phosphorylation in biochemical and biological processes caused or mediated by the activation of the eIF2alpha kinase PKR.

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CCR7 Is Recruited to the Immunological Synapse, Acts as Co-stimulatory Molecule and Drives LFA-1 Clustering for Efficient T Cell Adhesion Through ZAP70.

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Project description:T cells expressing tumor-specific T cell receptors are promising cancer therapeutic agents, but safety control switches are needed to manage potential side effects arising from overactivity. Here, we present the first dual small molecule-gated ZAP70 signaling switch for the regulation of T cell activity. We show that when an analogue-sensitive ZAP70 allele is fused to the engineered ligand binding domain of the estrogen receptor, ERT2, its activity can be upregulated to an extent by a metabolite of an FDA-approved tamoxifen, 4-hydroxy-tamoxifen, and downregulated by an ATP analogue, 3-MB-PP1. The strength of early T cell signaling can also be modulated by varying the concentrations of activator and inhibitor, and the switch exhibits temporal control on the time scale of minutes. Interestingly, the switch has the ability to control CD69 and calcium levels in T cells but has limited capabilities in the regulation of downstream cytokine release, suggesting further investigation is needed before it can be implemented in adoptive T cell therapy.

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