Dissecting protein tyrosine phosphatase signaling by engineered chemogenetic control of its activity.

Protein tyrosine phosphatases (PTPases) are critical mediators of dynamic cell signaling. A tool capable of identifying transient signaling events downstream of PTPases is essential to understand phosphatase function on a physiological time scale. We report a broadly applicable protein engineering method for allosteric regulation of PTPases. This method enables dissection of transient events and reconstruction of individual signaling pathways. Implementation of this approach for Shp2 phosphatase revealed parallel MAPK and ROCK II dependent pathways downstream of Shp2, mediating transient cell spreading and migration. Furthermore, we show that the N-SH2 domain of Shp2 regulates MAPK-independent, ROCK II-dependent cell migration. Engineered targeting of Shp2 activity to different protein complexes revealed that Shp2-FAK signaling induces cell spreading whereas Shp2-Gab1 or Shp2-Gab2 mediates cell migration. We identified specific transient morphodynamic processes induced by Shp2 and determined the role of individual signaling pathways downstream of Shp2 in regulating these events. Broad application of this approach is demonstrated by regulating PTP1B and PTP-PEST phosphatases.

Engineered Allosteric Regulation of Protein Function.

Allosteric regulation of proteins has been utilized to study various aspects of cell signaling, from unicellular events to organism-wide phenotypes. However, traditional methods of allosteric regulation, such as constitutively active mutants and inhibitors, lack tight spatiotemporal control. This often leads to unintended signaling consequences that interfere with data interpretation. To overcome these obstacles, researchers employed protein engineering approaches that enable tight control of protein function through allosteric mechanisms. These methods provide high specificity as well as spatial and temporal precision in regulation of protein activity in vitro and in vivo. In this review, we focus on the recent advancements in engineered allosteric regulation and discuss the various bioengineered allosteric techniques available now, from chimeric GPCRs to chemogenetic and optogenetic switches. We highlight the benefits and pitfalls of each of these techniques as well as areas in which future improvements can be made. Additionally, we provide a brief discussion on implementation of engineered allosteric regulation approaches, demonstrating that these tools can shed light on elusive biological events and have the potential to be utilized in precision medicine.

Förster resonance energy transfer biosensors for fluorescence and time-gated luminescence analysis of rac1 activity.

Genetically encoded, Förster resonance energy transfer (FRET) biosensors enable live-cell optical imaging of signaling molecules. Small conformational changes often limit the dynamic range of biosensors that combine fluorescent proteins (FPs) and sensing domains into a single polypeptide. To address this, we developed FRET and lanthanide-based FRET (LRET) biosensors of Rac1 activation with two key features that enhance sensitivity and dynamic range. For one, alpha helical linker domains separate FRET partners and ensure a large conformational change and FRET increase when activated Rac1 at the biosensor C-terminus interacts with an amino-terminal Rac binding domain. Incorporation of a luminescent Tb(III) complex with long (~ ms) excited state lifetime as a LRET donor enabled time-gated luminescence measurements of Rac1 activity in cell lysates. The LRET dynamic range increased with ER/K linker length up to 1100% and enabled robust detection of Rac1 inhibition in 96-well plates. The ER/K linkers had a less pronounced, but still significant, effect on conventional FRET biosensors (with FP donors and acceptors), and we were able to dynamically image Rac1 activation at cell edges using fluorescence microscopy. The results herein highlight the potential of FRET and LRET biosensors with ER/K linkers for cell-based imaging and screening of protein activities.

PKCθ-mediated serine/threonine phosphorylations of FAK govern adhesion and protrusion dynamics within the lamellipodia of migrating breast cancer cells.

The cytoskeleton and cell-matrix adhesions constitute a dynamic network that controls cellular behavior during development and cancer. The Focal Adhesion Kinase (FAK) is a central actor of these cell dynamics, promoting cell-matrix adhesion turnover and active membrane fluctuations. However, the initial steps leading to FAK activation and subsequent promotion of cell dynamics remain elusive. Here, we report that the serine/threonine kinase PKCθ participates in the initial steps of FAK activation. PKCθ, which is strongly expressed in aggressive human breast cancers, controls the dynamics of cell-matrix adhesions and active protrusions through direct FAK activation, thereby promoting cell invasion and lung metastases. Using various tools for in vitro and live cell studies, we precisely decipher the molecular mechanisms of FAK activation. PKCθ directly interacts with the FAK FERM domain to open FAK conformation through PKCθ's specific V3 domain, while phosphorylating FAK at newly identified serine/threonine residues within nascent adhesions, inducing cell dynamics and aggressive behavior. This study thus places PKCθ-directed FAK opening and phosphorylations as an original mechanism controlling dynamic, migratory, and invasive abilities of aggressive breast cancer cells, further strengthening the emerging oncogenic function of PKCθ.

Methods for assessment of membrane protrusion dynamics.

Membrane protrusions are a critical facet of cell function. Mediating fundamental processes such as cell migration, cell-cell interactions, phagocytosis, as well as assessment and remodeling of the cell environment. Different protrusion types and morphologies can promote different cellular functions and occur downstream of distinct signaling pathways. As such, techniques to quantify and understand the inner workings of protrusion dynamics are critical for a comprehensive understanding of cell biology. In this chapter, we describe approaches to analyze cellular protrusions and correlate physical changes in cell morphology with biochemical signaling processes. We address methods to quantify and characterize protrusion types and velocity, mathematical approaches to predictive models of cytoskeletal changes, and implementation of protein engineering and biosensor design to dissect cell signaling driving protrusive activity. Combining these approaches allows cell biologists to develop a comprehensive understanding of the dynamics of membrane protrusions.

Tyrosine phosphorylation of S1PR1 leads to chaperone BiP-mediated import to the endoplasmic reticulum.

Cell surface G protein-coupled receptors (GPCRs), upon agonist binding, undergo serine-threonine phosphorylation, leading to either receptor recycling or degradation. Here, we show a new fate of GPCRs, exemplified by ER retention of sphingosine-1-phosphate receptor 1 (S1PR1). We show that S1P phosphorylates S1PR1 on tyrosine residue Y143, which is associated with recruitment of activated BiP from the ER into the cytosol. BiP then interacts with endocytosed Y143-S1PR1 and delivers it into the ER. In contrast to WT-S1PR1, which is recycled and stabilizes the endothelial barrier, phosphomimicking S1PR1 (Y143D-S1PR1) is retained by BiP in the ER and increases cytosolic Ca2+ and disrupts barrier function. Intriguingly, a proinflammatory, but non-GPCR agonist, TNF-α, also triggered barrier-disruptive signaling by promoting S1PR1 phosphorylation on Y143 and its import into ER via BiP. BiP depletion restored Y143D-S1PR1 expression on the endothelial cell surface and rescued canonical receptor functions. Findings identify Y143-phosphorylated S1PR1 as a potential target for prevention of endothelial barrier breakdown under inflammatory conditions.

Optogenetics: The Art of Illuminating Complex Signaling Pathways.

Dissection of cell signaling requires tools that can mimic spatiotemporal dynamics of individual pathways in living cells. Optogenetic methods enable manipulation of signaling processes with precise timing and local control. In this review, we describe recent optogenetic approaches for regulation of cell signaling, highlight their advantages and limitations, and discuss examples of their application.

Light-regulated allosteric switch enables temporal and subcellular control of enzyme activity.

Engineered allosteric regulation of protein activity provides significant advantages for the development of robust and broadly applicable tools. However, the application of allosteric switches in optogenetics has been scarce and suffers from critical limitations. Here, we report an optogenetic approach that utilizes an engineered Light-Regulated (LightR) allosteric switch module to achieve tight spatiotemporal control of enzymatic activity. Using the tyrosine kinase Src as a model, we demonstrate efficient regulation of the kinase and identify temporally distinct signaling responses ranging from seconds to minutes. LightR-Src off-kinetics can be tuned by modulating the LightR photoconversion cycle. A fast cycling variant enables the stimulation of transient pulses and local regulation of activity in a selected region of a cell. The design of the LightR module ensures broad applicability of the tool, as we demonstrate by achieving light-mediated regulation of Abl and bRaf kinases as well as Cre recombinase.

Cells need to sense and respond to their environment. To do this, they have dedicated proteins that interpret outside signals and convert them into appropriate responses that are only active at a specific time and location within the cell. However, in many diseases, including cancer, these signaling proteins are switched on for too long or are active in the wrong place. To better understand why this is the case, researchers manipulate proteins to identify the processes they regulate. One way to do this is to engineer proteins so that they can be controlled by light, turning them either on or off. Ideally, a light-controlled tool can activate proteins at defined times, control proteins in specific locations within the cell and regulate any protein of interest. However, current methods do not combine all of these requirements in one tool, and scientists often have to use different methods, depending on the topic they are researching. Now, Shaaya et al. set out to develop a single tool that combines all required features. The researchers engineered a light-sensitive 'switch' that allowed them to activate a specific protein by illuminating it with blue light and to deactivate it by turning the light off. Unlike other methods, the new tool uses a light-sensitive switch that works like a clamp. In the dark, the clamp is open, which 'stretches' and distorts the protein, rendering it inactive. In light, however, the clamp closes and the structure of the protein and its activity are restored. Moreover, it can activate proteins multiple times, control proteins in specific locations within the cell and it can be applied to a variety of proteins. This specific design makes it possible to combine multiple features in one tool that will both simplify and broaden its use to investigate specific proteins and signaling pathways in a broad range of diseases.

Noncanonical function of long myosin light chain kinase in increasing ER-PM junctions and augmentation of SOCE.

Increased endothelial permeability leads to excessive exudation of plasma proteins and leukocytes in the interstitium, which characterizes several vascular diseases including acute lung injury. The myosin light chain kinase long (MYLK-L) isoform is canonically known to regulate the endothelial permeability by phosphorylating myosin light chain (MLC-P). Compared to the short MYLK isoform, MYLK-L contains an additional stretch of ~919 amino acid at the N-terminus of unknown function. We show that thapsigargin and thrombin-induced SOCE was markedly reduced in Mylk-L-/- endothelial cells (EC) or MYLK-L-depleted human EC. These agonists also failed to increase endothelial permeability in MYLK-L-depleted EC and Mylk-L-/- lungs, thus demonstrating the novel role of MYLK-L-induced SOCE in increasing vascular permeability. MYLK-L augmented SOCE by increasing endoplasmic reticulum (ER)-plasma membrane (PM) junctions and STIM1 translocation to these junctions. Transduction of N-MYLK domain (amino acids 1-919 devoid of catalytic activity) into Mylk-L-/- EC rescued SOCE to the level seen in control EC in a STIM1-dependent manner. N-MYLK-induced SOCE augmented endothelial permeability without MLC-P via an actin-binding motif, DVRGLL. Liposomal-mediated delivery of N-MYLK mutant but not ∆DVRGLL-N-MYLK mutant in Mylk-L-/- mice rescued vascular permeability increase in response to endotoxin, indicating that targeting of DVRGLL motif within MYLK-L may limit SOCE-induced vascular hyperpermeability.

Septin2 mediates podosome maturation and endothelial cell invasion associated with angiogenesis.

Podosomes are compartmentalized actin-rich adhesions, defined by their ability to locally secrete proteases and remodel extracellular matrix. Matrix remodeling by endothelial podosomes facilitates invasion and thereby vessel formation. However, the mechanisms underlying endothelial podosome formation and function remain unclear. Here, we demonstrate that Septin2, Septin6, and Septin7 are required for maturation of nascent endothelial podosomes into matrix-degrading organelles. We show that podosome development occurs through initial mobilization of the scaffolding protein Tks5 and F-actin accumulation, followed by later recruitment of Septin2. Septin2 localizes around the perimeter of podosomes in close proximity to the basolateral plasma membrane, and phosphoinositide-binding residues of Septin2 are required for podosome function. Combined, our results suggest that the septin cytoskeleton forms a diffusive barrier around nascent podosomes to promote their maturation. Finally, we show that Septin2-mediated regulation of podosomes is critical for endothelial cell invasion associated with angiogenesis. Therefore, targeting of Septin2-mediated podosome formation is a potentially attractive anti-angiogenesis strategy.

Time-Variant SRC Kinase Activation Determines Endothelial Permeability Response.

In the current model of endothelial barrier regulation, the tyrosine kinase SRC is purported to induce disassembly of endothelial adherens junctions (AJs) via phosphorylation of VE cadherin, and thereby increase junctional permeability. Here, using a chemical biology approach to temporally control SRC activation, we show that SRC exerts distinct time-variant effects on the endothelial barrier. We discovered that the immediate effect of SRC activation was to transiently enhance endothelial barrier function as the result of accumulation of VE cadherin at AJs and formation of morphologically distinct reticular AJs. Endothelial barrier enhancement via SRC required phosphorylation of VE cadherin at Y731. In contrast, prolonged SRC activation induced VE cadherin phosphorylation at Y685, resulting in increased endothelial permeability. Thus, time-variant SRC activation differentially phosphorylates VE cadherin and shapes AJs to fine-tune endothelial barrier function. Our work demonstrates important advantages of synthetic biology tools in dissecting complex signaling systems.

Piezo1 mediates angiogenesis through activation of MT1-MMP signaling.

Angiogenesis is initiated in response to a variety of external cues, including mechanical and biochemical stimuli; however, the underlying signaling mechanisms remain unclear. Here, we investigated the proangiogenic role of the endothelial mechanosensor Piezo1. Genetic deletion and pharmacological inhibition of Piezo1 reduced endothelial sprouting and lumen formation induced by wall shear stress and proangiogenic mediator sphingosine 1-phosphate, whereas Piezo1 activation by selective Piezo1 activator Yoda1 enhanced sprouting angiogenesis. Similarly to wall shear stress, sphingosine 1-phosphate functioned by activating the Ca2+ gating function of Piezo1, which in turn signaled the activation of the matrix metalloproteinase-2 and membrane type 1 matrix metalloproteinase during sprouting angiogenesis. Studies in mice in which Piezo1 was conditionally deleted in endothelial cells demonstrated the requisite role of sphingosine 1-phosphate-dependent activation of Piezo1 in mediating angiogenesis in vivo. These results taken together suggest that both mechanical and biochemical stimuli trigger Piezo1-mediated Ca2+ influx and thereby activate matrix metalloproteinase-2 and membrane type 1 matrix metalloproteinase and synergistically facilitate sprouting angiogenesis.

Sphingolipids Signaling in Lamellipodia Formation and Enhancement of Endothelial Barrier Function.

Sphingolipids, first described in the brain in 1884, are important structural components of biological membranes of all eukaryotic cells. In recent years, several lines of evidence support the critical role of sphingolipids such as sphingosine, sphingosine-1-phosphate (S1P), and ceramide as anti- or pro-inflammatory bioactive lipid mediators in a variety of human pathologies including pulmonary and vascular disorders. Among the sphingolipids, S1P is a naturally occurring agonist that exhibits potent barrier enhancing property in the endothelium by signaling via G protein-coupled S1P1 receptor. S1P, S1P analogs, and other barrier enhancing agents such as HGF, oxidized phospholipids, and statins also utilize the S1P/S1P1 signaling pathway to generate membrane protrusions or lamellipodia, which have been implicated in resealing of endothelial gaps and maintenance of barrier integrity. A better understanding of sphingolipids mediated regulation of lamellipodia formation and barrier enhancement of the endothelium will be critical for the development of sphingolipid-based therapies to alleviate pulmonary disorders such as sepsis-, radiation-, and mechanical ventilation-induced acute lung injury.

Inhibition of Clostridium perfringens epsilon toxin by ß-cyclodextrin derivatives.

Clostridium perfringens epsilon toxin (ETX) is considered as one of the most dangerous potential biological weapons. The goal of this work was to identify inhibitors of ETX using a novel approach for the inactivation of pore-forming toxins. The approach is based on the blocking of the target pore with molecules having the same symmetry as the pore itself. About 200 various ß-cyclodextrin derivatives were screened for inhibitors of ETX activity using a colorimetric cell viability assay. Several compounds with dose-dependent activities at low micromolar concentrations have been identified. The same compounds were also able to inhibit lethal toxin of Bacillus anthracis.

Dissecting Kinase Effector Signaling Using the RapRTAP Methodology.

Kinases are involved in a broad spectrum of cell behaviors. A single kinase can interact with different ligands each eliciting a specific cellular response. Dissecting downstream signaling pathways of kinases is a key step to understanding physiological and pathological cell process. However, directing kinase activity to specific substrates remains challenging. Here, we present a new tool to selectively activate a kinase in a specific protein complex in living cells. This technology uses a rapamycin-inducible kinase activation coupled to interaction with FKBP12-binding domain (FRB) tagged protein. Here, we demonstrate application of this method by targeting Src to either p130Cas or FAK and discriminating cell mophodynamic changes downstream each of these signaling complexes.

Engineering Pak1 Allosteric Switches.

P21-activated kinases (PAKs) are important regulators of cell motility and morphology. It has been challenging to interrogate their functions because cells adapt to genetic manipulation of PAK, and because inhibitors act on multiple PAK isoforms. Here we describe genetically encoded PAK1 analogues that can be selectively activated by the membrane-permeable small molecule rapamycin. An engineered domain inserted away from the active site responds to rapamycin to allosterically control activity of the PAK1 isoform. To examine the mechanism of rapamycin-induced PAK1 activation, we used molecular dynamics with graph theory to predict amino acids involved in allosteric communication with the active site. This analysis revealed allosteric pathways that were exploited to generate kinase switches. Activation of PAK1 resulted in transient cell spreading in metastatic breast cancer cells, and long-term dendritic spine enlargement in mouse hippocampal CA1 neurons.

Mimicking transient activation of protein kinases in living cells.

Physiological stimuli activate protein kinases for finite periods of time, which is critical for specific biological outcomes. Mimicking this transient biological activity of kinases is challenging due to the limitations of existing methods. Here, we report a strategy enabling transient kinase activation in living cells. Using two protein-engineering approaches, we achieve independent control of kinase activation and inactivation. We show successful regulation of tyrosine kinase c-Src (Src) and Ser/Thr kinase p38α (p38), demonstrating broad applicability of the method. By activating Src for finite periods of time, we reveal how the duration of kinase activation affects secondary morphological changes that follow transient Src activation. This approach highlights distinct roles for sequential Src-Rac1- and Src-PI3K-signaling pathways at different stages during transient Src activation. Finally, we demonstrate that this method enables transient activation of Src and p38 in a specific signaling complex, providing a tool for targeted regulation of individual signaling pathways.

Src-dependent phosphorylation of caveolin-1 Tyr-14 promotes swelling and release of caveolae.

Caveolin 1 (Cav1) is a required structural component of caveolae, and its phosphorylation by Src is associated with an increase in caveolae-mediated endocytosis. Here we demonstrate, using quantitative live-cell 4D, TIRF, and FRET imaging, that endocytosis and trafficking of caveolae are associated with a Cav1 Tyr-14 phosphorylation-dependent conformational change, which spatially separates, or loosens, Cav1 molecules within the oligomeric caveolar coat. When tracked by TIRF and spinning-disk microscopy, cells expressing phosphomimicking Cav1 (Y14D) mutant formed vesicles that were greater in number and volume than with Y14F-Cav1-GFP. Furthermore, we observed in HEK cells cotransfected with wild-type, Y14D, or Y14F Cav1-CFP and -YFP constructs that FRET efficiency was greater with Y14F pairs than with Y14D, indicating that pY14-Cav1 regulates the spatial organization of Cav1 molecules within the oligomer. In addition, albumin-induced Src activation or direct activation of Src using a rapamycin-inducible Src construct (RapR-Src) led to an increase in monomeric Cav1 in Western blots, as well as a simultaneous increase in vesicle number and decrease in FRET intensity, indicative of a Src-mediated conformational change in CFP/YFP-tagged WT-Cav1 pairs. We conclude that phosphorylation of Cav1 leads to separation or "spreading" of neighboring negatively charged N-terminal phosphotyrosine residues, promoting swelling of caveolae, followed by their release from the plasma membrane.

Characterization of an Engineered Src Kinase to Study Src Signaling and Biology.

Pharmacologic inhibitors of protein kinases comprise the vast majority of approved signal transduction inhibitors for cancer treatment. An important facet of their clinical development is the identification of the key substrates critical for their driver role in cancer. One approach for substrate identification involves evaluating the phosphorylation events associated with stable expression of an activated protein kinase. Another involves genetic or pharmacologic inhibition of protein kinase expression or activity. However, both approaches are limited by the dynamic nature of signaling, complicating whether phosphorylation changes are primary or secondary activities of kinase function. We have developed rapamycin-regulated (RapR) protein kinases as molecular tools that allow for the study of spatiotemporal regulation of signaling. Here we describe the application of this technology to the Src tyrosine kinase and oncoprotein (RapR-Src). We describe how to achieve stable expression of this tool in cell lines and how to subsequently activate the tool and determine its function in signaling and morphology.

The interaction of Gα13 with integrin ß1 mediates cell migration by dynamic regulation of RhoA.

Heterotrimeric G protein Gα13 is known to transmit G protein-coupled receptor (GPCR) signals leading to activation of RhoA and plays a role in cell migration. The mechanism underlying the role of Gα13 in cell migration, however, remains unclear. Recently we found that Gα13 interacts with the cytoplasmic domain of integrin ß3 subunits in platelets via a conserved ExE motif. Here we show that a similar direct interaction between Gα13 and the cytoplasmic domain of the integrin ß1 subunit plays a critical role in ß1-dependent cell migration. Point mutation of either glutamic acid in the Gα13-binding (767)EKE motif in ß1 or treatment with a peptide derived from the Gα13-binding sequence of ß1 abolished Gα13-ß1 interaction and inhibited ß1 integrin-dependent cell spreading and migration. We further show that the Gα13-ß1 interaction mediates ß1 integrin-dependent Src activation and transient RhoA inhibition during initial cell adhesion, which is in contrast to the role of Gα13 in mediating GPCR-dependent RhoA activation. These data indicate that Gα13 plays dynamic roles in both stimulating RhoA via a GPCR pathway and inhibiting RhoA via an integrin signaling pathway. This dynamic regulation of RhoA activity is critical for cell migration on ß1 integrin ligands.