Recent advances in the use of genetically encodable optical tools to elicit and monitor signaling events

Cells rely on a complex network of spatiotemporally regulated signaling activities to effectively transduce information from extracellular cues to intracellular machinery. To probe this activity architecture, researchers have developed an extensive molecular tool kit of fluorescent biosensors and optogenetic actuators capable of monitoring and manipulating various signaling activities with high spatiotemporal precision. The goal of this review is to provide readers with an overview of basic concepts and recent advances in the development and application of genetically encodable biosensors and optogenetic tools for understanding signaling activity.     

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using Förster resonance energy transfer (FRET)^1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.     

Protein kinase sensors: an overview of new designs for visualizing kinase dynamics in single plant cells

Protein kinase dynamics play key roles in regulation of cell differentiation, growth, development and in diverse cell signaling networks. Protein kinase sensors enable visualization of protein kinase activity in living cells and tissues in time and space. These sensors have therefore become important and powerful molecular tools for investigation of diverse kinase activities and can resolve long-standing and challenging biological questions. In the present Update, we review new advanced approaches for genetically encoded protein kinase biosensor designs developed in animal systems together with the basis of each biosensor’s working principle and components. In addition, we review recent first examples of real time plant protein kinase activity biosensor development and application. We discuss how these sensors have helped to resolve how stomatal signal transduction in response to elevated CO₂ merges with abscisic acid signaling downstream of a resolved basal SnRK2 kinase activity in guard cells. Furthermore, recent advances, combined with the new strategies described in this Update, can help deepen the understanding of how signaling networks regulate unique functions and responses in distinct plant cell types and tissues and how different stimuli and signaling pathways can interact.

New genetically encoded biosensor design approaches and visualization of protein kinase activity in living plant cells and tissues in time and space are reviewed.     

Structural basis for the autoinhibition of focal adhesion kinase

Appropriate tyrosine kinase signaling depends on coordinated sequential coupling of protein-protein interactions with catalytic activation. Focal adhesion kinase (FAK) integrates signals from integrin and growth factor receptors to regulate cellular responses including cell adhesion, migration, and survival. Here, we describe crystal structures representing both autoinhibited and active states of FAK. The inactive structure reveals a mechanism of inhibition in which the N-terminal FERM domain directly binds the kinase domain, blocking access to the catalytic cleft and protecting the FAK activation loop from Src phosphorylation. Additionally, the FERM domain sequesters the Tyr397 autophosphorylation and Src recruitment site, which lies in the linker connecting the FERM and kinase domains. The active phosphorylated FAK kinase adopts a conformation that is immune to FERM inhibition. Our biochemical and structural analysis shows how the architecture of autoinhibited FAK orchestrates an activation sequence of FERM domain displacement, linker autophosphorylation, Src recruitment, and full catalytic activation.     

Intermolecular Autophosphorylation Regulates Myosin IIIa Activity and Localization in Parallel Actin Bundles

Myosin IIIa (Myo3A) transports cargo to the distal end of actin protrusions and contains a kinase domain that is thought to autoregulate its activity. Because Myo3A tends to cluster at the tips of actin protrusions, we investigated whether intermolecular phosphorylation could regulate Myo3A biochemical activity, cellular localization, and cellular function. Inactivation of Myo3A 2IQ kinase domain with the point mutation K50R did not alter maximal ATPase activity, whereas phosphorylation of Myo3A 2IQ resulted in reduced maximal ATPase activity and actin affinity. The rate and degree of Myo3A 2IQ autophosphorylation was unchanged by the presence of actin but was found to be dependent upon Myo3A 2IQ concentration within the range of 0.1 to 1.2 μm, indicating intermolecular autophosphorylation. In cultured cells, we observed that the filopodial tip localization of Myo3A lacking the kinase domain decreased when co-expressed with kinase-active, full-length Myo3A. The cellular consequence of reduced Myo3A tip localization was decreased filopodial density along the cell periphery, identifying a novel cellular function for Myo3A in mediating the formation and stability of actin-based protrusions. Our results suggest that Myo3A motor activity is regulated through a mechanism involving concentration-dependent autophosphorylation. We suggest that this regulatory mechanism plays an essential role in mediating the transport and actin bundle formation/stability functions of Myo3A.     

A role for protein tyrosine kinase activity in natural cytotoxicity as well as antibody-dependent cellular cytotoxicity. Effects of herbimycin A.

NK cells, CD3- large granular lymphocytes, have diverse means by which they lyse targets, including antibody-dependent cellular cytotoxicity. The low affinity receptor for the Fc portion of Ig (Fc gamma RIIIA), like the TCR, is a multimeric receptor complex coupled to a protein tyrosine kinase. In the present study, we observed that inhibition of tyrosine kinase activity by herbimycin A interferes with receptor-mediated phosphorylation of a variety of substrates and mobilization of intracellular calcium. Fc gamma RIIIA induced IL-2R alpha-chain expression was also extremely sensitive to herbimycin A as was antibody-dependent cellular cytotoxicity, in fact more so than receptor-mediated phosphorylation and calcium mobilization. In contrast to Fc gamma RIIIA, the surface molecules and biochemical mechanisms involved in NK cytotoxicity and lymphokine-activated killing are not well characterized. Interestingly, however, herbimycin A also blocks these modes of cytolysis, implicating a role for tyrosine kinase function in these processes. Whether FcR-mediated signaling and receptor-mediated signaling involved in NK activity share specific biochemical intermediates is not known, but the involvement of tyrosine kinase function in the latter means of cytotoxicity may provide novel avenues for understanding the biochemical basis of this perplexing cellular function.     

Optogenetic reporters

The discovery of naturally evolved fluorescent proteins and their subsequent tuning by protein engineering provided the basis for a large family of genetically encoded biosensors that report a variety of physicochemical processes occurring in living tissue. These optogenetic reporters are powerful tools for live‐cell microscopy and quantitative analysis at the subcellular level. In this review, we present an overview of the transduction mechanisms that have been exploited for engineering these genetically encoded reporters. Finally, we discuss current and future efforts towards the combined use of various optogenetic actuators and reporters for simultaneously controlling and imaging the physiology of cells and tissues.     

MEKK3 directly regulates MEK5 activity as part of the big mitogen-activated protein kinase 1 (BMK1) signaling pathway

Big mitogen-activated protein (MAP) kinase (BMK1), also known as ERK5, is a member of the MAP kinase family whose cellular activity is elevated in response to growth factors, oxidative stress, and hyperosmolar conditions. Previous studies have identified MEK5 as a cellular kinase directly regulating BMK1 activity; however, signaling molecules that directly regulate MEK5 activity have not yet been defined. Through utilization of a yeast two-hybrid screen, we have identified MEKK3 as a molecule that physically interacts with MEK5. This interaction appears to take place in mammalian cells as evidenced by the fact that cellular MEK5 and MEKK3 co-immunoprecipitate. In addition, we show that a dominant active form of MEKK3 stimulates BMK1 activity through MEK5. Moreover, we demonstrate that MEKK3 activity is required for growth factor mediated cellular activation of endogenous BMK1. Taken together, these results identify MEKK3 as a kinase that regulates the activity of MEK5 and BMK1 during growth factor-induced cellular stimulation.     

AMPK-dependent phosphorylation of ULK1 regulates ATG9 localization

Autophagy is activated in response to a variety of cellular stresses including metabolic stress. While elegant genetic studies in yeast have identified the core autophagy machinery, the signaling pathways that regulate this process are less understood. AMPK is an energy sensing kinase and several studies have suggested that AMPK is required for autophagy. The biochemical connections between AMPK and autophagy, however, have not been elucidated. In this report, we identify a biochemical connection between a critical regulator of autophagy, ULK1, and the energy sensing kinase, AMPK. ULK1 forms a complex with AMPK, and AMPK activation results in ULK1 phosphorylation. Moreover, we demonstrate that the immediate effect of AMPK-dependent phosphorylation of ULK1 results in enhanced binding of the adaptor protein YWHAZ/14-3-3ζ; and this binding alters ULK1 phosphorylation in vitro. Finally, we provide evidence that both AMPK and ULK1 regulate localization of a critical component of the phagophore, ATG9, and that some of the AMPK phosphorylation sites on ULK1 are important for regulating ATG9 localization. Taken together these data identify an ULK1-AMPK signaling cassette involved in regulation of the autophagy machinery.     

AMPK: An odyssey of a metabolic regulator,a tumor suppressor,and now a contextual oncogene

Metabolic reprogramming is a unique but complex biochemical adaptation that allows solid tumors to tolerate various stresses that challenge cancer cells for survival. Under conditions of metabolic stress, mammalian cells employ adenosine monophosphate (AMP)-activated protein kinase (AMPK) to regulate energy homeostasis by controlling cellular metabolism. AMPK has been described as a cellular energy sensor that communicates with various metabolic pathways and networks to maintain energy balance. Earlier studies characterized AMPK as a tumor suppressor in the context of cancer. Later, a paradigm shift occurred in support of the oncogenic nature of AMPK, considering it a contextual oncogene. In support of this, various cellular and mouse models of tumorigenesis and clinicopathological studies demonstrated increased AMPK activity in various cancers. This review will describe AMPK's pro-tumorigenic activity in various malignancies and explain the rationale and context for using AMPK inhibitors in combination with anti-metabolite drugs to treat AMPK-driven cancers.     

Illuminating the kinome: Visualizing real-time kinase activity in biological systems using genetically encoded fluorescent protein-based biosensors

Genetically encoded fluorescent protein-based kinase biosensors are a central tool for illumination of the kinome. The adaptability and versatility of biosensors have allowed for spatiotemporal observation of real-time kinase activity in living cells and organisms. In this review, we highlight various types of kinase biosensors, along with their burgeoning applications in complex biological systems. Specifically, we focus on kinase activity reporters used in neuronal systems and whole animal settings. Genetically encoded kinase biosensors are key for elucidation of the spatiotemporal regulation of protein kinases, with broader applications beyond the Petri dish.     

Imaging voltage and brain chemistry with genetically encoded sensors and modulators

Neurons and glia are functionally organized into circuits and higher-order structures that allow the precise information processing required for complex behaviors. To better understand the structure and function of the brain, we must understand synaptic connectivity, action potential generation and propagation, as well as well-orchestrated molecular signaling. Recently, dramatically improved sensors for voltage, intracellular calcium, and neurotransmitters/modulators, combined with advanced microscopy provide new opportunities for in vivo dissection of cellular and circuit activity in awake, behaving animals. This review focuses on the current trends in genetically encoded sensors for molecules and cellular events and their potential applicability to the study of nervous system in health and disease.     

The Rho GEFs LARG and GEF-H1 regulate the mechanical response to force on integrins

How individual cells respond to mechanical forces is of considerable interest to biologists as force affects many aspects of cell behaviour. The application of force on integrins triggers cytoskeletal rearrangements and growth of the associated adhesion complex, resulting in increased cellular stiffness, also known as reinforcement. Although RhoA has been shown to play a role during reinforcement, the molecular mechanisms that regulate its activity are unknown. By combining biochemical and biophysical approaches, we identified two guanine nucleotide exchange factors (GEFs), LARG and GEF-H1, as key molecules that regulate the cellular adaptation to force. We show that stimulation of integrins with tensional force triggers activation of these two GEFs and their recruitment to adhesion complexes. Surprisingly, activation of LARG and GEF-H1 involves distinct signalling pathways. Our results reveal that LARG is activated by the Src family tyrosine kinase Fyn, whereas GEF-H1 catalytic activity is enhanced by ERK downstream of a signalling cascade that includes FAK and Ras.     

Web-based applications for building, managing and analysing kinetic models of biological systems

Mathematical modelling and computational analysis play an essentialrole in improving our capability to elucidate the functionsand characteristics of complex biological systems such as metabolic,regulatory and cell signalling pathways. The modelling and concomitantsimulation render it possible to predict the cellular behaviourof systems under various genetically and/or environmentallyperturbed conditions. This motivates systems biologists/bioengineers/bioinformaticiansto develop new tools and applications, allowing non-expertsto easily conduct such modelling and analysis. However, amonga multitude of systems biology tools developed to date, onlya handful of projects have adopted a web-based approach to kineticmodelling. In this report, we evaluate the capabilities andcharacteristics of current web-based tools in systems biologyand identify desirable features, limitations and bottlenecksfor further improvements in terms of usability and functionality.A short discussion on software architecture issues involvedin web-based applications and the approaches taken by existingtools is included for those interested in developing their ownsimulation applications.      

Regulation of cellular functions by the ERK5 signalling pathway

Extracellular-regulated protein kinase 5 (ERK5) is a mitogen-activated protein kinase (MAPK) regulated by a wide range of mitogens and cellular stresses. Since its cloning in 1995, the lack of biological tools, including antibodies and specific inhibitors, have made it one of the least studied MAPK subfamilies. The discovery that ERK5 was an important contributor to cell survival mechanisms has increased interest in this signalling pathway. The ability of inhibitors of the classical MAPK (ERK1/2) cascade to block ERK5 activation suggested that ERK5 might regulate some cellular functions originally attributed to ERK1/2. For example, ERK5 is suspected to mediate the effects of numerous oncogenes. A link between abnormal levels of ERK5 expression and cancers was established by the analysis of human tumours. Recently, the targeted deletions of the erk5 and the mek5 genes in mice have provided genetic evidence that the ERK5 cascade is a non-redundant signalling pathway essential for normal cardiovascular development. The analysis of genetically modified mice in which the erk5 gene can be specifically deleted in certain tissues is shedding light into the physiological function of the ERK5 pathway during development and pathogenesis.     

Drosophila sticky/citron kinase is a regulator of cell-cycle progression, genetically interacts with Argonaute 1 and modulates epigenetic gene silencing

The sticky/citron kinase protein is a conserved regulator of cell-cycle progression from invertebrates to humans. While this kinase is essential for completion of cytokinesis, sticky/citron kinase phenotypes disrupting neurogenesis and cell differentiation suggest additional non-cell-cycle functions. However, it is not known whether these phenotypes are an indirect consequence of sticky mutant cell-cycle defects or whether they define a novel function for this kinase. We have isolated a temperature-sensitive allele of the Drosophila sticky gene and we show that sticky/citron kinase is required for histone H3-K9 methylation, HP1 localization, and heterochromatin-mediated gene silencing. sticky genetically interacts with Argonaute 1 and sticky mutants exhibit context-dependent Su(var) and E(var) activity. These observations indicate that sticky/citron kinase functions to regulate both actin-myosin-mediated cytokinesis and epigenetic gene silencing, possibly linking cell-cycle progression to heterochromatin assembly and inheritance of gene expression states.