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The articles in this special issue highlight how modern cellular, biochemical, biophysical and computational techniques are allowing deeper and more detailed studies of allosteric kinase regulation.

The production of heterologous proteins is an important procedure for biologists in basic and applied sciences. A variety of cell-based and cell-free protein expression systems are available to achieve this. The expression system must be selected carefully, especially for target proteins that require post-translational modifications. In this study, human Src family kinases were prepared using six different protein expression systems: 293 human embryonic kidney cells, Escherichia coli, and cell-free expression systems derived from rabbit reticulocytes, wheat germ, insect cells, or Escherichia coli . The phosphorylation status of each kinase was analyzed by Phos-tag SDS-PAGE. The kinase activities were also investigated. In the eukaryotic systems, multiple phosphorylated forms of the expressed kinases were observed. In the rabbit reticulocyte lysate system and 293 cells, differences in phosphorylation status between the wild-type and kinase-dead mutants were observed. Whether the expressed kinase was active depended on the properties of both the kinase and each expression system. In the prokaryotic systems, Src and Hck were expressed in autophosphorylated active forms. Clear differences in post-translational phosphorylation among the protein expression systems were revealed. These results provide useful information for preparing functional proteins regulated by phosphorylation.

Focal adhesion kinase (FAK) relays integrin signaling from outside to inside cells and contributes to cell adhesion and motility. However, the spatiotemporal dynamics of FAK activity in single FAs is unclear due to the lack of a robust FAK reporter, which limits our understanding of these essential biological processes. Here we have engineered a genetically encoded FAK activity sensor, dubbed FAK-separation of phases-based activity reporter of kinase (SPARK), which visualizes endogenous FAK activity in living cells and vertebrates. Our work reveals temporal dynamics of FAK activity during FA turnover. Most importantly, our study unveils polarized FAK activity at the distal tip of newly formed single FAs in the leading edge of a migrating cell. By combining FAK-SPARK with DNA tension probes, we show that tensions applied to FAs precede FAK activation and that FAK activity is proportional to the strength of tension. These results suggest tension-induced polarized FAK activity in single FAs, advancing the mechanistic understanding of cell migration.

HIPK2 is a highly conserved, constitutively active Ser/Thr protein kinase that is involved in a broad spectrum of biological processes. We have previously reported that the expression of HIPK2 is auto-regulated by a mechanism that depends on the activity of its kinase domain, leading to decreased expression of kinase-dead versus wild-type HIPK2. We have now explored this mechanism in more detail. Differential expression of wild-type and kinase-dead HIPK2 is dependent on sequences located in the C-terminal part of HIPK2, but is only observed when this part of HIPK2 is translated together with the defective kinase domain. On their own, both the defective kinase domain and the C-terminal amino acid sequences are expressed at normal levels and independently of kinase activity. Insertion of a 2A-ribosomal skipping sequence into the HIPK2 coding sequence revealed that the differential expression of wild-type and kinase-dead HIPK2 is caused by degradation of nascent kinase-dead HIPK2. Because HIPK2 is constitutively active and auto-activates its kinase domain already during its translation we speculate that the regulatory mechanism discovered here serves as a quality control mechanism that leads to degradation of nascent kinase molecules with defective kinase domains. Overall our work provides insight into a novel auto-regulatory mechanism of HIPK2 expression, thereby adding a new layer of control to the regulation of HIPK2., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Determining cellular activities of protein kinases is a fundamental step for characterizing pathophysiological cell signaling pathways. Here, we optimized a nonradioactive method that detects protein kinases in tissues or cells after separation by SDS-PAGE and transfer onto polyvinylidene fluoride membranes. The method, kinase activity-tagged western blotting (KAT-WB), consists of five steps: electrophoresis of cell extracts that contain protein kinases, electroblotting proteins onto polyvinylidene fluoride membrane, denaturation-renaturation, phosphorylation, with or without an added substrate protein and immunodetection using anti-phospho-specific antibodies. KAT-WB detected autophosphorylation of one Tyr-kinase and site-specific phosphorylation of added substrate by multiple kinases. KAT-WB assay enables us to interrogate multiple kinase signaling pathways without using radioactive ATP.

The serine/threonine protein kinase Akt integrates diverse upstream inputs to regulate cell survival, growth, metabolism, migration, and differentiation. Mounting evidence suggests that Akt activity is differentially regulated depending on its subcellular location, which can include the plasma membrane, endomembrane, and nuclear compartment. This spatial control of Akt activity is critical for achieving signaling specificity and proper physiological functions, and deregulation of compartment-specific Akt signaling is implicated in various diseases, including cancer and diabetes. Understanding the spatial coordination of the signaling network centered around this key kinase and the underlying regulatory mechanisms requires precise tracking of Akt activity at distinct subcellular compartments within its native biological contexts. To address this challenge, new molecular tools are being developed, enabling us to directly interrogate the spatiotemporal regulation of Akt in living cells. These include, for instance, the newly developed genetically encodable fluorescent-protein-based Akt kinase activity reporter (AktAR2), which serves as a substrate surrogate of Akt kinase and translates Akt-specific phosphorylation into a quantifiable change in Förster resonance energy transfer (FRET). In addition, we developed the Akt substrate tandem occupancy peptide sponge (Akt-STOPS), which allows biochemical perturbation of subcellular Akt activity. Both molecular tools can be readily targeted to distinct subcellular localizations. Here, we describe a workflow to study Akt kinase activity at different subcellular locations in living cells. We provide a protocol for using genetically targeted AktAR2 and Akt-STOPS, along with fluorescence imaging in living NIH3T3 cells, to visualize and perturb, respectively, the activity of endogenous Akt kinase at different subcellular compartments. We further describe a protocol for using chemically inducible dimerization (CID) to control the plasma membrane-specific inhibition of Akt activity in real time. Lastly, we describe a protocol for maintaining NIH3T3 cells in culture, a cell line known to exhibit robust Akt activity. In all, this approach enables interrogation of spatiotemporal regulation and functions of Akt, as well as the intricate signaling networks in which it is embedded, at specific subcellular locations. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Visualizing and perturbing subcellular Akt kinase activity using AktAR and Akt-STOPS Basic Protocol 2: Using chemically inducible dimerization (CID) to control inhibition of Akt at the plasma membrane Support Protocol: Maintaining NIH3T3 cells in culture.

AMP-activated protein kinase (AMPK) is a master regulator of cellular energetics which coordinates metabolism by phosphorylating a plethora of substrates throughout the cell. But how AMPK activity is regulated at different subcellular locations for precise spatiotemporal control over metabolism is unclear. Here we present a sensitive, single-fluorophore AMPK activity reporter (ExRai AMPKAR), which reveals distinct kinetic profiles of AMPK activity at the mitochondria, lysosome, and cytoplasm. Genetic deletion of the canonical upstream kinase liver kinase B1 (LKB1) results in slower AMPK activity at lysosomes but does not affect the response amplitude at lysosomes or mitochondria, in sharp contrast to the necessity of LKB1 for maximal cytoplasmic AMPK activity. We further identify a mechanism for AMPK activity in the nucleus, which results from cytoplasmic to nuclear shuttling of AMPK. Thus, ExRai AMPKAR enables illumination of the complex subcellular regulation of AMPK signaling.

Neuroblastoma is the most common extracranial solid tumor in childhood. Gain of chromosome 17q material is found in >60% of neuroblastoma tumors and is associated with poor patient prognosis. The NME1 gene is located in the 17q21.3 region, and high NME1 expression is correlated with poor neuroblastoma patient outcomes. However, the functional roles and signaling activity of NME1 in neuroblastoma cells and tumors are unknown. NME1 and NME2 have been shown to possess histidine (His) kinase activity. Using anti-1- and 3-pHis specific monoclonal antibodies and polyclonal anti-pH118 NME1/2 antibodies, we demonstrated the presence of pH118-NME1/2 and multiple additional pHis-containing proteins in all tested neuroblastoma cell lines and in xenograft neuroblastoma tumors, supporting the presence of histidine kinase activity in neuroblastoma cells and demonstrating the potential significance of histidine kinase signaling in neuroblastoma pathogenesis. We have also demonstrated associations between NME1 expression and neuroblastoma cell migration and differentiation. Our demonstration of NME1 histidine phosphorylation in neuroblastoma and of the potential role of NME1 in neuroblastoma cell migration and differentiation suggest a functional role for NME1 in neuroblastoma pathogenesis and open the possibility of identifying new therapeutic targets and developing novel approaches to neuroblastoma therapy.

Phosphorylation reactions performed by protein kinases are one of the most studied post-translational modifications within cells. Much is understood about conserved residues within protein kinase domains that perform catalysis of the phosphotransfer reaction, yet the identity of the target substrates and downstream biological effects vary widely among cells, tissues, and organisms. Here, we characterize key residues essential for NUAK kinase activity in Drosophila melanogaster myogenesis and homeostasis. Creation of a NUAK kinase-dead mutation using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 results in lethality at the embryo to larval transition, while loss of NUAK catalytic function later in development produces aggregation of the chaperone protein αB-crystallin/CryAB in muscle tissue. Yeast 2-hybrid assays demonstrate a physical interaction between NUAK and CryAB. We further show that a phosphomimetic version of NUAK promotes the phosphorylation of CryAB and this post-translational modification occurs at 2 previously unidentified phosphosites that are conserved in the primary sequence of human CryAB. Mutation of these serine residues in D. melanogaster NUAK abolishes CryAB phosphorylation, thus, proving their necessity at the biochemical level. These studies together highlight the importance of kinase activity regulation and provide a platform to further explore muscle tissue proteostasis. [ABSTRACT FROM AUTHOR]

In the active HER receptor dimers, kinases play distinct roles; one is the catalytically active kinase and the other is its allosteric activator. This specialization enables signaling by the catalytically inactive HER3, which functions exclusively as an allosteric activator upon heterodimerization with other HER receptors. It is unclear whether the allosteric activation mechanism evolved before HER receptors functionally specialized. We determined the crystal structure of the kinase domain of the only EGF receptor in Caenorhabditis elegans, LET-23. Our structure of a non-human EGFR kinase reveals autoinhibitory features conserved in the human counterpart. Strikingly, mutations within the putative allosteric dimer interface abrogate activity of the isolated LET-23 kinase and of the full-length receptor despite these regions being only partially conserved with human EGFR. Our results indicate that ancestral EGFRs have built-in features that poise them for allosteric activation that could facilitate emergence of the catalytically dead, yet functional, orthologs.

The function of the amyloid precursor protein (APP) in brain health remains unclear. This study elucidated a novel cytoprotective signaling pathway initiated by the APP transcriptionally active intracellular domain (AICD) in response to 27-hydroxycholesterol (27OHC), an oxidized cholesterol metabolite associated with neurodegeneration. The cellular response to 27OHC was hormetic, such that low, but not high, doses promoted AICD transactivation of microtubule associated serine/threonine kinase family member 4 (MAST4). MAST4 in turn phosphorylated and inhibited FOXO1-dependent transcriptional repression of rhotekin 2 (RTKN2), an oxysterol stress responder, to optimize cell survival. A palmitate-rich diet, which increases serum 27OHC, or APP ablation, abrogated this response in vivo. Further, this pathway was downregulated in human Alzheimer's Disease (AD) brains but not in frontotemporal dementia brains. These results unveil MAST4 as functional kinase of FOXO1 in a 27OHC AICD-driven, hormetic pathway providing insight for therapeutic approaches against cholesterol associated neuronal disorders.

Extensive research has been conducted for decades to elucidate the molecular and regulatory mechanisms for phytochrome-mediated light signaling in plants. As a result, tens of downstream signaling components that physically interact with phytochromes are identified, among which negative transcription factors for photomorphogenesis, PHYTOCHROME-INTERACTING FACTORs (PIFs), are well known to be regulated by phytochromes. In addition, phytochromes are also shown to inactivate an important E3 ligase complex consisting of CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) and SUPPRESSORs OF phyA-105 (SPAs). This inactivation induces the accumulation of positive transcription factors for plant photomorphogenesis, such as ELONGATED HYPOCOTYL 5 (HY5). Although many downstream components of phytochrome signaling have been studied thus far, it is not fully elucidated which intrinsic activity of phytochromes is necessary for the regulation of these components. It should be noted that phytochromes are autophosphorylating protein kinases. Recently, the protein kinase activity of phytochrome A (phyA) has shown to be important for its function in plant light signaling using Avena sativa phyA mutants with reduced or increased kinase activity. In this review, we highlight the function of phyA as a protein kinase to explain the regulation of plant photoresponses by phyA.

Constitutive activation of the non-receptor tyrosine kinase c-Abl (cellular Abelson tyrosine protein kinase 1, Abl1) in the Bcr (breakpoint cluster region)-Abl1 fusion oncoprotein is the molecular cause of chronic myeloid leukaemia (CML). Recent studies have indicated that an interaction between the SH2 (Src-homology 2) domain and the N-lobe (N-terminal lobe) of the c-Abl kinase domain (KD) has a critical role in leukaemogenesis [Grebien et al. (2011) Cell 147, 306-319; Sherbenou et al. (2010) Blood 116, 3278-3285]. To dissect the structural basis of this phenomenon, we studied c-Abl constructs comprising the SH2 and KDs in vitro. We present a crystal structure of an SH2-KD construct bound to dasatinib, which contains the relevant interface between the SH2 domain and the N-lobe of the KD. We show that the presence of the SH2 domain enhances kinase activity moderately and that this effect depends on contacts in the SH2/N-lobe interface and is abrogated by specific mutations. Consistently, formation of the interface decreases slightly the association rate of imatinib with the KD. That the effects are small compared with the dramatic in vivo consequences suggests an important function of the SH2-N-lobe interaction might be to help disassemble the auto-inhibited conformation of c-Abl and promote processive phosphorylation, rather than substantially stimulate kinase activity.

Plant phytochromes are known as autophosphorylating serine/threonine protein kinases. However, the functional importance of their kinase activity is not fully elucidated. Previously, the kinase activity is shown to be necessary for the function of Avena sativa phytochrome A (AsphyA) using transgenic plants with mutants displaying reduced kinase activity, such as K411L and T418D. In this study, we isolated and analyzed two AsphyA mutants, K411R and T418V, that showed increased kinase activity. Transgenic phyA-201 plants with these mutants showed hypersensitive responses to far-red (FR) light, such as shorter hypocotyls and more expanded cotyledons than those of control plant (i.e., transgenic phyA-201 plant with wild-type AsphyA). Contrary to the mutants with reduced kinase activity, these mutants accelerated FR-induced phosphorylation and subsequent degradation of phytochrome-interacting factor 3 (PIF3) in Arabidopsis. Moreover, elongated hypocotyl 5 (HY5), a critical positive regulator of photoresponses in plants, accumulated in higher amounts in the transgenic plants under FR light than in the control plant. In addition, PIF1 degradation was accelerated in the transgenic plants. Consequently, the transgenic plants exhibit higher germination frequencies than the control plant. Collectively, our results demonstrate that the AsphyA mutants with increased kinase activity are hyperactive in plants, supporting a positive relationship between the kinase activity of phytochromes and photoresponses in plants.

In Saccharomyces cerevisiae, the p21-activated kinase Cla4p regulates polarized morphogenesis and cytokinesis. However, it remains unknown how Cla4p kinase activity is regulated. After pheromone exposure, yeast cells temporally separate the mitotic and mating programs by sequestering Fus2p in the nucleus until cell cycle completion, after which Fus2p exits to facilitate cell fusion. Previously, we showed that sequestration is regulated by two opposing protein kinases, Cla4p and Fus3p. Phosphorylation of Fus2p-S67 by Cla4p promotes nuclear localization by both activating nuclear import and blocking export. During mating, phosphorylation of Fus2p-S85 and Fus2p-S100 by Fus3p promotes nuclear export and blocks import. Here, we find that Cla4p kinase activity is itself down-regulated during mating. Pheromone exposure causes Cla4p hyper-phosphorylation and reduced Fus2p-S67 phosphorylation, dependent on Fus3p. Multiple phosphorylation sites in Cla4p are mating- and/or Fus3p-specific. Of these, Cla4p-S186 phosphorylation reduced the kinase activity of Cla4p, in vitro. A phosphomimetic cla4-S186E mutation caused a strong reduction in Fus2p-S67 phosphorylation and nuclear localization, in vivo. More generally, a non-phosphorylatable mutation, cla4-S186A, caused failure to maintain pheromone arrest and delayed formation of the mating-specific septin morphology. Thus, as cells enter the mating pathway, Fus3p counteracts Cla4p kinase activity to allow proper mating differentiation.