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Phosphorylation is a ubiquitous mechanism by which signals are transduced in cells. Protein kinases, enzymes that catalyze the phosphotransfer reaction are, themselves, often regulated by phosphorylation. Paradoxically, however, a substantial fraction of more than 500 human protein kinases are capable of catalyzing their own activation loop phosphorylation. Commonly, these kinases perform this autophosphorylation reaction in trans, whereby transient dimerization leads to the mutual phosphorylation of the activation loop of the opposing protomer. In this study, we demonstrate that protein kinase D (PKD) is regulated by the inverse mechanism of dimerization-mediated trans-autoinhibition, followed by activation loop autophosphorylation in cis. We show that PKD forms a stable face-to-face homodimer that is incapable of either autophosphorylation or substrate phosphorylation. Dissociation of this trans-autoinhibited dimer results in activation loop autophosphorylation, which occurs exclusively in cis. Phosphorylation serves to increase PKD activity and prevent trans-autoinhibition, thereby switching PKD on. Our findings not only reveal the mechanism of PKD regulation but also have profound implications for the regulation of many other eukaryotic kinases.

In this article the calcium/calmodulin-dependent protein kinases are reviewed. The primary focus is on the structure and function of this diverse family of enzymes, and the elegant regulation of their activity. Structures are compared in order to highlight the conserved architecture of their catalytic domains with respect to each other as well as protein kinase A, a prototype for kinase structure. In addition to reviewing structure and function in these enzymes, the variety of biological processes for which they play a mediating role are also examined. Finally, how the enzymes become activated in the intracellular setting is considered by exploring the reciprocal interactions that exist between calcium binding to calmodulin when interacting with the CaM-kinases.

CaMKII is a remarkably complex protein kinase, known to have a fundamental role in synaptic plasticity and memory formation. Further, CaMKII has also been suggested to be a tau kinase. CaMKII dysregulation may therefore be a modulator of toxicity in Alzheimer’s disease, a dementia characterised by aberrant calcium signalling, synapse and neuronal loss, and impaired memory. Here, we first examine the evidence for CaMKII dysregulation in Alzheimer’s patients and draw parallels to findings in disease models which recapitulate key aspects of the disease. We then put forward the hypothesis that these changes critically contribute to neurodegeneration and memory impairment in Alzheimer’s disease.

In this article the calcium/calmodulin-dependent protein kinases are reviewed. The primary focus is on the structure and function of this diverse family of enzymes, and the elegant regulation of their activity. Structures are compared in order to highlight the conserved architecture of their catalytic domains with respect to each other as well as protein kinase A, a prototype for kinase structure. In addition to reviewing structure and function in these enzymes, the variety of biological processes for which they play a mediating role are also examined. Finally, how the enzymes become activated in the intracellular setting is considered by exploring the reciprocal interactions that exist between calcium binding to calmodulin when interacting with the CaM-kinases.

Cytosolic hybrid histidine kinases (HHKs) constitute major signaling nodes that control various biological processes, but their input signals and how these are processed are largely unknown. In Caulobacter crescentus, the HHK ShkA is essential for accurate timing of the G1-S cell cycle transition and is regulated by the corresponding increase in the level of the second messenger c-di-GMP. Here, we use a combination of X-ray crystallography, NMR spectroscopy, functional analyses, and kinetic modeling to reveal the regulatory mechanism of ShkA. In the absence of c-di-GMP, ShkA predominantly adopts a compact domain arrangement that is catalytically inactive. C-di-GMP binds to the dedicated pseudoreceiver domain Rec1, thereby liberating the canonical Rec2 domain from its central position where it obstructs the large-scale motions required for catalysis. Thus, c-di-GMP cannot only stabilize domain interactions, but also engage in domain dissociation to allosterically invoke a downstream effect. Enzyme kinetics data are consistent with conformational selection of the ensemble of active domain constellations by the ligand and show that autophosphorylation is a reversible process.

Many CMGC group kinases require phosphorylation of a conserved tyrosine residue in the activation loop to achieve catalytic activity. DYRK family members use a distinctive mechanism involving constitutive cis -autophosphorylation of this tyrosine. The structural basis of this process has remained unclear, as it occurs while the kinase is still in an inactive conformation, and the tyrosine does not match the known substrate consensus of DYRKs. Here, we exploited the different autophosphorylation capacities of the paralogs DYRK1A and DYRK1B to define structural determinants of this process. DYRK1A efficiently autophosphorylates even in cell-free systems, whereas DYRK1B does not. Using domain swaps and point mutations, we identify the CMGC insert in the C-terminal lobe and two adjacent proline residues (P332/P333 in DYRK1B) as critical for proper folding and activation. Mutation of either proline impaired DYRK1B autophosphorylation and nuclear localization but had no effect in DYRK1A. Substitution of the DYRK1B CMGC insert with that of DYRK1A rescued the maturation defect, demonstrating functional interplay between the insert and flanking prolines. Furthermore, the pathogenic R349W mutation in DYRK1B, associated with monogenic obesity and type 2 diabetes, also disrupted autophosphorylation. These findings highlight the role of the CMGC insert and adjacent prolines in DYRK kinase maturation and autoactivation.

Two-component signal transduction systems (TCSs) are widely conserved in bacteria to respond to and adapt to the changing environment. Since TCSs are also involved in controlling the expression of virulence, biofilm formation, quorum sensing, and antimicrobial resistance in pathogens, they serve as candidates for novel drug targets. TCSs consist of a sensor histidine kinase (HK) and its cognate response regulator (RR). Upon perception of a signal, HKs autophosphorylate their conserved histidine residues, followed by phosphotransfer to their partner RRs. The phosphorylated RRs mostly function as transcriptional regulators and control the expression of genes necessary for stress response. HKs sense their specific signals not only in their extracytoplasmic sensor domain but also in their cytoplasmic and transmembrane domains. The signals are sensed either directly or indirectly via cofactors and accessory proteins. Accumulating evidence shows that a single HK can sense and respond to multiple signals in different domains. The underlying molecular mechanisms of how HK activity is controlled by these signals have been extensively studied both biochemically and structurally. In this article, we introduce the wide diversity of signal perception in different domains of HKs, together with their recently clarified structures and molecular mechanisms.

Extensive research over several decades in plant light signaling mediated by photoreceptors has identified the molecular mechanisms for how phytochromes regulate photomorphogenic development, which includes degradation of phytochrome-interacting factors (PIFs) and inactivation of COP1-SPA complexes with the accumulation of master transcription factors for photomorphogenesis, such as HY5. However, the initial biochemical mechanism for the function of phytochromes has not been fully elucidated. Plant phytochromes have long been known as phosphoproteins, and a few protein phosphatases that directly interact with and dephosphorylate phytochromes have been identified. However, there is no report thus far of a protein kinase that acts on phytochromes. On the other hand, plant phytochromes have been suggested as autophosphorylating serine/threonine protein kinases, proposing that the kinase activity might be important for their functions. Indeed, the autophosphorylation of phytochromes has been reported to play an important role in the regulation of plant light signaling. More recently, evidence that phytochromes function as protein kinases in plant light signaling has been provided using phytochrome mutants displaying reduced kinase activities. In this review, we highlight recent advances in the reversible phosphorylation of phytochromes and their functions as protein kinases in plant light signaling.

The many variants of human Ca2+/calmodulin-dependent protein kinase II (CaMKII) differ in the lengths and sequences of disordered linkers connecting the kinase domains to the oligomeric hubs of the holoenzyme. CaMKII activity depends on the balance between activating and inhibitory autophosphorylation (on Thr 286 and Thr 305/306, respectively, in the human α isoform). Variation in the linkers could alter transphosphorylation rates within a holoenzyme and the balance of autophosphorylation outcomes. We show, using mammalian cell expression and a single-molecule assay, that the balance of autophosphorylation is flipped between CaMKII variants with longer and shorter linkers. For the principal isoforms in the brain, CaMKII-α, with a ~30 residue linker, readily acquires activating autophosphorylation, while CaMKII-β, with a ~200 residue linker, is biased towards inhibitory autophosphorylation. Our results show how the responsiveness of CaMKII holoenzymes to calcium signals can be tuned by varying the relative levels of isoforms with long and short linkers.

Ire1 (Ern1) is an unusual transmembrane protein kinase essential for the endoplasmic reticulum (ER) unfolded protein response (UPR). Activation of Ire1 by association of its N‐terminal ER luminal domains promotes autophosphorylation by its cytoplasmic kinase domain, leading to activation of the C‐terminal ribonuclease domain, which splices Xbp1 mRNA generating an active Xbp1s transcriptional activator. We have determined the crystal structure of the cytoplasmic portion of dephosphorylated human Ire1α bound to ADP, revealing the ‘phosphoryl‐transfer’ competent dimeric face‐to‐face complex, which precedes and is distinct from the back‐to‐back RNase ‘active’ conformation described for yeast Ire1. We show that the Xbp1‐specific ribonuclease activity depends on autophosphorylation, and that ATP‐competitive inhibitors staurosporin and sunitinib, which inhibit autophosphorylation in vitro , also inhibit Xbp1 splicing in vivo . Furthermore, we demonstrate that activated Ire1α is a competent protein kinase, able to phosphorylate a heterologous peptide substrate. These studies identify human Ire1α as a target for development of ATP‐competitive inhibitors that will modulate the UPR in human cells, which has particular relevance for myeloma and other secretory malignancies. In the endoplasmic reticulum, unfolded proteins stimulate Ire1 autophosphorylation and RNase activity. The crystal structure of the dephosphorylated kinase/RNase domain of human Ire1 bound to ADP provides insight into the autophosphorylation reaction.