Protein kinases have evolved in eukaryotes to be highly dynamic molecular switches that regulate a plethora of biological processes. Two motifs, a dynamic activation segment and a GHI helical ...subdomain, distinguish the eukaryotic protein kinases (EPKs) from the more primitive eukaryotic-like kinases. The EPKs are themselves highly regulated, typically by phosphorylation, and this allows them to be rapidly turned on and off. The EPKs have a novel hydrophobic architecture that is typically regulated by the dynamic assembly of two hydrophobic spines that is usually mediated by the phosphorylation of an activation loop phosphate. Cyclic AMP-dependent protein kinase (protein kinase A (PKA)) is used as a prototype to exemplify these features of the PKA superfamily. Specificity in PKA signalling is achieved in large part by packaging the enzyme as inactive tetrameric holoenzymes with regulatory subunits that then are localized to macromolecular complexes in close proximity to dedicated substrates by targeting scaffold proteins. In this way, the cell creates discrete foci that most likely represent the physiological environment for cyclic AMP-mediated signalling.
The Hippo tumor suppressor pathway plays an important role in tissue homeostasis that ensures development of functional organs at proper size. The YAP transcription coactivator is a major effector of ...the Hippo pathway and is phosphorylated and inactivated by the Hippo pathway kinases Lats1/2. It has recently been shown that YAP activity is regulated by G-protein-coupled receptor signaling. Here we demonstrate that cyclic adenosine monophosphate (cAMP), a second messenger downstream from Gαs-coupled receptors, acts through protein kinase A (PKA) and Rho GTPases to stimulate Lats kinases and YAP phosphorylation. We also show that inactivation of YAP is crucial for PKA-induced adipogenesis. In addition, PKA activation in Drosophila inhibits the expression of Yorki (Yki, a YAP ortholog) target genes involved in cell proliferation and death. Taken together, our study demonstrates that Hippo-YAP is a key signaling branch of cAMP and PKA and reveals new insight into mechanisms of PKA in regulating a broad range of cellular functions.
Allosteric regulation of proteins continues to be an engaging research topic for the scientific community. Models describing allosteric communication have evolved from focusing on conformation-based ...descriptors of protein structural changes to appreciating the role of internal protein dynamics as a mediator of allostery. Here, we explain a "violin model" for allostery as a contemporary method for approaching the Cooper-Dryden model based on redistribution of protein thermal fluctuations. Based on graph theory, the violin model makes use of community network analysis to functionally cluster correlated protein motions obtained from molecular dynamics simulations. This Review provides the theory and workflow of the methodology and explains the application of violin model to unravel the workings of protein kinase A.
Matrix stiffness potently regulates cellular behaviour in various biological contexts. In breast tumours, the presence of dense clusters of collagen fibrils indicates increased matrix stiffness and ...correlates with poor survival. It is unclear how mechanical inputs are transduced into transcriptional outputs to drive tumour progression. Here we report that TWIST1 is an essential mechanomediator that promotes epithelial-mesenchymal transition (EMT) in response to increasing matrix stiffness. High matrix stiffness promotes nuclear translocation of TWIST1 by releasing TWIST1 from its cytoplasmic binding partner G3BP2. Loss of G3BP2 leads to constitutive TWIST1 nuclear localization and synergizes with increasing matrix stiffness to induce EMT and promote tumour invasion and metastasis. In human breast tumours, collagen fibre alignment, a marker of increasing matrix stiffness, and reduced expression of G3BP2 together predict poor survival. Our findings reveal a TWIST1-G3BP2 mechanotransduction pathway that responds to biomechanical signals from the tumour microenvironment to drive EMT, invasion and metastasis.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SBMB, UILJ, UKNU, UL, UM, UPUK
OBJECTIVE:To evaluate the literature and identify important aspects of insulin therapy that facilitate safe and effective infusion therapy for a defined glycemic end point.
METHODS:Where available, ...the literature was evaluated using Grades of Recommendation, Assessment, Development, and Evaluation (GRADE) methodology to assess the impact of insulin infusions on outcome for general intensive care unit patients and those in specific subsets of neurologic injury, traumatic injury, and cardiovascular surgery. Elements that contribute to safe and effective insulin infusion therapy were determined through literature review and expert opinion. The majority of the literature supporting the use of insulin infusion therapy for critically ill patients lacks adequate strength to support more than weak recommendations, termed suggestions, such that the difference between desirable and undesirable effect of a given intervention is not always clear.
RECOMMENDATIONS:The article is focused on a suggested glycemic control end point such that a blood glucose ≥150 mg/dL triggers interventions to maintain blood glucose below that level and absolutely <180 mg/dL. There is a slight reduction in mortality with this treatment end point for general intensive care unit patients and reductions in morbidity for perioperative patients, postoperative cardiac surgery patients, post-traumatic injury patients, and neurologic injury patients. We suggest that the insulin regimen and monitoring system be designed to avoid and detect hypoglycemia (blood glucose ≤70 mg/dL) and to minimize glycemic variability.Important processes of care for insulin therapy include use of a reliable insulin infusion protocol, frequent blood glucose monitoring, and avoidance of finger-stick glucose testing through the use of arterial or venous glucose samples. The essential components of an insulin infusion system include use of a validated insulin titration program, availability of appropriate staffing resources, accurate monitoring technology, and standardized approaches to infusion preparation, provision of consistent carbohydrate calories and nutritional support, and dextrose replacement for hypoglycemia prevention and treatment. Quality improvement of glycemic management programs should include analysis of hypoglycemia rates, run charts of glucose values <150 and 180 mg/dL. The literature is inadequate to support recommendations regarding glycemic control in pediatric patients.
CONCLUSIONS:While the benefits of tight glycemic control have not been definitive, there are patients who will receive insulin infusion therapy, and the suggestions in this article provide the structure for safe and effective use of this therapy.
Structures of set of serine-threonine and tyrosine kinases were investigated by the recently developed bioinformatics tool Local Spatial Patterns (LSP) alignment. We report a set of conserved motifs ...comprised mostly of hydrophobic residues. These residues are scattered throughout the protein sequence and thus were not previously detected by traditional methods. These motifs traverse the conserved protein kinase core and play integrating and regulatory roles. They are anchored to the F-helix, which acts as an organizing "hub" providing precise positioning of the key catalytic and regulatory elements. Consideration of these discovered structures helps to explain previously inexplicable results.
Mitochondrial fission and fusion impact numerous cellular functions and neurons are particularly sensitive to perturbations in mitochondrial dynamics. Here we describe that male mice lacking the ...mitochondrial A-kinase anchoring protein 1 (AKAP1) exhibit increased sensitivity in the transient middle cerebral artery occlusion model of focal ischemia. At the ultrastructural level, AKAP1
mice have smaller mitochondria and increased contacts between mitochondria and the endoplasmic reticulum in the brain. Mechanistically, deletion of AKAP1 dysregulates complex II of the electron transport chain, increases superoxide production, and impairs Ca
homeostasis in neurons subjected to excitotoxic glutamate. Ca
deregulation in neurons lacking AKAP1 can be attributed to loss of inhibitory phosphorylation of the mitochondrial fission enzyme dynamin-related protein 1 (Drp1) at the protein kinase A (PKA) site Ser637. Our results indicate that inhibition of Drp1-dependent mitochondrial fission by the outer mitochondrial AKAP1/PKA complex protects neurons from ischemic stroke by maintaining respiratory chain activity, inhibiting superoxide production, and delaying Ca
deregulation. They also provide the first genetic evidence that Drp1 inhibition may be of therapeutic relevance for the treatment of stroke and neurodegeneration.
Previous work suggests that activation of dynamin-related protein 1 (Drp1) and mitochondrial fission contribute to ischemic injury in the brain. However, the specificity and efficacy of the pharmacological Drp1 inhibitor mdivi-1 that was used has now been discredited by several high-profile studies. Our report is timely and highly impactful because it provides the first evidence that genetic disinhibition of Drp1 via knock-out of the mitochondrial protein kinase A (PKA) scaffold AKAP1 exacerbates stroke injury in mice. Mechanistically, we show that electron transport deficiency, increased superoxide production, and Ca
overload result from genetic disinhibition of Drp1. In summary, our work settles current controversies regarding the role of mitochondrial fission in neuronal injury, provides mechanisms, and suggests that fission inhibitors hold promise as future therapeutic agents.
Leucine-rich repeat kinase 2 (LRRK2) is a large multidomain protein with both a Ras of complex (ROC) domain and a kinase domain (KD) and, therefore, exhibits both GTPase and kinase activities. Human ...genetics studies have linked LRRK2 as a major genetic contributor to familial and sporadic Parkinson’s disease (PD), a neurodegenerative movement disorder that inflicts millions worldwide. The C-terminal region of LRRK2 is a Trp-Asp-40 (WD40) domain with poorly defined biological functions but has been implicated in microtubule interaction. Here, we present the crystal structure of the WD40 domain of human LRRK2 at 2.6-Å resolution, which reveals a seven-bladed WD40 fold. The structure displays a dimeric assembly in the crystal, which we further confirm by measurements in solution. We find that structure-based and PD-associated disease mutations in the WD40 domain including the common G2385R polymorphism mainly compromise dimer formation. Assessment of full-length LRRK2 kinase activity by measuring phosphorylation of Rab10, a member of the family of Rab GTPases known to be important kinase substrates of LRRK2, shows enhancement of kinase activity by several dimerization-defective mutants including G2385R, although dimerization impairment does not always result in kinase activation. Furthermore, mapping of phylogenetically conserved residues onto the WD40 domain structure reveals surface patches that may be important for additional functions of LRRK2. Collectively, our analyses provide insights for understanding the structures and functions of LRRK2 and suggest the potential utility of LRRK2 kinase inhibitors in treating PD patients with WD40 domain mutations.
Dynamic allostery-based molecular workings of kinase Ahuja, Lalima G.; Aoto, Phillip C.; Kornev, Alexandr P. ...
Proceedings of the National Academy of Sciences - PNAS,
07/2019, Letnik:
116, Številka:
30
Journal Article
Recenzirano
Odprti dostop
A dense interplay between structure and dynamics underlies the working of proteins, especially enzymes. Protein kinases are molecular switches that are optimized for their regulation rather than ...catalytic turnover rates. Using long-simulations dynamic allostery analysis, this study describes an exploration of the dynamic kinase:peptide complex. We have used protein kinase A (PKA) as a model system as a generic prototype of the protein kinase superfamily of signaling enzymes. Our results explain the role of dynamic coupling of active-site residues that must work in coherence to provide for a successful activation or inhibition response from the kinase. Amino acid networks-based community analysis allows us to ponder the conformational entropy of the kinase:nucleotide:peptide ternary complex. We use a combination of 7 peptides that include 3 types of PKA-binding partners: Substrates, products, and inhibitors. The substrate peptides provide for dynamic insights into the enzyme:substrate complex, while the product phospho-peptide allows for accessing modes of enzyme: product release. Mapping of allosteric communities onto the PKA structure allows us to locate the more unvarying and flexible dynamic regions of the kinase. These distributions, when correlated with the structural elements of the kinase core, allow for a detailed exploration of key dynamics-based signatures that could affect peptide recognition and binding at the kinase active site. These studies provide a unique dynamic allostery-based perspective to kinase:peptide complexes that have previously been explored only in a structural or thermodynamic context.
Fatalska et al.1 use an interdisciplinary strategy to elucidate how an intrinsically disordered regulatory subunit of protein phosphatase 1 binds trimeric eIF2 and positions the phosphatase-substrate ...complex for dephosphorylation. As validation, they show that a disease mutation abolishes the interaction.
Fatalska et al. use an interdisciplinary strategy to elucidate how an intrinsically disordered regulatory subunit of protein phosphatase 1 binds trimeric eIF2 and positions the phosphatase-substrate complex for dephosphorylation. As validation, they show that a disease mutation abolishes the interaction.
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