scholarly journals The multifaceted allosteric regulation of Aurora kinase A

2018 ◽  
Vol 475 (12) ◽  
pp. 2025-2042 ◽  
Author(s):  
Nicholas Mark Levinson
Keyword(s):  
Molecular Level ◽  
Aurora Kinase ◽  
Kinase Domain ◽  
Regulatory Control ◽  
Activation Loop ◽  
Mitotic Spindles ◽  
Kinase Activation ◽  
Structure And Dynamics ◽  
Spindle Microtubules ◽  
Eukaryotic Organisms

The protein kinase Aurora A (AurA) is essential for the formation of bipolar mitotic spindles in all eukaryotic organisms. During spindle assembly, AurA is activated through two different pathways operating at centrosomes and on spindle microtubules. Recent studies have revealed that these pathways operate quite differently at the molecular level, activating AurA through multifaceted changes to the structure and dynamics of the kinase domain. These advances provide an intimate atomic-level view of the finely tuned regulatory control operating in protein kinases, revealing mechanisms of allosteric cooperativity that provide graded levels of regulatory control, and a previously unanticipated mechanism for kinase activation by phosphorylation on the activation loop. Here, I review these advances in our understanding of AurA function, and discuss their implications for the use of allosteric small molecule inhibitors to address recently discovered roles of AurA in neuroblastoma, prostate cancer and melanoma.

scholarly journals dDYRK2: a novel dual-specificity tyrosine-phosphorylation-regulated kinase in Drosophila

2003 ◽  
Vol 374 (2) ◽  
pp. 381-391 ◽  
Author(s):  
Pamela A. LOCHHEAD ◽  
Gary SIBBET ◽  
Ross KINSTRIE ◽  
Tava CLEGHON ◽  
Margie RYLATT ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Protein Kinases ◽  
Mutational Analysis ◽  
Kinase Domain ◽  
Activation Loop ◽  
Embryonic Neurogenesis ◽  
Founding Family ◽  
Dual Specificity ◽  
Eukaryotic Organisms

Dual-specificity tyrosine-phosphorylation-regulated kinases (DYRKs) are an emerging family of protein kinases that have been identified in all eukaryotic organisms examined to date. DYRK family members are involved in regulating key developmental and cellular processes such as neurogenesis, cell proliferation, cytokinesis and cellular differentiation. Two distinct subgroups exist, nuclear and cytosolic. In Drosophila, the founding family member minibrain, whose human orthologue maps to the Down syndrome critical region, belongs to the nuclear subclass and affects post-embryonic neurogenesis. In the present paper, we report the isolation of dDYRK2, a cytosolic DYRK and the putative product of the smell-impaired smi35A gene. This is the second such kinase described in Drosophila, but the first to be characterized at the molecular and biochemical level. dDYRK2 is an 81 kDa dual-specificity kinase that autophosphorylates on tyrosine and serine/threonine residues, but appears to phosphorylate exogenous substrates only on serine/threonine residues. It contains a YXY motif in the activation loop of the kinase domain in the same location as the TXY motif in mitogenactivated protein kinases. dDYRK2 is tyrosine-phosphorylated in vivo, and mutational analysis reveals that the activation loop tyrosines are phosphorylated and are essential for kinase activity. Finally, dDYRK2 is active at all stages of fly development, with elevated levels observed during embryogenesis and pupation.

scholarly journals Attenuation of yeast UPR is essential for survival and is mediated by IRE1 kinase

2011 ◽  
Vol 193 (1) ◽  
pp. 41-50 ◽  
Author(s):  
Aditi Chawla ◽  
Sutapa Chakrabarti ◽  
Gourisankar Ghosh ◽  
Maho Niwa
Keyword(s):  
Er Stress ◽  
Kinase Domain ◽  
Activation Loop ◽  
Kinase Activation ◽  
Messenger Ribonucleic Acid ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response ◽  
In Cells

The unfolded protein response (UPR) activates Ire1, an endoplasmic reticulum (ER) resident transmembrane kinase and ribonuclease (RNase), in response to ER stress. We used an in vivo assay, in which disappearance of the UPR-induced spliced HAC1 messenger ribonucleic acid (mRNA) correlates with the recovery of the ER protein-folding capacity, to investigate the attenuation of the UPR in yeast. We find that, once activated, spliced HAC1 mRNA is sustained in cells expressing Ire1 carrying phosphomimetic mutations within the kinase activation loop, suggesting that dephosphorylation of Ire1 is an important step in RNase deactivation. Additionally, spliced HAC1 mRNA is also sustained after UPR induction in cells expressing Ire1 with mutations in the conserved DFG kinase motif (D828A) or a conserved residue (F842) within the activation loop. The importance of proper Ire1 RNase attenuation is demonstrated by the inability of cells expressing Ire1-D828A to grow under ER stress. We propose that the activity of the Ire1 kinase domain plays a role in attenuating its RNase activity when ER function is recovered.

scholarly journals A dynamic mechanism for allosteric activation of Aurora kinase A by activation loop phosphorylation

2017 ◽  
Author(s):  
Emily F. Ruff ◽  
Joseph M. Muretta ◽  
Andrew Thompson ◽  
Eric Lake ◽  
Soreen Cyphers ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Aurora Kinase ◽  
Kinase Domain ◽  
Activation Loop ◽  
Post Translational Modification ◽  
Paramagnetic Resonance ◽  
Mitotic Kinase ◽  
Dynamics Simulations ◽  
Electron Paramagnetic ◽  
Kinetics Of

AbstractMany eukaryotic protein kinases are activated by phosphorylation on a specific conserved residue in the regulatory activation loop, a post-translational modification thought to stabilize the active DFG-In state of the catalytic domain. Here we use a battery of spectroscopic methods that track different catalytic elements of the kinase domain to show that the ~100-fold activation of the mitotic kinase Aurora A (AurA) by phosphorylation occurs without a population shift to the DFG-In state, and that the activation loop of the activated kinase remains highly dynamic. Instead, molecular dynamics simulations and electron paramagnetic resonance experiments show that phosphorylation profoundly alters the structure and dynamics of the DFG-In subpopulation, leading to activation of the kinase. Kinetics experiments tracking structural transitions during nucleotide binding suggest that a substantial DFG-Out subpopulation is an important feature of activated AurA that evolved to optimize the kinetics of substrate binding and product release.

scholarly journals Quantitative conformational profiling of kinase inhibitors reveals origins of selectivity for Aurora kinase activation states

2018 ◽  
Vol 115 (51) ◽  
pp. E11894-E11903 ◽  
Author(s):  
Eric W. Lake ◽  
Joseph M. Muretta ◽  
Andrew R. Thompson ◽  
Damien M. Rasmussen ◽  
Abir Majumdar ◽  
...  
Keyword(s):  
Large Scale ◽  
Kinase Inhibitors ◽  
Conformational Dynamics ◽  
Aurora Kinase ◽  
Activation Loop ◽  
Aurora A ◽  
Kinase Activation ◽  
Mitotic Kinase ◽  
Conformational Preferences ◽  
Wide Range

Protein kinases undergo large-scale structural changes that tightly regulate function and control recognition by small-molecule inhibitors. Methods for quantifying the conformational effects of inhibitors and linking them to an understanding of selectivity patterns have long been elusive. We have developed an ultrafast time-resolved fluorescence methodology that tracks structural movements of the kinase activation loop in solution with angstrom-level precision, and can resolve multiple structural states and quantify conformational shifts between states. Profiling a panel of clinically relevant Aurora kinase inhibitors against the mitotic kinase Aurora A revealed a wide range of conformational preferences, with all inhibitors promoting either the active DFG-in state or the inactive DFG-out state, but to widely differing extents. Remarkably, these conformational preferences explain broad patterns of inhibitor selectivity across different activation states of Aurora A, with DFG-out inhibitors preferentially binding Aurora A activated by phosphorylation on the activation loop, which dynamically samples the DFG-out state, and DFG-in inhibitors binding preferentially to Aurora A constrained in the DFG-in state by its allosteric activator Tpx2. The results suggest that many inhibitors currently in clinical development may be capable of differentiating between Aurora A signaling pathways implicated in normal mitotic control and in melanoma, neuroblastoma, and prostate cancer. The technology is applicable to a wide range of clinically important kinases and could provide a wealth of valuable structure–activity information for the development of inhibitors that exploit differences in conformational dynamics to achieve enhanced selectivity.

scholarly journals Profound ligand-independent kinase activation of fibroblast growth factor receptor 3 by the activation loop mutation responsible for a lethal skeletal dysplasia, thanatophoric dysplasia type II.

1996 ◽  
Vol 16 (8) ◽  
pp. 4081-4087 ◽  
Author(s):  
M K Webster ◽  
P Y D'Avis ◽  
S C Robertson ◽  
D J Donoghue
Keyword(s):  
Fibroblast Growth Factor Receptor ◽  
Growth Factor Receptor ◽  
Kinase Domain ◽  
Activation Loop ◽  
Type Ii ◽  
Fibroblast Growth ◽  
Thanatophoric Dysplasia ◽  
Kinase Activation ◽  
Constitutive Activation ◽  
Lethal Skeletal Dysplasia

Thanatophoric dysplasia type II (TDII) is a neonatal lethal skeletal dysplasia caused by a recurrent Lys-650-->Glu mutation within the highly conserved activation loop of the kinase domain of fibroblast growth factor receptor 3 (FGFR3). We demonstrate here that this mutation results in profound constitutive activation of the FGFR3 tyrosine kinase, approximately 100-fold above that of wild-type FGFR3. The mechanism of FGFR3 activation in TDII was probed by constructing various point mutations in the activation loop. Substitutions at position 650 indicated that not only Glu but also Asp and, to a lesser extent, Gln and Leu result in pronounced constitutive activation of FGFR3. Additional mutagenesis within the beta10-beta11 loop region (amino acids Tyr-647 to Leu-656) demonstrated that amino acid 650 is the only residue which can activate the receptor when changed to a Glu, indicating a specificity of position as well as charge for mutations which can give rise to kinase activation. Furthermore, when predicted sites of autophosphorylation at Tyr-647 and Tyr-648 were mutated to Phe, either singly or in combination, constitutive kinase activity was still observed in response to the Lys-650-->Glu mutation, although the effect of these mutations on downstream signalling was not investigated. Our data suggest that the molecular effect of the TDII activation loop mutation is to mimic the conformational changes that activate the tyrosine kinase domain, which are normally initiated by ligand binding and autophosphorylation. These results have broad implications for understanding the molecular basis of other human developmental syndromes that involve mutations in members of the FGFR family. Moreover, these findings are relevant to the study of kinase regulation and the design of activating mutations in related tyrosine kinases.

scholarly journals A dynamic mechanism for allosteric activation of Aurora kinase A by activation loop phosphorylation

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Emily F Ruff ◽  
Joseph M Muretta ◽  
Andrew R Thompson ◽  
Eric W Lake ◽  
Soreen Cyphers ◽  
...  
Keyword(s):  
Protein Kinases ◽  
Catalytic Domain ◽  
Aurora Kinase ◽  
Kinase Domain ◽  
Activation Loop ◽  
Post Translational Modification ◽  
Paramagnetic Resonance ◽  
Mitotic Kinase ◽  
Catalytic Activation ◽  
Dynamics Simulations

Many eukaryotic protein kinases are activated by phosphorylation on a specific conserved residue in the regulatory activation loop, a post-translational modification thought to stabilize the active DFG-In state of the catalytic domain. Here we use a battery of spectroscopic methods that track different catalytic elements of the kinase domain to show that the ~100 fold activation of the mitotic kinase Aurora A (AurA) by phosphorylation occurs without a population shift from the DFG-Out to the DFG-In state, and that the activation loop of the activated kinase remains highly dynamic. Instead, molecular dynamics simulations and electron paramagnetic resonance experiments show that phosphorylation triggers a switch within the DFG-In subpopulation from an autoinhibited DFG-In substate to an active DFG-In substate, leading to catalytic activation. This mechanism raises new questions about the functional role of the DFG-Out state in protein kinases.

scholarly journals Role of the kinase activation loop on protein kinase C θ activity and intracellular localisation

FEBS Letters ◽  
2003 ◽  
Vol 554 (1-2) ◽  
pp. 35-40 ◽  
Author(s):  
Bianca Sparatore ◽  
Mario Passalacqua ◽  
Marco Pedrazzi ◽  
Sabina Ledda ◽  
Mauro Patrone ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Activation Loop ◽  
Kinase Activation ◽  
Kinase C ◽  
Intracellular Localisation

Structural differences between repressed and derepressed forms of p60c-src

1989 ◽  
Vol 9 (6) ◽  
pp. 2648-2656
Author(s):  
A MacAuley ◽  
J A Cooper
Keyword(s):  
Kinase Activity ◽  
Kinase Domain ◽  
Terminal Region ◽  
Phosphorylation Sites ◽  
Carboxy Terminus ◽  
Kinase Activation ◽  
Amino Terminal ◽  
Structural Differences ◽  
Carboxy Terminal ◽  
Pronase E

The kinase activity of p60c-src is derepressed by removal of phosphate from Tyr-527, mutation of this residue to Phe, or binding of a carboxy-terminal antibody. We have compared the structures of repressed and active p60c-src, using proteases. All forms of p60c-src are susceptible to proteolysis at the boundary between the amino-terminal region and the kinase domain, but there are several sites elsewhere that are more sensitive to trypsin digestion in repressed than in derepressed forms of p60c-src. The carboxy-terminal tail (containing Tyr-527) is more sensitive to digestion by pronase E and thermolysin when Tyr-527 is not phosphorylated. The kinase domain fragment released with trypsin has kinase activity. Relative to intact p60c-src, the kinase domain fragment shows altered substrate specificity, diminished regulation by the phosphorylated carboxy terminus, and novel phosphorylation sites. The results identify parts of p60c-src that change conformation upon kinase activation and suggest functions for the amino-terminal region.

The Src family kinase Fgr is a transforming oncoprotein that functions independently of SH3-SH2 domain regulation

2018 ◽  
Vol 11 (553) ◽  
pp. eaat5916 ◽  
Author(s):  
Kexin Shen ◽  
Jamie A. Moroco ◽  
Ravi K. Patel ◽  
Haibin Shi ◽  
John R. Engen ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Colony Formation ◽  
Family Members ◽  
Cellular Transformation ◽  
Soft Agar ◽  
Kinase Domain ◽  
Sh2 Domain ◽  
Mass Spectrometry Data ◽  
Activation Loop ◽  
Exchange Mass Spectrometry

Fgr is a member of the Src family of nonreceptor tyrosine kinases, which are overexpressed and constitutively active in many human cancers. Fgr expression is restricted to myeloid hematopoietic cells and is markedly increased in a subset of bone marrow samples from patients with acute myeloid leukemia (AML). Here, we investigated the oncogenic potential of Fgr using Rat-2 fibroblasts that do not express the kinase. Expression of either wild-type or regulatory tail-mutant constructs of Fgr promoted cellular transformation (inferred from colony formation in soft agar), which was accompanied by phosphorylation of the Fgr activation loop, suggesting that the kinase domain of Fgr functions independently of regulation by its noncatalytic SH3-SH2 region. Unlike other family members, recombinant Fgr was not activated by SH3-SH2 domain ligands. However, hydrogen-deuterium exchange mass spectrometry data suggested that the regulatory SH3 and SH2 domains packed against the back of the kinase domain in a Src-like manner. Sequence alignment showed that the activation loop of Fgr was distinct from that of all other Src family members, with proline rather than alanine at the +2 position relative to the activation loop tyrosine. Substitution of the activation loop of Fgr with the sequence from Src partially inhibited kinase activity and suppressed colony formation. Last, Fgr expression enhanced the sensitivity of human myeloid progenitor cells to the cytokine GM-CSF. Because its kinase domain is not sensitive to SH3-SH2–mediated control, simple overexpression of Fgr without mutation may contribute to oncogenic transformation in AML and other blood cancers.

scholarly journals A Redox-Sensitive Thiol in Wis1 Modulates the Fission Yeast Mitogen-Activated Protein Kinase Response to H2O2 and Is the Target of a Small Molecule

2020 ◽  
Vol 40 (7) ◽  
Author(s):  
Johanna J. Sjölander ◽  
Agata Tarczykowska ◽  
Cecilia Picazo ◽  
Itziar Cossio ◽  
Itedale Namro Redwan ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Fission Yeast ◽  
Inhibitory Effect ◽  
Mitogen Activated Protein Kinase ◽  
Activation Loop ◽  
Content Type ◽  
Kinase Activation ◽  
Mitogen Activated Protein

ABSTRACT Oxidation of a highly conserved cysteine (Cys) residue located in the kinase activation loop of mitogen-activated protein kinase kinases (MAPKK) inactivates mammalian MKK6. This residue is conserved in the fission yeast Schizosaccharomyces pombe MAPKK Wis1, which belongs to the H2O2-responsive MAPK Sty1 pathway. Here, we show that H2O2 reversibly inactivates Wis1 through this residue (C458) in vitro. We found that C458 is oxidized in vivo and that serine replacement of this residue significantly enhances Wis1 activation upon addition of H2O2. The allosteric MAPKK inhibitor INR119, which binds in a pocket next to the activation loop and C458, prevented the inhibition of Wis1 by H2O2 in vitro and significantly increased Wis1 activation by low levels of H2O2 in vivo. We propose that oxidation of C458 inhibits Wis1 and that INR119 cancels out this inhibitory effect by binding close to this residue. Kinase inhibition through the oxidation of a conserved Cys residue in MKK6 (C196) is thus conserved in the S. pombe MAPKK Wis1.

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