Light-induced Conformational Changes of LOV1 (Light Oxygen Voltage-sensing Domain 1) and LOV2 Relative to the Kinase Domain and Regulation of Kinase Activity inChlamydomonasPhototropin

To explore how pathogenic mutations of the multidomain leucine-rich repeat kinase 2 (LRRK2) hijack its finely tuned activation process and drive Parkinson’s disease (PD), we used a multitiered approach. Most mutations mimic Rab-mediated activation by “unleashing” kinase activity, and many, like the kinase inhibitor MLi-2, trap LRRK2 onto microtubules. Here we mimic activation by simply deleting the inhibitory N-terminal domains and then characterize conformational changes induced by MLi-2 and PD mutations. After confirming that LRRK2RCKW retains full kinase activity, we used hydrogen-deuterium exchange mass spectrometry to capture breathing dynamics in the presence and absence of MLi-2. Solvent-accessible regions throughout the entire protein are reduced by MLi-2 binding. With molecular dynamics simulations, we created a dynamic portrait of LRRK2RCKW and demonstrate the consequences of kinase domain mutations. Although all domains contribute to regulating kinase activity, the kinase domain, driven by the DYGψ motif, is the allosteric hub that drives LRRK2 regulation.

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The contribution of individual subunits to the coupling of the voltage sensor to pore opening in Shaker K channels: effect of ILT mutations in heterotetramers

The Journal of General Physiology ◽

10.1085/jgp.201010487 ◽

2010 ◽

Vol 136 (5) ◽

pp. 555-568 ◽

Cited By ~ 16

Author(s):

Dominique G. Gagnon ◽

Francisco Bezanilla

Keyword(s):

Conformational Changes ◽

Ionic Conduction ◽

Voltage Sensing ◽

K Channels ◽

Thermodynamical Properties ◽

Pore Opening ◽

Voltage Gated Ion Channels ◽

Voltage Sensing Domain ◽

S4 Segment ◽

Voltage Relationship

Voltage-gated ion channels couple conformational change(s) of the voltage-sensing domain to those of the opening of an intracellular gate to allow ionic conduction. Much larger positive potentials are required to couple these conformational changes to the opening of the gate of Shaker K+ channels with the concurrent mutations V369I, I372L, and S376T (ILT) at the N-terminal end of the S4 segment. We used cut-open oocyte voltage clamp to study the biophysical and thermodynamical properties of heterotetrameric concatemerized channels with different stoichiometries of ILT mutations. The voltage-sensing domains of ILT mutant channels require smaller depolarization to activate but their intracellular gate does not immediately follow the movement of the voltage-sensing domain, requiring larger depolarization to open. Our results demonstrate that each subunit contributes equally to the rightward shift of the conductance–voltage relationship and that a single ILT-containing subunit is sufficient to induce a large enthalpic and entropic barrier, limiting opening of the intracellular gate.

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Ruxolitinib Mediated Paradox JAK2 Hyperphosphorylation Is Due To The Protection Of Activation Loop Phosphotyrosines From Phosphatases

Blood ◽

10.1182/blood.v122.21.2847.2847 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2847-2847 ◽

Cited By ~ 2

Author(s):

Sivahari P. Gorantla ◽

Kolluri Suresh Babu ◽

Anna Lena Illert ◽

Nikolas von Bubnoff ◽

Christian Peschel ◽

...

Keyword(s):

Conformational Changes ◽

Tyrosine Kinases ◽

Kinase Activity ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Specific Inhibitor ◽

Jak2 V617f ◽

Kinase Domain ◽

Activation Loop ◽

Kinase Domain Mutation

Abstract The JAK2 V617F mutation has been reported in the majority of bcr-abl negative myeloproliferative neoplasms (MPNs) including polycythemia vera (PV:90%), essential thrombocythemia (ET:50%) and idiopathic myelofibrosis (IMF:50%). Recently, ruxolitinib (Jakavi, INCB018424) a JAK2/JAK1 specific inhibitor has been approved by the FDA for the treatment of myelofibrosis. Unexpectedly, treatment of V617FJAK2 expressing cells with ruxolitinib causes paradoxical hyperphosphorylation of JAK2 at its two critical sites (tyr1007/tyr1008). The mechanism of ruxolitinib induced JAK2 hyperphosphorylation is not understood. It has been demonstrated that also the pseudokinase domain of JAK2 display kinase activity and plays a major role in regulating the kinase activity of JAK2. Deletion of the pseudokinase domain leads to hyperphosphorylation of JAK2. We thus hypothesized that ATP-competitive inhibitors might target the kinase activity of the pseudokinase domain. However, treatment of kinase dead pseudokinase domain JAK2V617F mutants with ruxolitinib also causes JAK2 hyperphosphorylation indicating that ruxolitinib mediated JAK2 hyperphosphorylation is not due to inhibition of the pseudokinase domain. We then reasoned that treatment with ruxolitinib might protect JAK2 from tyrosine phosphatases. Co-immunoprecipitation results however did not suggested that ruxolitinib treatment leads to the dissociation of phosphatases such as SHP-2. A Ruxolitinib resistant JAK2V617F mutant (V617F+L902QJAK2) and a JAK2V617F kinase dead mutant (V617F+K882RJAK2) both did not show paradox hyperphoshorylation after ruxolitinib treatment. This indicated that ruxolitinib mediated JAK2 hyperphosphorylation is mediated by a JAK2 intrinsic mechanism. Finally, native-immunocomplexes (non-denaturing IP) of JAK2V617F generated with a monoclonal antibody against ptyr1007/1008 could be blocked by the presence of ruxolitinib, although JAK2V617F was hyperphosphorylated at these sites. We therefore believe now that in the presence of ruxolitinib the JAK2 activation loop folds inside the kinase domain. This leads to the protection of ptyr1007/1008 sites from phosphatases. Most tyrosine kinases are structurally very plastic enzymes with large conformational differences observed between the inactive and active states of the kinase. These conformational changes are largely governed by disorder to order changes in N-lobe, activation loop and hydrophobic motif. When JAK2 is in an inhibited conformational restricted state, the phosphate group of tyr1007 and tyr1008 have an intermolecular interaction with Arg975 and Lys999. These interactions play a major role in the stabilization of the activation loop conformation inside the kinase domain. Mutation of Arg975 and Lys999 to Ala (V617F+R975A), (V617F+L999A) reduced the phosphorylation at both tyr1007/1008 residues. Importantly, ruxolitinib treatment did not lead to JAK2 hyperphosphorylation in this mutants. Taken together our results suggest a novel mode of kinase regulation by modulating kinase activity through conformational changes induced by ruxolutinib. Disclosures: No relevant conflicts of interest to declare.

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Regulation of the Src Family Kinase Lck by Hsp90 and Ubiquitination

Molecular and Cellular Biology ◽

10.1128/mcb.24.13.5667-5676.2004 ◽

2004 ◽

Vol 24 (13) ◽

pp. 5667-5676 ◽

Cited By ~ 38

Author(s):

Ana Giannini ◽

Marie-José Bijlmakers

Keyword(s):

Conformational Changes ◽

Kinase Activity ◽

Cell Receptor ◽

Kinase Domain ◽

Hsp90 Inhibitor ◽

Intramolecular Interactions ◽

Atp Binding Site ◽

Regulatory Processes ◽

The Stability ◽

Lck Kinase

ABSTRACT Regulation of the Src-related tyrosine kinase Lck is crucial to the outcome of T-cell receptor (TCR) stimulation. It was previously shown that the stability of the constitutively active mutant LckY505F is controlled by Hsp90 (M. J. Bijlmakers and M. Marsh, Mol. Biol. Cell. 11:1585-1595, 2000). Here we establish that following TCR stimulation, endogenous activated Lck in T cells is also degraded in the presence of the Hsp90 inhibitor geldanamycin. Using Lck constructs expressed in COS-7 cells, we show that the presence of activating Lck mutations results not only in the enhanced dependence on Hsp90 but also in enhanced ubiquitination of Lck. Although both processes were induced by mutations Y505F and W97A that release the SH2 and SH3 inhibitory intramolecular interactions, respectively, neither process required Lck kinase activity or activation-dependent phosphorylation at serines 42 and 59 or tyrosine 394. By binding to the ATP-binding site, the Src family inhibitor PP2 reduced ubiquitination and overcame the need for Hsp90 monitoring of active Lck. We conclude that the levels of active Lck are influenced by two opposing processes, targeting for degradation by ubiquitination and rescue from degradation by Hsp90 monitoring. Based on the PP2 result, we propose that activation-induced conformational changes of the Lck kinase domain instigate both regulatory processes.

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New Molecular Scaffolds for Fluorescent Voltage Indicators

10.26434/chemrxiv.7338683.v1 ◽

2018 ◽

Author(s):

Steven Boggess ◽

Shivaani Gandhi ◽

Brian Siemons ◽

Nathaniel Huebsch ◽

Kevin Healy ◽

...

Keyword(s):

Membrane Potential ◽

Action Potentials ◽

Induced Pluripotent Stem Cell ◽

Molecular Wire ◽

Voltage Sensing ◽

Design Synthesis ◽

Cardiac Action ◽

Action Potential Waveform ◽

Induced Pluripotent ◽

Voltage Sensing Domain

<div> <p>The ability to non-invasively monitor membrane potential dynamics in excitable cells like neurons and cardiomyocytes promises to revolutionize our understanding of the physiology and pathology of the brain and heart. Here, we report the design, synthesis, and application of a new class of fluorescent voltage indicator that makes use of a fluorene-based molecular wire as a voltage sensing domain to provide fast and sensitive measurements of membrane potential in both mammalian neurons and human-derived cardiomyocytes. We show that the best of the new probes, fluorene VoltageFluor 2 (fVF 2) readily reports on action potentials in mammalian neurons, detects perturbations to cardiac action potential waveform in human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes, shows a substantial decrease in phototoxicity compared to existing molecular wire-based indicators, and can monitor cardiac action potentials for extended periods of time. Together, our results demonstrate the generalizability of a molecular wire approach to voltage sensing and highlights the utility of fVF 2 for interrogating membrane potential dynamics.</p> </div>

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Quantum Calculation of Proton and Other Charge Transfer Steps in Voltage Sensing in the Kv1.2 Channel

10.26434/chemrxiv.8251073 ◽

2019 ◽

Author(s):

Alisher M Kariev ◽

Michael Green

Keyword(s):

Charge Transfer ◽

Energy Charge ◽

Quantum Calculation ◽

Quantum Calculations ◽

Gating Current ◽

Transmembrane Segment ◽

Voltage Sensing ◽

Standard Models ◽

Charge Displacement ◽

Voltage Sensing Domain

Quantum calculations on 976 atoms of the voltage sensing domain of the K<sub>v</sub>1.2 channel, with protons in several positions, give energy, charge transfer, and other properties. Motion of the S4 transmembrane segment that accounts for gating current in standard models is shown not to occur; there is H<sup>+ </sup>transfer instead. The potential at which two proton positions cross in energy approximately corresponds to the gating potential for the channel. The charge displacement seems approximately correct for the gating current. Two mutations are accounted for (Y266F, R300cit, cit =citrulline). The primary conclusion is that voltage sensing depends on H<sup>+</sup> transfer, not motion of arginine charges.

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