Biology subjects

Polley, S.

Publications and source records attributed to Polley, S..

9 recordsLinked to original sources

bioRxiv2026

IKK2/β mediated phosphorylation of transcription factor Ets2 at site(s) distal to DNA binding domain negatively modulates its DNA binding activity

Transcription factor Ets2 coalesce with the NF-{kappa}B pathway to regulate gene expression in specific signaling contexts. IKK2/{beta}-mediated phosphorylation events critically regulate the NF-{kappa}B pathway. However, any link between Ets2 and IKK2 remains elusive. Here we report Ets2 as a direct substrate of IKK2. In-vitro kinase assays using deletion constructs, high resolution MS-MS and site directed mutagenesis identified S295 as a prominent phosphorylation site distal to the DNA binding domain, substitution of which to phosphor-mimetic Glutamate triggers further phosphorylation of Ets2. MD simulations clearly indicate conformational constriction of the otherwise disordered N-terminal region and inhibition of DNA binding activity upon phosphorylation, which was further confirmed by Electrophoretic mobility shift assays. Our results uncover a phosphoregulatory connection between Ets2 and IKK2.

biochemistry↗

bioRxiv2024

Activation and regulation of a p38α MAPK by its downstream MAPKAP kinase through feedback phosphorylation and LLPS-driven condensate formation.

MAP kinases (MAPKs) represent a class of evolutionarily conserved stress and extracellular stimuli responsive signaling molecules. p38 group of MAPKs known to be activated by dual-specific upstream MAPK kinases and also by autophosphorylation. They activate MAPK activated protein kinases (MAPKAPKs) in a context dependent manner by specific phosphorylation, and together, they play crucial biological roles. One such pair is PMK3, p38-MAPK and its cognate MAPKAPK, MAK2, downstream of DLK1 (MAPK kinase kinase) and MKK4 (MAPK kinase) in C. elegans. They are implicated in axonal regeneration, degeneration and synaptic pruning in response to neuronal injury. Here, we report that PMK3 and MAK2 engage in a feedback relationship, leading to phosphorylation-mediated activation of both kinases. Interaction of PMK3 with MAK2 through the canonical docking site leads to negligible de novo autophosphorylation of PMK3. Phosphorylation of both Thr and Tyr residues on the TxY-motif necessary for the full activation of PMK3 requires catalytic activity of MAK2 as confirmed by mass spectrometry. Distribution of phosphorylation sites on either kinase when incubated together, and presence of long intrinsically disordered regions in each kinase indicate that PMK3:MAK2 complex is conformationally plastic in nature. MAK2 increases bioavailability of aggregation-prone PMK3 by virtue of its ability to form LLPS-driven condensates in vitro, wherein they retain enzymatic activities and phosphorylate each other. Furthermore, experiments with transgenic C. elegans reveal that MAK2 controls stability of PMK3 and localization of PMK3 puncta in touch neuron. Our observations offer an unreported activation mechanism of a p38-MAPK by its downstream MAPKAPK. Significance StatementMAPK kinases are evolutionarily conserved, and are key players in stress response, cell survival, differentiation, metabolic processes and neuronal response to injury. MAPK pathways are primarily activated through unidirectional flow of phosphorylation-signal MAP3K to MAP2K to MAPK, and in some cases to a downstream MAPKAPK. We found that a C. elegans MAPK (PMK3) can also be activated by its downstream MAPKAPK (MAK2) through feedback phosphorylation that ensures robust activation of PMK3 by MAK2 without requiring the MAP2K. Furthermore, MAK2 increases bioavailability of activation-competent and active PMK3 by preventing its aggregation through LLPS-driven condensate formation in vitro and in the worm neuron. This feedback relationship might ensure rapid activation of such MAPK pathways in response to nervous system injury or stress.

biochemistry↗

bioRxiv2023

Insights into human outer kinetochore assembly and force transmission from a structure-function analysis of the KMN network

The biorientation of chromosomes during cell division is necessary for precise dispatching of a mother cells chromosomes into its two daughters. Kinetochores, large layered structures built on specialized chromosome loci named centromeres, promote biorientation by binding and sensing spindle microtubules. The kinetochore outer layer consists of a 10-subunit apparatus comprising Knl1C, Mis12C, and Ndc80C subcomplexes (KMN network). The KMN network is highly elongated and docks on kinetochores and microtubules using interfaces at its opposite extremes. Here, we combine cryo-EM reconstructions and AlphaFold2 predictions to generate a model of the KMN network that reveals all intra-KMN interfaces. We identify and functionally validate two interaction interfaces that link Mis12C to Ndc80C and Knl1C. Through targeted interference experiments and molecular dynamics simulations we demonstrate this mutual organization stabilizes the KMN network. Our work reports the first comprehensive structural and functional analysis of the microtubule binding machinery of kinetochores and elucidates a path of microtubule-generated force transmission

molecular biology↗

bioRxiv2023

Dual-specific autophosphorylation of kinase IKK2 enables phosphorylation of substrate IkappaBalpha without requiring ATP

Rapid and high-fidelity phosphorylation of serine residues at positions 32 and 36 of I{kappa}B by IKK2, a prototypical Ser/Thr kinase, is critical for canonical NF-{kappa}B activation. Here, we report that IKK2 not only phosphorylates substrate serine residues and autophosphorylates its own activation loop, but also autophosphorylates at a tyrosine residue proximal to the active site and is, therefore, a dual-specificity kinase. We observed that mutation of Y169, an autophosphorylatable tyrosine located at the DFG+1 (DLG in IKK1) position, to phenylalanine renders IKK2 incapable of catalyzing phosphorylation at S32 within its I{kappa}B substrate. We also observed that mutation of the phylogenetically conserved ATP-contacting residue K44 in IKK2 to methionine converts IKK2 to an enzyme that no longer catalyzes specific phosphorylation of I{kappa}B at S32 or S36, but rather directs phosphorylation of I{kappa}B at other residues. Lastly, we report evidence of a phospho-relay from autophosphorylated IKK2 to I{kappa}B in the presence of ADP. These observations suggest an unusual evolution of IKK2, in which autophosphorylation of tyrosine(s) in the activation loop, and the conserved ATP-contacting K44 residue provide its signal-responsive substrate specificity and ensure fidelity during NF-{kappa}B activation.

biochemistry↗

bioRxiv2022

Stable kinetochore-microtubule attachment requires loop-dependent Ndc80-Ndc80 binding

During cell division, kinetochores link chromosomes to spindle microtubules. The Ndc80 complex, a crucial microtubule binder, populates each kinetochore with dozens of copies. Whether adjacent Ndc80 complexes cooperate to promote microtubule binding remains unclear. Here we demonstrate that the Ndc80 loop, a short sequence identified across eukaryotes and predicted to interrupt the Ndc80 coiled-coil, promotes direct interactions between full-length Ndc80 complexes. Both in dividing cells and in a fully reconstituted system, these interactions are essential for the formation of force-resistant kinetochore-microtubule attachments, explaining why deletion of the loop ablates chromosome congression. The loop may fold into a more rigid structure than previously assumed and we identify point mutations that impair Ndc80-Ndc80 interactions and recapitulate the effects of loop depletion. The loop also promotes spindle checkpoint signaling, suggesting that the organisation of adjacent Ndc80 complexes is crucial to couple kinetochore-microtubule binding to cell cycle control.

biochemistry↗

bioRxiv2021

Discovery of an IKK2 Site that Allosterically Controls Its Activation

I{kappa}B kinase 2/{beta} (IKK2) is a critical regulator of inflammation which is inducibly activated by a host of stimuli. Aberrant activation of IKK2 is the leading cause of most inflammatory diseases and many associated cancers. Efforts to prevent these diseases by small-molecule inhibitors of IKK2 activity have not been successful. Most inhibitors developed for IKK2 are ATP-competitive, and they are toxic in vivo due to their off-target effects. Here we focused on identifying inhibitors to block IKK2 activity from an allosteric site, not the ATP-binding pocket. Using virtual screening, we first identified several candidate allosteric sites and screened for potential small-molecule binders, and then selected candidates inhibitory to IKK2 activity using cell-based functional assays. Hydrogen deuterium exchange coupled to mass-spectrometry (HDX-MS) and MS-MS assays revealed that a class of benzoyl conjugates of pyrrolidinedione covalently bound to a site located at the interface of the kinase domain (KD) and the helical domain (SDD), and inhibited IKK2 activation allosterically by preventing phosphorylation of its activation loop serines. Additionally, this class of inhibitor partially blocks IKK2s catalytic activity by enhancing dynamics within the ATP binding pocket and likely the general active site. Hydrogen deuterium exchange (HDX) experiments further revealed that while binding of substrate ATP perturbs only the local structure surrounding its binding site, binding to ATP-competitive or allosteric inhibitors induces structural perturbations in an expansive area including the helical domain. We propose that these allosteric sites can act as specific targets for the development of novel potent IKK inhibitors. SIGNIFICANCEAberrant activation of IKK2 is the leading cause of most inflammatory diseases and many associated cancers. Most inhibitors developed for IKK2 are ATP-competitive, and they are toxic in vivo due to their off-target effects. By combination of virtual screening and cell-based functional assays, we identified small-molecule binders of the class of benzoyl conjugates of pyrrolidinedione that block IKK2 activity from an allosteric site through covalent attachment and could be specific only for IKK2. HDX-MS and MS-MS assays identified a binding pocket with a Cys-Cys motif for these inhibitors, and revealed specific differences in IKK2 dynamics upon binding to substrate ATP vs ATP-competitive and allosteric inhibitors. Present work provides a framework for the development of allosteric inhibitors to combat IKK2-induced diseases inhibitors.

biochemistry↗

bioRxiv2019

Electroporated recombinant proteins as tools for in vivo functional complementation, imaging, and chemical biology

Delivery of native or chemically modified recombinant proteins into mammalian cells shows promise for functional investigations and various technological applications, but concerns that sub-cellular localization and functional integrity of delivered proteins may be affected remain high. Here, we surveyed batch electroporation as a delivery tool for single polypeptides and multi-subunit protein assemblies of kinetochores, a spatially confined and well-studied subcellular structures. After electroporation in human cells, recombinant fluorescent Ndc80 and Mis12 multi-subunit complexes displayed native localization, physically interacted with endogenous binding partners, and functionally complemented depleted endogenous counterparts to promote mitotic checkpoint signaling and chromosome segregation. Farnesylation is required for kinetochore localization of the Dynein adaptor Spindly. In cells with chronically inhibited farnesyl transferase activity, in vitro farnesylation and electroporation reconstituted robust kinetochore localization of Spindly. Thus, electroporation is uniquely versatile for delivering synthetic and, as required, chemically modified functional mimics of endogenous proteins, and is therefore a promising tool for chemical and synthetic biology.

cell biology↗

bioRxiv2019

Indirect Readout of DNA Controls Filamentation and Activation of a Sequence-Specific Endonuclease

Filament or run-on oligomer formation by enzymes is increasingly recognized as an important phenomenon with potentially unique regulatory properties and biological roles. SgrAI is an allosterically regulated type II restriction endonuclease that forms run-on oligomeric (ROO) filaments with enhanced DNA cleavage activity and altered sequence specificity. Here, we present the 3.5 [A] cryo-electron microscopy structure of the ROO filament of SgrAI bound to a mimic of cleaved primary site DNA and Mg2+. Large conformational changes stabilize a second metal ion cofactor binding site within the catalytic pocket and facilitate assembling a higher-order enzyme form that is competent for rapid DNA cleavage. The structural changes illuminate the mechanistic origin of hyper-accelerated DNA cleavage activity within the filamentous SgrAI form. An analysis of the protein-DNA interface and the stacking of individual nucleotides reveals how indirect DNA readout within filamentous SgrAI enables recognition of substantially more nucleotide sequences than its low-activity form, thereby expanding DNA sequence specificity. Together, substrate DNA binding, indirect readout, and filamentation simultaneously enhance SgrAIs catalytic activity and modulate substrate preference. This unusual enzyme mechanism may have evolved to perform the specialized functions of bacterial innate immunity in rapid defense against invading phage DNA without causing damage to the host DNA.

biochemistry↗

bioRxiv2019

A staphylococcal cyclophilin carries a single domain and unfolds via the formation of an intermediate that preserves cyclosporin A binding activity

Cyclophilin (Cyp), a peptidyl-prolyl cis-trans isomerase (PPIase), acts as a virulence factor in many bacteria including Staphylococcus aureus. The enzymatic activity of Cyp is inhibited by cyclosporin A (CsA), an immunosuppressive drug. To precisely determine the unfolding mechanism and the domain structure of Cyp, we have investigated a chimeric S. aureus Cyp (rCyp) using various probes. Our limited proteolysis and the consequent analysis of the proteolytic fragments indicate that rCyp is composed of one domain with a short flexible tail at the C-terminal end. We also show that the urea-induced unfolding of both rCyp and rCyp-CsA is completely reversible and proceeds via the synthesis of at least one stable intermediate. The secondary structure, tertiary structure, and the hydrophobic surface area of no intermediate are fully identical to those of other intermediate or the related native protein. Further analyses reveal no loss of CsA binding activity in rCyp intermediate. The thermodynamic stability of rCyp was also significantly increased in the presence of CsA, recommending that this protein could be employed to screen new CsA derivatives in future.

biochemistry↗