A5025805311- Microtubule and mitosis dynamics
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Novo Nordisk Foundation
2014-2024
Foundation Center
2024
Amgen (Germany)
2024
University of Copenhagen
2014-2023
Stanford University
2020-2023
Universität Hamburg
2010
University Medical Center Hamburg-Eppendorf
2010
Article13 May 2020Open Access Source DataTransparent process Mechanisms of site-specific dephosphorylation and kinase opposition imposed by PP2A regulatory subunits Thomas Kruse orcid.org/0000-0002-2619-7388 Novo Nordisk Foundation Center for Protein Research, Faculty Health Medical Sciences, University Copenhagen, Denmark Search more papers this author Sebastian Peter Gnosa Biotech Research Innovation Centre (BRIC), Isha Nasa Biochemistry Cell Biology, Geisel School Medicine at Dartmouth...
Abstract Progression through the cell cycle is controlled by regulated and abrupt changes in phosphorylation 1 . Mitotic entry initiated increased of mitotic proteins, a process driven kinases 2 , whereas exit achieved counteracting dephosphorylation, phosphatases, especially PP2A:B55 3 Although role well established, recent data have shown that mitosis only successfully when counterbalancing phosphatases are also inhibited 4 Inhibition intrinsically disordered proteins ARPP19 5,6 FAM122A 7...
Proper cell-cycle progression requires tight temporal control of the Anaphase Promoting Complex/Cyclosome (APC/C), a large ubiquitin ligase that is activated by one two co-activators, Cdh1 or Cdc20. APC/C and Cdc20 are already present during interphase but APC/C-Cdc20 regulation this window cell cycle, if any, unknown. Here we show cyclin A2-Cdk2 binds phosphorylates in inhibits activity. Preventing phosphorylation results pre-mature activation several substrates, including B1 A2,...
The PP2A-B55 phosphatase regulates a plethora of signaling pathways throughout eukaryotes. How selects its substrates presents severe knowledge gap. By integrating AlphaFold modeling with comprehensive high-resolution mutational scanning, we show that α helices in bind B55 through an evolutionary conserved mechanism. Despite large diversity sequence and composition, these share key amino acid determinants engage discrete hydrophobic electrostatic patches. Using deep learning protein design,...
Phosphoprotein phosphatases (PPPs) regulate major signaling pathways, but the determinants of phosphatase specificity are poorly understood. This is because methods to investigate this at scale lacking. Here, we develop a novel in vitro assay, MRBLE:Dephos, that allows multiplexing dephosphorylation reactions determine preferences. Using establish amino acid preferences residues surrounding site for PP1 and PP2A-B55, which reveals common unique To compare MRBLE:Dephos results cellular...
The widespread adoption of bead-based multiplexed bioassays requires the ability to easily synthesize encoded microspheres and conjugate analytes interest their surface. Here, we present a simple method (MRBLEs 2.0) for efficient high-throughput generation with ratiometric barcode lanthanide encoding (MRBLEs) that bear functional groups downstream surface bioconjugation. Bead production in MRBLEs 2.0 relies on manual mixing lanthanide/polymer mixtures (each which comprises unique spectral...
Abstract The PP2A-B55 phosphatase regulates a plethora of signaling pathways throughout eukaryotes. How selects its substrates presents severe knowledge gap. By integrating AlphaFold modelling with comprehensive high resolution mutational scanning, we show that α-helices in bind B55 through an evolutionary conserved mechanism. Despite large diversity sequence and composition, these share key amino acid determinants engage discrete hydrophobic electrostatic patches. Using deep learning...
Recent studies have shown that inositol 1,4,5-trisphosphate 3-kinase isoform B (IP3KB) possesses important roles in the development of immune cells. IP3KB can be targeted to multiple cellular compartments, among them nuclear localization and binding close proximity plasma membrane. The is only IP3K almost ubiquitously expressed mammalian Detailed mechanisms its targeting regulation will understanding role Ins(1,4,5)P(3) phosphorylation on subcellular calcium signaling compartment-specific...
Tight regulation of the APC/C-Cdc20 ubiquitin ligase that targets cyclin B1 for degradation is important mitotic fidelity. The spindle assembly checkpoint (SAC) inhibits Cdc20 through complex (MCC). In addition, phosphorylation by B1–Cdk1 independently APC/C–Cdc20 activation. This creates a conundrum how activated before degradation. Here, we show MCC component BubR1 harbors both inhibition and activation activities, allowing cross-talk between two pathways. Specifically, acts as substrate...
Abstract Phosphoprotein phosphatases (PPPs) dephosphorylate Serine (Ser)/Threonine (Thr) residues to regulate major signaling pathways and cellular transitions. Despite the central role of PPPs substrates in most processes determinants phosphatase specificity are poorly understood. This is because methods investigate this at scale lacking. Here we develop a novel vitro assay, MRBLE:Dephos, that allows multiplexing dephosphorylation reactions determine preferences. Using establish amino acid...
Every living cell relies on signal transduction pathways comprised of protein-protein interactions (PPIs).In many cases, these PPIs are between a folded protein domain and short linear motif (SLiM) within an unstructured region protein.As result this small interaction interface (3-10 amino acids), the affinities SLiM-mediated typically weak (Kds ~1-10 µM), allowing physiologically relevant changes in cellular concentrations either partner to dictate occupancy thereby transmit...
Signal transduction pathways rely on dynamic interactions between protein globular domains and short linear motifs (SLiMs). The weak affinities of these are essential to allow fast rewiring signaling downstream responses but also pose technical challenges for interaction detection measurement. We recently developed a technique (MRBLE-pep) that leverages spectrally encoded hydrogel beads measure binding single interest 48 different peptide sequences in small volume. In prior work, we applied...
Abstract Progression through the cell cycle is controlled by regulated and abrupt changes in phosphorylation. 1 Mitotic entry initiated increased phosphorylation of mitotic proteins, a process driven kinases, 2 while exit achieved counteracting dephosphorylation, phosphatases, especially PP2A:B55. 3 While role kinases well-established, recent data have shown that mitosis only successfully when counterbalancing phosphatases are also inhibited. 4 For PP2A:B55, inhibition two intrinsically...
Abstract Widespread adoption of bead-based multiplexed bioassays requires the ability to easily synthesize encoded microspheres and conjugate analytes interest their surface. Here, we present a simple method (MRBLEs 2.0) for efficient high-throughput generation with ratiometric barcode lanthanide encoding (MRBLEs) bearing functional groups downstream bioconjugation. Bead production in MRBLEs 2.0 relies on manual mixing lanthanide/polymer mixtures (each which comprises unique spectral code)...
Tight regulation of the APC/C-Cdc20 ubiquitin ligase that targets Cyclin B1 for degradation is important mitotic fidelity. The spindle assembly checkpoint (SAC) inhibits Cdc20 through complex (MCC). In addition, phosphorylation by B1-Cdk1 independently activation. This creates a conundrum how gets activated prior to degradation. Here we show MCC component BubR1 harbours both inhibition and activation activities, allowing cross-talk between two pathways. Specifically acts as substrate...
Abstract Signal transduction pathways rely on dynamic interactions between protein globular domains and short linear motifs (SLiMs). The weak affinities of these are essential to allow fast rewiring signaling downstream responses, but pose technical challenges for interaction detection measurement. We recently developed a technique (MRBLE-pep) that leverages spectrally encoded hydrogel beads measure binding single 48 different peptide sequences in small volume. In prior work, we applied it...