Stimulation of the catalytic activity of the tyrosine kinase Btk by the adaptor protein Grb2

Nocka, Laura M.; Eisen, Timothy J.; Iavarone, Anthony T.; Groves, Jay T.; Kuriyan, John

doi:10.7554/elife.82676

Cited by 5 publications

(9 citation statements)

References 72 publications

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“…The extent to which the intrinsically disordered PRR region of BTK participates in additional, perhaps fuzzy, interactions with the BTK SH3–SH2–kinase region remains to be investigated. As well, interactions of the N-terminal region of BTK with both positive and negative regulators ( Liu et al, 2001 ; Nocka et al, 2023 ; Tsukada et al, 1994 ) may alter the dynamic characteristics observed here. It is also possible that the dynamic linker between the PHTH and SH3 domains of BTK may mediate phase separation ( Shelby et al, 2023 ) and clustering into signaling competent condensates.…”

Section: Discussionmentioning

confidence: 90%

Conformational heterogeneity of the BTK PHTH domain drives multiple regulatory states

Lin

Kueffer

Juneja

et al. 2024

eLife

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Full-length BTK has been refractory to structural analysis. The nearest full-length structure of BTK to date consists of the autoinhibited SH3-SH2-kinase core. Precisely how the BTK N-terminal domains (the Pleckstrin homology/Tec homology (PHTH) domain and proline-rich regions (PRR) contain linker) contribute to BTK regulation remains unclear. We have produced crystals of full-length BTK for the first time but despite efforts to stabilize the autoinhibited state, the diffraction data still reveals only the SH3-SH2-kinase core with no electron density visible for the PHTH-PRR segment. CryoEM data of full-length BTK, on the other hand, provide the first view of the PHTH domain within full-length BTK. CryoEM reconstructions support conformational heterogeneity in the PHTH-PRR region wherein the globular PHTH domain adopts a range of states arrayed around the autoinhibited SH3-SH2-kinase core. On the way to activation, disassembly of the SH3-SH2-kinase core opens a new autoinhibitory site on the kinase domain for PHTH domain binding that is ultimately released upon interaction of PHTH with PIP3. Membrane-induced dimerization activates BTK and we present here a crystal structure of an activation loop swapped BTK kinase domain dimer that likely represents the conformational state leading to trans-autophosphorylation. Together, these data provide the first structural elucidation of full-length BTK and allow a deeper understanding of allosteric control over the BTK kinase domain during distinct stages of activation.

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Section: Discussionmentioning

confidence: 90%

Conformational heterogeneity of the BTK PHTH domain drives multiple regulatory states

Lin

Kueffer

Juneja

et al. 2024

eLife

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“…Peptides were extracted in 50% acetonitrile, 5% formic acid, 45% water, and dried with a vacuum centrifuge before mass spectrometric analysis. Samples of purified intact proteins and trypsin-digested proteins were submitted to the QB3/Chemistry Mass Spectrometry Facility at the University of California, Berkeley and analyzed using Synapt and Orbitrap instruments, as described elsewhere 46,47 .…”

Section: Methodsmentioning

confidence: 99%

Dissecting neurofilament tail sequence-phosphorylation-structure relationships with multicomponent reconstituted protein brushes

Ding,

Yokokura,

Wang

et al. 2024

Preprint

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Neurofilaments (NFs) are multi-subunit, bottlebrush-shaped intermediate filaments abundant in the axonal cytoskeleton, with "bristles" composed of the subunits' disordered tail domains. Precisely how the tails' variable charge patterns and repetitive phosphorylation sites mediate their conformation within the brush remains an open question in axonal biology. We address this problem by grafting recombinant NF tail protein constructs (NFL, NFM, and NFH) to functionalized substrates, forming phosphorylatable brushes of defined stoichiometry. Atomic force microscopy reveals that NFM-based brushes are highly extended, while brushes incorporating the much larger NFH are surprisingly compact even after multisite phosphorylation. A self-consistent field theory predicts multilayered brush morphologies for NFM and phosphorylated NFH brushes. Further experiments with designed mutants reveal that N-terminal negative charges in NFH repel phosphorylated residues to generate the multilayer morphology and that charge segregation in NFM promotes collapsed conformations, lending new insight into how NF tail sequence features determine protein brush conformation.

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“…We purified three variants of the BTK PH-TH module (residues 1-171): PH-TH WT , PH-TH E45K (the activating variant discussed above), and PH-TH Y42K (the mutation at the dimer interface that we suspect destabilizes the PH domain), all tagged with mNeon-Green at the C terminus. Bilayer adsorption was quantified by imaging using total internal reflection fluorescence (TIRF) microscopy, which can discriminate between protein in solution and on the bilayer, as described previously for the BTK PH-TH module [62] (Figure 8A).…”

Section: The Mutation E45k In the Btk Ph-th Module Promotes Membrane ...mentioning

confidence: 99%

“…80 µL of the SUV mixture was added to each well of an etched glass slide mounted onto a Sticky Slide VI 0.4 (Ibidi GMBH) and incubated for 30 min at room temperature. Each well was then washed with 1x hanks balanced salt solution (HBSS) under vacuum as described [62],…”

Section: Supported-lipid Bilayer Preparationmentioning

confidence: 99%

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Conditional Requirement for Dimerization of the Membrane-Binding Module of BTK

Eisen,

Ghaffari-Kashani,

Groves

et al. 2023

Preprint

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Bruton’s tyrosine kinase (BTK) functions in many types of immune cells and is a major chemotherapeutic target. BTK is activated at the plasma membrane and can dimerize through its pleckstrin-homology and contiguous tec-homology domains (the PH–TH module). The features that support dimerization of the PH–TH module are unique to BTK among the Tec kinases and appeared relatively recently in evolutionary history. Previous studies suggest that dimerization enhances kinase activity by promotingtransautophosphorylation, but whether BTK dimerizes in cells via the PH–TH module, and whether this dimerization controls signaling, is unknown. To address this question, we developed a high-throughput mutagenesis assay for BTK function in B cells and T cells. We measured the fitness costs for thousands of point mutations in the PH–TH module and kinase domain, allowing us to assess whether dimerization of the PH–TH module and BTK kinase activity are necessary for function. In Ramos B cells we find that neither dimerization nor kinase activity is required for BTK signaling. Instead, in Ramos cells, BTK signaling is enhanced by mutations that increase membrane adsorption, even at the cost of reduced PH–TH dimerization. In contrast, in Jurkat T cells, we find that BTK signaling depends on PH–TH dimerization and also on kinase activity. These results provide a framework for understanding why PH–TH dimerization is a more recent evolutionary innovation that is not conserved across the Tec family.

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Stimulation of the catalytic activity of the tyrosine kinase Btk by the adaptor protein Grb2

Cited by 5 publications

References 72 publications

Conformational heterogeneity of the BTK PHTH domain drives multiple regulatory states

Conformational heterogeneity of the BTK PHTH domain drives multiple regulatory states

Dissecting neurofilament tail sequence-phosphorylation-structure relationships with multicomponent reconstituted protein brushes

Conditional Requirement for Dimerization of the Membrane-Binding Module of BTK

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