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Autoinhibition of Bruton's tyrosine kinase (Btk) and activation by soluble inositol hexakisphosphate.

Wang Q, Vogan EM, Nocka LM, Rosen CE, Zorn JA, Harrison SC, Kuriyan J - Elife (2015)

Bottom Line: In addition to the expected activation of Btk by membranes containing phosphatidylinositol triphosphate (PIP3), we found that inositol hexakisphosphate (IP6), a soluble signaling molecule found in both animal and plant cells, also activates Btk.This activation is a consequence of a transient PH-TH dimerization induced by IP6, which promotes transphosphorylation of the kinase domains.Sequence comparisons with other Tec-family kinases suggest that activation by IP6 is unique to Btk.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States.

ABSTRACT
Bruton's tyrosine kinase (Btk), a Tec-family tyrosine kinase, is essential for B-cell function. We present crystallographic and biochemical analyses of Btk, which together reveal molecular details of its autoinhibition and activation. Autoinhibited Btk adopts a compact conformation like that of inactive c-Src and c-Abl. A lipid-binding PH-TH module, unique to Tec kinases, acts in conjunction with the SH2 and SH3 domains to stabilize the inactive conformation. In addition to the expected activation of Btk by membranes containing phosphatidylinositol triphosphate (PIP3), we found that inositol hexakisphosphate (IP6), a soluble signaling molecule found in both animal and plant cells, also activates Btk. This activation is a consequence of a transient PH-TH dimerization induced by IP6, which promotes transphosphorylation of the kinase domains. Sequence comparisons with other Tec-family kinases suggest that activation by IP6 is unique to Btk.

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Autoinhibition of Btk.(A) Activation of full-length bovine Btk (residues 1 to 659, 2 μM). Reactions are carried out in the presence of 10 mM Mg2+, 150 mM NaCl, 1 mM ATP, 25 mM Tris-HCl pH 8.0. The level of autophosphorylation is assayed by immunoblotting an SDS-PAGE gel with a non-specific, anti-phosphotyrosine antibody (4G10, EMD Millipore) (upper panel). The amount of total protein loaded on the gel is measured by coomassie-blue staining. The kinase activity of Btk is assayed by a continuous kinase-coupled colorimetric assay, in the presence of 1 mM PLC-γ2 peptide substrate. See methods for detailed experimental procedures. (B) Comparison of the activation of the Btk Src-like module (residues 217 to 659), SH2-kinase (residues 270 to 659), and the kinase domain (residues 394 to 659). The SH2-kinase construct activates substantially faster than full-length Btk and the Src-like module of Btk. Activated full-length Btk degrades to a small extent over time, which results in some lower molecule-weight bands being detected on the western blot. (C) Activation of full-length Btk with mutations Y223A and Y268A. Tyr 223 and Tyr 268 are on the SH3/SH2-linker interface, and the two mutants activate faster than wild-type Btk. (D) Activation of full-length Btk with a double mutation (R134E/Y133E). Arg 134 and Tyr 133 are located at the PH-TH/kinase interface.DOI:http://dx.doi.org/10.7554/eLife.06074.012
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fig4: Autoinhibition of Btk.(A) Activation of full-length bovine Btk (residues 1 to 659, 2 μM). Reactions are carried out in the presence of 10 mM Mg2+, 150 mM NaCl, 1 mM ATP, 25 mM Tris-HCl pH 8.0. The level of autophosphorylation is assayed by immunoblotting an SDS-PAGE gel with a non-specific, anti-phosphotyrosine antibody (4G10, EMD Millipore) (upper panel). The amount of total protein loaded on the gel is measured by coomassie-blue staining. The kinase activity of Btk is assayed by a continuous kinase-coupled colorimetric assay, in the presence of 1 mM PLC-γ2 peptide substrate. See methods for detailed experimental procedures. (B) Comparison of the activation of the Btk Src-like module (residues 217 to 659), SH2-kinase (residues 270 to 659), and the kinase domain (residues 394 to 659). The SH2-kinase construct activates substantially faster than full-length Btk and the Src-like module of Btk. Activated full-length Btk degrades to a small extent over time, which results in some lower molecule-weight bands being detected on the western blot. (C) Activation of full-length Btk with mutations Y223A and Y268A. Tyr 223 and Tyr 268 are on the SH3/SH2-linker interface, and the two mutants activate faster than wild-type Btk. (D) Activation of full-length Btk with a double mutation (R134E/Y133E). Arg 134 and Tyr 133 are located at the PH-TH/kinase interface.DOI:http://dx.doi.org/10.7554/eLife.06074.012

Mentions: The fluctuations in the PH-TH orientation generate transient structures in which there would be essentially no clashes between the SH3 domain and the PH-TH module in the merged structures. We initiated multiple MD trajectories from such a composite model (Figure 3A). The inhibitory interactions between each of the regulatory modules and the kinase domain were preserved, but various intra-domain rearrangements occurred during the course of these trajectories. The overall conformation of Btk at the end of the trajectories is very similar to the initial structure. The averaged r.m.s. deviations of the PH-TH module and the SH3 domain between the crystal structures and the end of trajectories, aligned on the Src-like module of the kinase, were ∼4 Å and ∼2.5 Å, respectively. The former is comparable to the value found in simulations of the PH-TH-kinase, but the latter is larger than in simulations of the Src-like module, probably because the SH3 domain reorients with respect to the kinase domain. The SH3 rotation results in a polar interface between SH3 and PH-TH, with good electrostatic complementarity, which preserves the interactions between the SH3 domain, the linker and the N lobe of the kinase (Figure 4B). In these simulations, the r.m.s. deviations of the SH2 domain and the kinase N lobe are ∼2.6 Å and 2.0 Å, respectively, after 360 ns, similar to those of the Src-like module simulations. We emphasize, however, that restrictions in the time scale of the simulations allow only qualitative conclusions and do not give a high-resolution picture of the intact molecule. Structures from several time points in the simulation of the composite model are deposited on the eLife website (Figure 3—source data 1).10.7554/eLife.06074.012Figure 4.Autoinhibition of Btk.


Autoinhibition of Bruton's tyrosine kinase (Btk) and activation by soluble inositol hexakisphosphate.

Wang Q, Vogan EM, Nocka LM, Rosen CE, Zorn JA, Harrison SC, Kuriyan J - Elife (2015)

Autoinhibition of Btk.(A) Activation of full-length bovine Btk (residues 1 to 659, 2 μM). Reactions are carried out in the presence of 10 mM Mg2+, 150 mM NaCl, 1 mM ATP, 25 mM Tris-HCl pH 8.0. The level of autophosphorylation is assayed by immunoblotting an SDS-PAGE gel with a non-specific, anti-phosphotyrosine antibody (4G10, EMD Millipore) (upper panel). The amount of total protein loaded on the gel is measured by coomassie-blue staining. The kinase activity of Btk is assayed by a continuous kinase-coupled colorimetric assay, in the presence of 1 mM PLC-γ2 peptide substrate. See methods for detailed experimental procedures. (B) Comparison of the activation of the Btk Src-like module (residues 217 to 659), SH2-kinase (residues 270 to 659), and the kinase domain (residues 394 to 659). The SH2-kinase construct activates substantially faster than full-length Btk and the Src-like module of Btk. Activated full-length Btk degrades to a small extent over time, which results in some lower molecule-weight bands being detected on the western blot. (C) Activation of full-length Btk with mutations Y223A and Y268A. Tyr 223 and Tyr 268 are on the SH3/SH2-linker interface, and the two mutants activate faster than wild-type Btk. (D) Activation of full-length Btk with a double mutation (R134E/Y133E). Arg 134 and Tyr 133 are located at the PH-TH/kinase interface.DOI:http://dx.doi.org/10.7554/eLife.06074.012
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4384635&req=5

fig4: Autoinhibition of Btk.(A) Activation of full-length bovine Btk (residues 1 to 659, 2 μM). Reactions are carried out in the presence of 10 mM Mg2+, 150 mM NaCl, 1 mM ATP, 25 mM Tris-HCl pH 8.0. The level of autophosphorylation is assayed by immunoblotting an SDS-PAGE gel with a non-specific, anti-phosphotyrosine antibody (4G10, EMD Millipore) (upper panel). The amount of total protein loaded on the gel is measured by coomassie-blue staining. The kinase activity of Btk is assayed by a continuous kinase-coupled colorimetric assay, in the presence of 1 mM PLC-γ2 peptide substrate. See methods for detailed experimental procedures. (B) Comparison of the activation of the Btk Src-like module (residues 217 to 659), SH2-kinase (residues 270 to 659), and the kinase domain (residues 394 to 659). The SH2-kinase construct activates substantially faster than full-length Btk and the Src-like module of Btk. Activated full-length Btk degrades to a small extent over time, which results in some lower molecule-weight bands being detected on the western blot. (C) Activation of full-length Btk with mutations Y223A and Y268A. Tyr 223 and Tyr 268 are on the SH3/SH2-linker interface, and the two mutants activate faster than wild-type Btk. (D) Activation of full-length Btk with a double mutation (R134E/Y133E). Arg 134 and Tyr 133 are located at the PH-TH/kinase interface.DOI:http://dx.doi.org/10.7554/eLife.06074.012
Mentions: The fluctuations in the PH-TH orientation generate transient structures in which there would be essentially no clashes between the SH3 domain and the PH-TH module in the merged structures. We initiated multiple MD trajectories from such a composite model (Figure 3A). The inhibitory interactions between each of the regulatory modules and the kinase domain were preserved, but various intra-domain rearrangements occurred during the course of these trajectories. The overall conformation of Btk at the end of the trajectories is very similar to the initial structure. The averaged r.m.s. deviations of the PH-TH module and the SH3 domain between the crystal structures and the end of trajectories, aligned on the Src-like module of the kinase, were ∼4 Å and ∼2.5 Å, respectively. The former is comparable to the value found in simulations of the PH-TH-kinase, but the latter is larger than in simulations of the Src-like module, probably because the SH3 domain reorients with respect to the kinase domain. The SH3 rotation results in a polar interface between SH3 and PH-TH, with good electrostatic complementarity, which preserves the interactions between the SH3 domain, the linker and the N lobe of the kinase (Figure 4B). In these simulations, the r.m.s. deviations of the SH2 domain and the kinase N lobe are ∼2.6 Å and 2.0 Å, respectively, after 360 ns, similar to those of the Src-like module simulations. We emphasize, however, that restrictions in the time scale of the simulations allow only qualitative conclusions and do not give a high-resolution picture of the intact molecule. Structures from several time points in the simulation of the composite model are deposited on the eLife website (Figure 3—source data 1).10.7554/eLife.06074.012Figure 4.Autoinhibition of Btk.

Bottom Line: In addition to the expected activation of Btk by membranes containing phosphatidylinositol triphosphate (PIP3), we found that inositol hexakisphosphate (IP6), a soluble signaling molecule found in both animal and plant cells, also activates Btk.This activation is a consequence of a transient PH-TH dimerization induced by IP6, which promotes transphosphorylation of the kinase domains.Sequence comparisons with other Tec-family kinases suggest that activation by IP6 is unique to Btk.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States.

ABSTRACT
Bruton's tyrosine kinase (Btk), a Tec-family tyrosine kinase, is essential for B-cell function. We present crystallographic and biochemical analyses of Btk, which together reveal molecular details of its autoinhibition and activation. Autoinhibited Btk adopts a compact conformation like that of inactive c-Src and c-Abl. A lipid-binding PH-TH module, unique to Tec kinases, acts in conjunction with the SH2 and SH3 domains to stabilize the inactive conformation. In addition to the expected activation of Btk by membranes containing phosphatidylinositol triphosphate (PIP3), we found that inositol hexakisphosphate (IP6), a soluble signaling molecule found in both animal and plant cells, also activates Btk. This activation is a consequence of a transient PH-TH dimerization induced by IP6, which promotes transphosphorylation of the kinase domains. Sequence comparisons with other Tec-family kinases suggest that activation by IP6 is unique to Btk.

Show MeSH