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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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The Saraste dimer of the PH-TH module is critical for Btk activation by IP6.(A) Molecular details of the Saraste dimer interface in the crystal structure of the IP6-bound PH-TH module. (B) End-point autophosphorylation assays for wild-type Btk, Btk L29R/I9R mutant, Btk I94R/I95R mutant, and Btk Y42R/F44R mutant in the presence and absence of IP6 (100 μM). The measurements were made after incubating the proteins with and without IP6 in the presence of ATP/Mg2+ for 5 min. Residues Leu 29, Ile 9, IIe 94, IIe 95 Tyr 42 and Phe 44 are located in the Saraste dimer interface, as shown in panel A. Activation of the Btk Y42R/F44R mutant is shown in the lower panel. (C) Activation of the Btk-Abl fusion construct (1 μM) and that of a variant of this fusion protein in which the Btk PH-TH module is mutated (Y42R/F44R). Measurements were made with 1 μM protein in the presence and absence of IP6 (100 μM).DOI:http://dx.doi.org/10.7554/eLife.06074.020
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fig8: The Saraste dimer of the PH-TH module is critical for Btk activation by IP6.(A) Molecular details of the Saraste dimer interface in the crystal structure of the IP6-bound PH-TH module. (B) End-point autophosphorylation assays for wild-type Btk, Btk L29R/I9R mutant, Btk I94R/I95R mutant, and Btk Y42R/F44R mutant in the presence and absence of IP6 (100 μM). The measurements were made after incubating the proteins with and without IP6 in the presence of ATP/Mg2+ for 5 min. Residues Leu 29, Ile 9, IIe 94, IIe 95 Tyr 42 and Phe 44 are located in the Saraste dimer interface, as shown in panel A. Activation of the Btk Y42R/F44R mutant is shown in the lower panel. (C) Activation of the Btk-Abl fusion construct (1 μM) and that of a variant of this fusion protein in which the Btk PH-TH module is mutated (Y42R/F44R). Measurements were made with 1 μM protein in the presence and absence of IP6 (100 μM).DOI:http://dx.doi.org/10.7554/eLife.06074.020

Mentions: The Saraste dimer is formed by symmetric interactions between helix α1 and strands β3 and β4 of the PH-TH module (Figure 7A). The largely hydrophobic dimer interface has a buried surface area of ∼1000 Å2 on each molecule. Phe 44 and Tyr 42 on strand β3, Ile 9 on strand β1 and IIe 95 on helix α1 create a hydrophobic patch in one subunit of the dimer that packs tightly against the corresponding patch on the other subunit (Figure 8A).10.7554/eLife.06074.020Figure 8.The Saraste dimer of the PH-TH module is critical for Btk activation by IP6.


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)

The Saraste dimer of the PH-TH module is critical for Btk activation by IP6.(A) Molecular details of the Saraste dimer interface in the crystal structure of the IP6-bound PH-TH module. (B) End-point autophosphorylation assays for wild-type Btk, Btk L29R/I9R mutant, Btk I94R/I95R mutant, and Btk Y42R/F44R mutant in the presence and absence of IP6 (100 μM). The measurements were made after incubating the proteins with and without IP6 in the presence of ATP/Mg2+ for 5 min. Residues Leu 29, Ile 9, IIe 94, IIe 95 Tyr 42 and Phe 44 are located in the Saraste dimer interface, as shown in panel A. Activation of the Btk Y42R/F44R mutant is shown in the lower panel. (C) Activation of the Btk-Abl fusion construct (1 μM) and that of a variant of this fusion protein in which the Btk PH-TH module is mutated (Y42R/F44R). Measurements were made with 1 μM protein in the presence and absence of IP6 (100 μM).DOI:http://dx.doi.org/10.7554/eLife.06074.020
© Copyright Policy
Related In: Results  -  Collection

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

fig8: The Saraste dimer of the PH-TH module is critical for Btk activation by IP6.(A) Molecular details of the Saraste dimer interface in the crystal structure of the IP6-bound PH-TH module. (B) End-point autophosphorylation assays for wild-type Btk, Btk L29R/I9R mutant, Btk I94R/I95R mutant, and Btk Y42R/F44R mutant in the presence and absence of IP6 (100 μM). The measurements were made after incubating the proteins with and without IP6 in the presence of ATP/Mg2+ for 5 min. Residues Leu 29, Ile 9, IIe 94, IIe 95 Tyr 42 and Phe 44 are located in the Saraste dimer interface, as shown in panel A. Activation of the Btk Y42R/F44R mutant is shown in the lower panel. (C) Activation of the Btk-Abl fusion construct (1 μM) and that of a variant of this fusion protein in which the Btk PH-TH module is mutated (Y42R/F44R). Measurements were made with 1 μM protein in the presence and absence of IP6 (100 μM).DOI:http://dx.doi.org/10.7554/eLife.06074.020
Mentions: The Saraste dimer is formed by symmetric interactions between helix α1 and strands β3 and β4 of the PH-TH module (Figure 7A). The largely hydrophobic dimer interface has a buried surface area of ∼1000 Å2 on each molecule. Phe 44 and Tyr 42 on strand β3, Ile 9 on strand β1 and IIe 95 on helix α1 create a hydrophobic patch in one subunit of the dimer that packs tightly against the corresponding patch on the other subunit (Figure 8A).10.7554/eLife.06074.020Figure 8.The Saraste dimer of the PH-TH module is critical for Btk activation by IP6.

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
Related in: MedlinePlus