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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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Structure of the kinase domain of Btk and its interaction with the PH domain.(A) Crystal structure of the isolated Btk kinase domain with mutations in the activation loop. These mutations (L542M, S543T, V555T, R562K, S564A, and P565S) are based on a previous study (Joseph et al., 2013), and they improved the quality of the crystals of the PH-TH-kinase construct. The structure of the mutant kinase is very similar to that of the wild-type Btk kinase domain (PDB: 3OCS). (B) Comparison of the Btk PH-TH-kinase structure (left) and the Akt1 PH-kinase structure (right). In Akt1, The PH domain is docked between the N lobe and the C lobe of the kinase domain, and it blocks substrate access to the catalytic cleft. The lipid-binding site on the PH domain is buried at the PH-kinase interface, and binding of PIP3/IP4 breaks the PH-kinase interface and activates Akt1 (Wu et al., 2010; Calleja et al., 2009).DOI:http://dx.doi.org/10.7554/eLife.06074.008
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fig2s1: Structure of the kinase domain of Btk and its interaction with the PH domain.(A) Crystal structure of the isolated Btk kinase domain with mutations in the activation loop. These mutations (L542M, S543T, V555T, R562K, S564A, and P565S) are based on a previous study (Joseph et al., 2013), and they improved the quality of the crystals of the PH-TH-kinase construct. The structure of the mutant kinase is very similar to that of the wild-type Btk kinase domain (PDB: 3OCS). (B) Comparison of the Btk PH-TH-kinase structure (left) and the Akt1 PH-kinase structure (right). In Akt1, The PH domain is docked between the N lobe and the C lobe of the kinase domain, and it blocks substrate access to the catalytic cleft. The lipid-binding site on the PH domain is buried at the PH-kinase interface, and binding of PIP3/IP4 breaks the PH-kinase interface and activates Akt1 (Wu et al., 2010; Calleja et al., 2009).DOI:http://dx.doi.org/10.7554/eLife.06074.008

Mentions: The overall conformation of the kinase domain, as well as the specific conformation of the activation loop, is very similar in the two structures we have determined (Figure 2—figure supplement 1A). We also determined the structure of the isolated Btk kinase domain with the six mutations described above and found by comparing it to wild-type Btk that the mutations do not introduce major perturbations in the structure (Figure 2—figure supplement 1A). Details of expression, purification, crystallization, and structure determination are in the ‘Materials and methods’.


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)

Structure of the kinase domain of Btk and its interaction with the PH domain.(A) Crystal structure of the isolated Btk kinase domain with mutations in the activation loop. These mutations (L542M, S543T, V555T, R562K, S564A, and P565S) are based on a previous study (Joseph et al., 2013), and they improved the quality of the crystals of the PH-TH-kinase construct. The structure of the mutant kinase is very similar to that of the wild-type Btk kinase domain (PDB: 3OCS). (B) Comparison of the Btk PH-TH-kinase structure (left) and the Akt1 PH-kinase structure (right). In Akt1, The PH domain is docked between the N lobe and the C lobe of the kinase domain, and it blocks substrate access to the catalytic cleft. The lipid-binding site on the PH domain is buried at the PH-kinase interface, and binding of PIP3/IP4 breaks the PH-kinase interface and activates Akt1 (Wu et al., 2010; Calleja et al., 2009).DOI:http://dx.doi.org/10.7554/eLife.06074.008
© Copyright Policy
Related In: Results  -  Collection

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

fig2s1: Structure of the kinase domain of Btk and its interaction with the PH domain.(A) Crystal structure of the isolated Btk kinase domain with mutations in the activation loop. These mutations (L542M, S543T, V555T, R562K, S564A, and P565S) are based on a previous study (Joseph et al., 2013), and they improved the quality of the crystals of the PH-TH-kinase construct. The structure of the mutant kinase is very similar to that of the wild-type Btk kinase domain (PDB: 3OCS). (B) Comparison of the Btk PH-TH-kinase structure (left) and the Akt1 PH-kinase structure (right). In Akt1, The PH domain is docked between the N lobe and the C lobe of the kinase domain, and it blocks substrate access to the catalytic cleft. The lipid-binding site on the PH domain is buried at the PH-kinase interface, and binding of PIP3/IP4 breaks the PH-kinase interface and activates Akt1 (Wu et al., 2010; Calleja et al., 2009).DOI:http://dx.doi.org/10.7554/eLife.06074.008
Mentions: The overall conformation of the kinase domain, as well as the specific conformation of the activation loop, is very similar in the two structures we have determined (Figure 2—figure supplement 1A). We also determined the structure of the isolated Btk kinase domain with the six mutations described above and found by comparing it to wild-type Btk that the mutations do not introduce major perturbations in the structure (Figure 2—figure supplement 1A). Details of expression, purification, crystallization, and structure determination are in the ‘Materials and methods’.

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