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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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Activation of various deletion constructs of Btk.(A) End-point autophosphorylation assay for full-length Btk, the Src-like module, the SH2-kinase construct and the kinase domain in the presence/absence of IP6. The measurements are carried out after incubating proteins (2 μM) with and without IP6 (100 μM) for 2 min in the presence of ATP/Mg2+. (B) End-point autophosphorylation assay for Btk PH-TH-kinase constructs of various linker lengths in the presence/absence of IP6 (100 μM). The measurements are carried out after incubating proteins (2 μM) with and without IP6 (100 μM) for 2 min at the presence of ATP/Mg2+. (C) Activation of the PH-TH-kinase construct (2 μM) with a 13-residue linker in the presence and absence of IP6 (100 μM). (D) Activation of full-length Btk (2 μM) with mutations R133E/Y134E in the presence and absence of IP6 (100 μM). Arg 133 and Tyr 134 are located at the PH-TH/kinase interface.DOI:http://dx.doi.org/10.7554/eLife.06074.014
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fig5s1: Activation of various deletion constructs of Btk.(A) End-point autophosphorylation assay for full-length Btk, the Src-like module, the SH2-kinase construct and the kinase domain in the presence/absence of IP6. The measurements are carried out after incubating proteins (2 μM) with and without IP6 (100 μM) for 2 min in the presence of ATP/Mg2+. (B) End-point autophosphorylation assay for Btk PH-TH-kinase constructs of various linker lengths in the presence/absence of IP6 (100 μM). The measurements are carried out after incubating proteins (2 μM) with and without IP6 (100 μM) for 2 min at the presence of ATP/Mg2+. (C) Activation of the PH-TH-kinase construct (2 μM) with a 13-residue linker in the presence and absence of IP6 (100 μM). (D) Activation of full-length Btk (2 μM) with mutations R133E/Y134E in the presence and absence of IP6 (100 μM). Arg 133 and Tyr 134 are located at the PH-TH/kinase interface.DOI:http://dx.doi.org/10.7554/eLife.06074.014

Mentions: By comparing constructs, we determined which domains in Btk are responsible for the IP6 response. Activation by IP6 required only the PH-TH module and not the SH3 and SH2 domains (Figure 5—figure supplement 1A,B). A PH-TH-kinase construct that contained the PH-TH-SH3 linker and the SH2-kinase linker (with a total linker length of 45 residues) had the same response to IP6 as did full-length Btk (Figure 5—figure supplement 1B). PH-TH-kinase constructs with shorter linkers also responded to IP6. The shortest linker necessary for activation contained only 13 residues from the SH2-kinase linker; we used this construct to determine the crystal structure described earlier (Figure 5—figure supplement 1C). The catalytic activity of the fully activated PH-TH-kinase construct was about threefold lower than that of full-length Btk. This lower activity can be explained by the absence of the SH2 domain.


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)

Activation of various deletion constructs of Btk.(A) End-point autophosphorylation assay for full-length Btk, the Src-like module, the SH2-kinase construct and the kinase domain in the presence/absence of IP6. The measurements are carried out after incubating proteins (2 μM) with and without IP6 (100 μM) for 2 min in the presence of ATP/Mg2+. (B) End-point autophosphorylation assay for Btk PH-TH-kinase constructs of various linker lengths in the presence/absence of IP6 (100 μM). The measurements are carried out after incubating proteins (2 μM) with and without IP6 (100 μM) for 2 min at the presence of ATP/Mg2+. (C) Activation of the PH-TH-kinase construct (2 μM) with a 13-residue linker in the presence and absence of IP6 (100 μM). (D) Activation of full-length Btk (2 μM) with mutations R133E/Y134E in the presence and absence of IP6 (100 μM). Arg 133 and Tyr 134 are located at the PH-TH/kinase interface.DOI:http://dx.doi.org/10.7554/eLife.06074.014
© Copyright Policy
Related In: Results  -  Collection

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

fig5s1: Activation of various deletion constructs of Btk.(A) End-point autophosphorylation assay for full-length Btk, the Src-like module, the SH2-kinase construct and the kinase domain in the presence/absence of IP6. The measurements are carried out after incubating proteins (2 μM) with and without IP6 (100 μM) for 2 min in the presence of ATP/Mg2+. (B) End-point autophosphorylation assay for Btk PH-TH-kinase constructs of various linker lengths in the presence/absence of IP6 (100 μM). The measurements are carried out after incubating proteins (2 μM) with and without IP6 (100 μM) for 2 min at the presence of ATP/Mg2+. (C) Activation of the PH-TH-kinase construct (2 μM) with a 13-residue linker in the presence and absence of IP6 (100 μM). (D) Activation of full-length Btk (2 μM) with mutations R133E/Y134E in the presence and absence of IP6 (100 μM). Arg 133 and Tyr 134 are located at the PH-TH/kinase interface.DOI:http://dx.doi.org/10.7554/eLife.06074.014
Mentions: By comparing constructs, we determined which domains in Btk are responsible for the IP6 response. Activation by IP6 required only the PH-TH module and not the SH3 and SH2 domains (Figure 5—figure supplement 1A,B). A PH-TH-kinase construct that contained the PH-TH-SH3 linker and the SH2-kinase linker (with a total linker length of 45 residues) had the same response to IP6 as did full-length Btk (Figure 5—figure supplement 1B). PH-TH-kinase constructs with shorter linkers also responded to IP6. The shortest linker necessary for activation contained only 13 residues from the SH2-kinase linker; we used this construct to determine the crystal structure described earlier (Figure 5—figure supplement 1C). The catalytic activity of the fully activated PH-TH-kinase construct was about threefold lower than that of full-length Btk. This lower activity can be explained by the absence of the SH2 domain.

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