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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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Flexibility in the PH domain of Btk and simulation of the kinetics of Btk autophosphorylation.(A) Fluctuations in the β3/β4 loop of the wild-type PH-TH module and its I9A/Y42A/F44A/I95A variant. Each trajectory was sampled every 1 ns. The instantaneous structures are aligned using the crystal structure as the reference. The r.m.s. deviation was calculated for the backbone of residues 36 through 57 of the PH-TH domain. (B) Simulations of the kinetics of Btk autophosphorylation. We consider a very simple two-step Michaelis–Menten model for autophosphorylation, in which two Btk molecules first form an encounter complex, followed by phosphorylation of one of the two molecules in the complex. We assume that the rate of complex formation increases if IP6 is bound, and that the catalytic rate constant for phosphorylation of the other increases if one of the two Btk molecules is phosphorylated. This model is described by the following reaction scheme:(1)Btk+Btk⇌k−1k1Btk·Btk,(2)Btk·Btk→k2pBtk+Btk,(3)pBtk+Btk⇌k−3k3pBtk·pBtk,(4)pBtk·Btk→k4pBtk+pBtk.The on-rate for a diffusion-limited encounter between two proteins is in the range of 108 to 107 M−1s−1. We assume that in the absence of IP6 the on-rate for Btk dimer formation is much slower than this (k1 = k3 = 104 M−1s−1) and that IP6 binding increases the on-rate by 10-fold (k1 = k3 = 105 M−1s−1). The dissociation rate constant is chosen so that the dissociation constant is 2 mM and 200 μM in the absence and presence of IP6, respective (k-1 = k-3 = 20 s−1). The catalytic constant (k4) for activated Btk is set to 1.0 s−1, comparable to the values determined for other tyrosine kinases, such as Src and ZAP70 (Mukherjee et al., 2013). We assume that the catalytic constant is 10-fold smaller for unphosphorylated Btk (k2 = 0.1 s−1). To compare with our experimental data, the initial concentration of Btk was set at 1 μM. The integration of the differential kinetic equations was done using BerkeleyMadonna (http://www.berkeleymadonna.com/). As shown in the diagram, the autophosphorylation kinetics generated by this simple reaction scheme is consistent with the experimentally observed kinetics.DOI:http://dx.doi.org/10.7554/eLife.06074.022
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fig9s1: Flexibility in the PH domain of Btk and simulation of the kinetics of Btk autophosphorylation.(A) Fluctuations in the β3/β4 loop of the wild-type PH-TH module and its I9A/Y42A/F44A/I95A variant. Each trajectory was sampled every 1 ns. The instantaneous structures are aligned using the crystal structure as the reference. The r.m.s. deviation was calculated for the backbone of residues 36 through 57 of the PH-TH domain. (B) Simulations of the kinetics of Btk autophosphorylation. We consider a very simple two-step Michaelis–Menten model for autophosphorylation, in which two Btk molecules first form an encounter complex, followed by phosphorylation of one of the two molecules in the complex. We assume that the rate of complex formation increases if IP6 is bound, and that the catalytic rate constant for phosphorylation of the other increases if one of the two Btk molecules is phosphorylated. This model is described by the following reaction scheme:(1)Btk+Btk⇌k−1k1Btk·Btk,(2)Btk·Btk→k2pBtk+Btk,(3)pBtk+Btk⇌k−3k3pBtk·pBtk,(4)pBtk·Btk→k4pBtk+pBtk.The on-rate for a diffusion-limited encounter between two proteins is in the range of 108 to 107 M−1s−1. We assume that in the absence of IP6 the on-rate for Btk dimer formation is much slower than this (k1 = k3 = 104 M−1s−1) and that IP6 binding increases the on-rate by 10-fold (k1 = k3 = 105 M−1s−1). The dissociation rate constant is chosen so that the dissociation constant is 2 mM and 200 μM in the absence and presence of IP6, respective (k-1 = k-3 = 20 s−1). The catalytic constant (k4) for activated Btk is set to 1.0 s−1, comparable to the values determined for other tyrosine kinases, such as Src and ZAP70 (Mukherjee et al., 2013). We assume that the catalytic constant is 10-fold smaller for unphosphorylated Btk (k2 = 0.1 s−1). To compare with our experimental data, the initial concentration of Btk was set at 1 μM. The integration of the differential kinetic equations was done using BerkeleyMadonna (http://www.berkeleymadonna.com/). As shown in the diagram, the autophosphorylation kinetics generated by this simple reaction scheme is consistent with the experimentally observed kinetics.DOI:http://dx.doi.org/10.7554/eLife.06074.022

Mentions: Our preliminary studies indicate that the IP6-induced dimerization of the Btk PH-TH module is very weak, with a dissociation constant that is likely to be greater than 100 μM. It might appear counter-intuitive that IP6 can markedly accelerate activation at 2 μM Btk concentration, well below the dissociation constant. A simple calculation shows that even a modest increase in a small dimer population can lead to a sharp change in the rate of activation because the reaction is autocatalytic. The initial production of phosphorylated Btk drives the further production of this species at an accelerating rate (Figure 9—figure supplement 1B).


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)

Flexibility in the PH domain of Btk and simulation of the kinetics of Btk autophosphorylation.(A) Fluctuations in the β3/β4 loop of the wild-type PH-TH module and its I9A/Y42A/F44A/I95A variant. Each trajectory was sampled every 1 ns. The instantaneous structures are aligned using the crystal structure as the reference. The r.m.s. deviation was calculated for the backbone of residues 36 through 57 of the PH-TH domain. (B) Simulations of the kinetics of Btk autophosphorylation. We consider a very simple two-step Michaelis–Menten model for autophosphorylation, in which two Btk molecules first form an encounter complex, followed by phosphorylation of one of the two molecules in the complex. We assume that the rate of complex formation increases if IP6 is bound, and that the catalytic rate constant for phosphorylation of the other increases if one of the two Btk molecules is phosphorylated. This model is described by the following reaction scheme:(1)Btk+Btk⇌k−1k1Btk·Btk,(2)Btk·Btk→k2pBtk+Btk,(3)pBtk+Btk⇌k−3k3pBtk·pBtk,(4)pBtk·Btk→k4pBtk+pBtk.The on-rate for a diffusion-limited encounter between two proteins is in the range of 108 to 107 M−1s−1. We assume that in the absence of IP6 the on-rate for Btk dimer formation is much slower than this (k1 = k3 = 104 M−1s−1) and that IP6 binding increases the on-rate by 10-fold (k1 = k3 = 105 M−1s−1). The dissociation rate constant is chosen so that the dissociation constant is 2 mM and 200 μM in the absence and presence of IP6, respective (k-1 = k-3 = 20 s−1). The catalytic constant (k4) for activated Btk is set to 1.0 s−1, comparable to the values determined for other tyrosine kinases, such as Src and ZAP70 (Mukherjee et al., 2013). We assume that the catalytic constant is 10-fold smaller for unphosphorylated Btk (k2 = 0.1 s−1). To compare with our experimental data, the initial concentration of Btk was set at 1 μM. The integration of the differential kinetic equations was done using BerkeleyMadonna (http://www.berkeleymadonna.com/). As shown in the diagram, the autophosphorylation kinetics generated by this simple reaction scheme is consistent with the experimentally observed kinetics.DOI:http://dx.doi.org/10.7554/eLife.06074.022
© Copyright Policy
Related In: Results  -  Collection

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

fig9s1: Flexibility in the PH domain of Btk and simulation of the kinetics of Btk autophosphorylation.(A) Fluctuations in the β3/β4 loop of the wild-type PH-TH module and its I9A/Y42A/F44A/I95A variant. Each trajectory was sampled every 1 ns. The instantaneous structures are aligned using the crystal structure as the reference. The r.m.s. deviation was calculated for the backbone of residues 36 through 57 of the PH-TH domain. (B) Simulations of the kinetics of Btk autophosphorylation. We consider a very simple two-step Michaelis–Menten model for autophosphorylation, in which two Btk molecules first form an encounter complex, followed by phosphorylation of one of the two molecules in the complex. We assume that the rate of complex formation increases if IP6 is bound, and that the catalytic rate constant for phosphorylation of the other increases if one of the two Btk molecules is phosphorylated. This model is described by the following reaction scheme:(1)Btk+Btk⇌k−1k1Btk·Btk,(2)Btk·Btk→k2pBtk+Btk,(3)pBtk+Btk⇌k−3k3pBtk·pBtk,(4)pBtk·Btk→k4pBtk+pBtk.The on-rate for a diffusion-limited encounter between two proteins is in the range of 108 to 107 M−1s−1. We assume that in the absence of IP6 the on-rate for Btk dimer formation is much slower than this (k1 = k3 = 104 M−1s−1) and that IP6 binding increases the on-rate by 10-fold (k1 = k3 = 105 M−1s−1). The dissociation rate constant is chosen so that the dissociation constant is 2 mM and 200 μM in the absence and presence of IP6, respective (k-1 = k-3 = 20 s−1). The catalytic constant (k4) for activated Btk is set to 1.0 s−1, comparable to the values determined for other tyrosine kinases, such as Src and ZAP70 (Mukherjee et al., 2013). We assume that the catalytic constant is 10-fold smaller for unphosphorylated Btk (k2 = 0.1 s−1). To compare with our experimental data, the initial concentration of Btk was set at 1 μM. The integration of the differential kinetic equations was done using BerkeleyMadonna (http://www.berkeleymadonna.com/). As shown in the diagram, the autophosphorylation kinetics generated by this simple reaction scheme is consistent with the experimentally observed kinetics.DOI:http://dx.doi.org/10.7554/eLife.06074.022
Mentions: Our preliminary studies indicate that the IP6-induced dimerization of the Btk PH-TH module is very weak, with a dissociation constant that is likely to be greater than 100 μM. It might appear counter-intuitive that IP6 can markedly accelerate activation at 2 μM Btk concentration, well below the dissociation constant. A simple calculation shows that even a modest increase in a small dimer population can lead to a sharp change in the rate of activation because the reaction is autocatalytic. The initial production of phosphorylated Btk drives the further production of this species at an accelerating rate (Figure 9—figure supplement 1B).

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