Stimulation of the catalytic activity of the tyrosine kinase Btk by the adaptor protein Grb2
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eLife assessment
The authors' finding that Btk kinase activation relies on specific interactions with the Grb2 scaffold protein, including for recruitment into signaling clusters on membranes, is an unexpected finding that will potentially be of broad interest. The authors make a case for reinterpretation of the "Saraste dimer" of Btk as a signaling entity and assign roles to the component domains in the Src module in Btk activation, but the data provided are not yet fully convincing for this scenario.
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Abstract
The Tec-family kinase Btk contains a lipid-binding Pleckstrin homology and Tec homology (PH-TH) module connected by a proline-rich linker to a ‘Src module’, an SH3-SH2-kinase unit also found in Src-family kinases and Abl. We showed previously that Btk is activated by PH-TH dimerization, which is triggered on membranes by the phosphatidyl inositol phosphate PIP 3 , or in solution by inositol hexakisphosphate (IP 6 ) (Wang et al., 2015, https://doi.org/10.7554/eLife.06074 ). We now report that the ubiquitous adaptor protein growth-factor-receptor-bound protein 2 (Grb2) binds to and substantially increases the activity of PIP 3 -bound Btk on membranes. Using reconstitution on supported-lipid bilayers, we find that Grb2 can be recruited to membrane-bound Btk through interaction with the proline-rich linker in Btk. This interaction requires intact Grb2, containing both SH3 domains and the SH2 domain, but does not require that the SH2 domain be able to bind phosphorylated tyrosine residues – thus Grb2 bound to Btk is free to interact with scaffold proteins via the SH2 domain. We show that the Grb2-Btk interaction recruits Btk to scaffold-mediated signaling clusters in reconstituted membranes. Our findings indicate that PIP 3 -mediated dimerization of Btk does not fully activate Btk, and that Btk adopts an autoinhibited state at the membrane that is released by Grb2.
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Author Response
Reviewer #2 (Public Review):
The authors unexpectedly found that the protein Grb2, an adaptor protein that mediates the recruitment of the Ras guanine-nucleotide exchange factor, SOS, to the EGF receptor, can be recruited to membranes by the immune cell tyrosine kinase Btk. The authors show, using total internal reflection fluorescence (TIRF) microscopy that the interaction with Grb2 is reversible, dependent on the proline-rich region of Btk, and independent of PIP3. These experiments are well performed and unambiguous.
The authors next asked whether Grb2 binding to Btk influences its kinase activity, by evaluating (i) Btk autophosphorylation and (ii) the phosphorylation of a peptide from the endogenous substrate PLCy1. The readout relies on non-specific antibody-mediated detection of phosphotyrosine but …
Author Response
Reviewer #2 (Public Review):
The authors unexpectedly found that the protein Grb2, an adaptor protein that mediates the recruitment of the Ras guanine-nucleotide exchange factor, SOS, to the EGF receptor, can be recruited to membranes by the immune cell tyrosine kinase Btk. The authors show, using total internal reflection fluorescence (TIRF) microscopy that the interaction with Grb2 is reversible, dependent on the proline-rich region of Btk, and independent of PIP3. These experiments are well performed and unambiguous.
The authors next asked whether Grb2 binding to Btk influences its kinase activity, by evaluating (i) Btk autophosphorylation and (ii) the phosphorylation of a peptide from the endogenous substrate PLCy1. The readout relies on non-specific antibody-mediated detection of phosphotyrosine but nevertheless reveals a concentration-dependent increase in both Btk autophosphorylation and PLCy1 phosphorylation. The experiments, however, have only been performed in duplicate and, particularly in the case of PLCy1 phosphorylation, exhibit enormous variability which is not reflected in the example blot the authors have chosen to display in Figure 3C. Comparison of the same, duplicate experiment presented in Figure 3 Supplement 2 paints a very different picture.
We added an experiment wherein we measure phosphorylation of the PLC𝛾2-peptide fusion by Btk in the presence of different concentrations of Grb2, and we have carried out LC-MS/MS to probe which Tyr are phosphorylated in these experiments. We have also modified our presentation of the Western blot data to allow readers to view each replicate separately. We believe this makes it easier to evaluate the trends observed in each replicate, and because the intensity measured here is only semi-quantitative, due to limitations of the technique, we believe this is a more accurate way to present our results. Both Tyr of the PLC𝛾2-peptide are phosphorylated, as well as one Tyr at the very C-terminus of GFP (Figure 3 – Supplements 3-5).
The authors next sought to determine which domains of Grb2 are required for activation of Btk. Again, these experiments were only performed in duplicates, and the authors’ claims that Grb2 can moderately stimulate the SH3-SH2-kinase module of Grb2 are not well supported by their data (Figure 4C-D).
We have opted to remove the data for the activation of the SH3-SH2-kinase construct (Src module) from the revised manuscript. Upon further inspection, we agree that these experiments only showed a weak trend and believe that much more experimentation is needed to draw firm conclusions regarding this construct. We do still speculate that SH2 linker displacement may contribute to our observations of enhanced catalytic activity of Btk in the presence of Grb2, however this speculation is based solely on previous work with Btk and other kinases (Aryal et al., 2022; Moarefi et al., 1997).
The authors next asked whether Grb2 stimulates Btk by promoting its dimerization and trans- autophosphorylation. The authors measured the diffusion coefficient of Btk on PIP3- containing supported lipid bilayers in the presence and absence of Grb2. They noted that the diffusion coefficient of individual Btk particles decreases with increasing unlabeled Btk, which they interpret as Btk dimerization. Grb2 does not appear to influence the diffusion of Btk on the membrane (Figure 5A). Presumably, the diffusion coefficient reported here is the average of a number of single-molecule tracks, which should result in error bars. It is unclear why these have not been reported. Next, the authors assessed the ability of Grb2 to stimulate a mutant of Btk that is impaired in its ability to dimerize on PIP3-containing membranes. In contrast to wild-type Btk, autophosphorylation of dimerization-deficient Btk is not enhanced by Grb2. Whilst the data are consistent with this conclusion, again, the experiment has only been repeated once and the western blot presented in Figure 5 Supplement 2 is unreadable. It is also puzzling why Grb2 gets phosphorylated in this experiment, but not in the same experiment reported in Figure 3 Supplement 2.
The diffusion coefficient reported here is determined from a large number of single molecule tracks. We have expanded our explanation of how this is done in the Materials and Methods, as well as providing an example of the data and fits from one of the conditions in Figure 4 – Supplement 3. We are now including standard deviation for each diffusion coefficient determined from the fit of the step size distribution.
We have opted to remove the data involving the dimerization-deficient Btk construct. We agree that these results are difficult to interpret.
We have investigated the Grb2 phosphorylation signal and concluded that this is an off-target effect of the antibody. MS/MS cannot detect and phosphorylation on Grb2. We now comment on this in the figure legend of Figure 3 – Supplement 2.
Finally, the authors argue that Grb2 facilitates the recruitment of Btk to molecular condensates of adaptor and scaffold proteins immobilized on a supported lipid bilayer (SLB) (Figure 6). This is a highly complex series of experiments in which various components are added to supported lipid bilayers and the diffusion of labelled Btk is measured. When Btk is added to SLBs containing the LAT adaptor protein (phosphorylated in situ by Hck immobilized on the membrane via its His tag), it exhibits similar mobility to LAT alone, and its mobility is decreased by the addition of Grb2. The addition of the proline-rich region (PRR) of SOS further decreases this mobility. In this final condition, the authors incubate the reactions for 1 h until LAT undergoes a phase transition, forming gel-like, protein-rich domains on the membrane, shown in Figure 6B. The authors’ conclusion that Btk is recruited into these phase-separated domains based on a slow-down in its diffusion is not well supported by the data, which rather indicates that Btk is excluded from these domains (Figure 6B – Btk punctae (green) are almost exclusively found in between the LAT condensates (red)). As such, the restricted mobility of Btk that the authors report may simply reflect the influence of barriers to diffusion on the membrane that result from LAT condensation into phase- separated domains. The authors also present data in Figure 6 Supplement 1 indicating that Grb2 recruitment to Btk is out-competed by SOS-PRR and that Btk does not support the co- recruitment of Grb2 and SOS-PRR to the membrane. These data would appear to suggest that the authors’ interpretation of the decreased mobility of Btk on the membrane may not be correct.
We have now included an example of one of the single molecule videos, overlayed with the surrounding LAT phase, to more directly display the data that was recorded for this experiment. In this video, it is possible to see that the LAT dense phase occupies only some of the observed window, and although it is possible that these dense “islands” function as barriers to Btk diffusion, Btk would be expected to diffuse freely outside of the LAT dense areas of the bilayer. This property can be clearly seen in the video we have now included. This is reminiscent of what was observed previously during the LAT phase transition for tracking of LAT itself (Sun et al., 2022). Given the extensive previous analysis of LAT diffusion on supported lipid bilayers (Lin et al., 2022; Sun et al., 2022), we believe the necessary controls have been included to support our conclusions. However, we agree there is much to be learned about this interaction and we hope that future studies will further investigate the relationship between cytoplasmic kinases and plasma membrane associated signaling clusters.
Reviewer #3 (Public Review):
The study of Nocka and colleagues examines the role of membrane scaffolding in Btk kinase activation by the Grb2 adaptor protein. The studies appear to make a case for a reinterpretation of the "Saraste dimer" of Btk as a signaling entity and assigns roles to the component domains in the Src module in Btk activation. The point of distinction from earlier studies is that this work ascribes a function to an adaptor protein as promoting the kinase activation, rather than vice versa, and also illustrates why Btk can be activated via modes distinct from its close relative, such as Itk. Importantly, these studies address these key questions through membrane tethering of Btk, which is a successful, reductionist way to mimic cellular scenarios. The writing could be improved and can absolutely be more economical in word choice and use; currently, there is a good deal of background to each section that is not always comprehensive or crucial to contextualise the findings, while key information is often omitted. The results are currently not described in a detailed manner so there is an imbalance between the findings, which should be the focus, relative to background and interpretations or models.
We have assessed the manuscript and made many improvements to shift the focus to the findings, while providing only the necessary background for readers unfamiliar with the specifics of Btk and Grb2 signaling and structure.
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eLife assessment
The authors' finding that Btk kinase activation relies on specific interactions with the Grb2 scaffold protein, including for recruitment into signaling clusters on membranes, is an unexpected finding that will potentially be of broad interest. The authors make a case for reinterpretation of the "Saraste dimer" of Btk as a signaling entity and assign roles to the component domains in the Src module in Btk activation, but the data provided are not yet fully convincing for this scenario.
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Reviewer #1 (Public Review):
In this manuscript, Nocka and colleagues reveal a novel layer of regulation of the Btk tyrosine kinase, a key signaling protein in B lymphocyte signaling and an important drug target with 3 recently FDA-approved drugs, by the SH3-SH2 domain-containing adaptor protein Grb2. The authors nicely demonstrate a critical role of the interaction of the Grb2 SH3 domains with the Pro-rich linker C-terminal to the Btk PH-TH domains on membranes for full kinase activation of Btk. Hence this interaction recruits Btk to scaffold-mediated signaling clusters.
This is a technically sound paper with high-quality experiments. The manuscript is easy to follow and excellently written. The findings are novel and of high relevance towards a complete understanding of Btk regulation and signaling in cancer and normal cells.
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Reviewer #2 (Public Review):
The authors unexpectedly found that the protein Grb2, an adaptor protein that mediates the recruitment of the Ras guanine-nucleotide exchange factor, SOS, to the EGF receptor, can be recruited to membranes by the immune cell tyrosine kinase Btk. The authors show, using total internal reflection fluorescence (TIRF) microscopy that the interaction with Grb2 is reversible, dependent on the proline-rich region of Btk, and independent of PIP3. These experiments are well performed and unambiguous.
The authors next asked whether Grb2 binding to Btk influences its kinase activity, by evaluating (i) Btk autophosphorylation and (ii) the phosphorylation of a peptide from the endogenous substrate PLC1. The readout relies on non-specific antibody-mediated detection of phosphotyrosine but nevertheless reveals a …
Reviewer #2 (Public Review):
The authors unexpectedly found that the protein Grb2, an adaptor protein that mediates the recruitment of the Ras guanine-nucleotide exchange factor, SOS, to the EGF receptor, can be recruited to membranes by the immune cell tyrosine kinase Btk. The authors show, using total internal reflection fluorescence (TIRF) microscopy that the interaction with Grb2 is reversible, dependent on the proline-rich region of Btk, and independent of PIP3. These experiments are well performed and unambiguous.
The authors next asked whether Grb2 binding to Btk influences its kinase activity, by evaluating (i) Btk autophosphorylation and (ii) the phosphorylation of a peptide from the endogenous substrate PLC1. The readout relies on non-specific antibody-mediated detection of phosphotyrosine but nevertheless reveals a concentration-dependent increase in both Btk autophosphorylation and PLCy1 phosphorylation. The experiments, however, have only been performed in duplicate and, particularly in the case of PLCy1 phosphorylation, exhibit enormous variability which is not reflected in the example blot the authors have chosen to display in Figure 3C. Comparison of the same, duplicate experiment presented in Figure 3 Supplement 2 paints a very different picture.
The authors next sought to determine which domains of Grb2 are required for activation of Btk. Again, these experiments were only performed in duplicates, and the authors' claims that Grb2 can moderately stimulate the SH3-SH2-kinase module of Grb2 are not well supported by their data (Figure 4C-D).
The authors next asked whether Grb2 stimulates Btk by promoting its dimerization and trans-autophosphorylation. The authors measured the diffusion coefficient of Btk on PIP3-containing supported lipid bilayers in the presence and absence of Grb2. They noted that the diffusion coefficient of individual Btk particles decreases with increasing unlabeled Btk, which they interpret as Btk dimerization. Grb2 does not appear to influence the diffusion of Btk on the membrane (Figure 5A). Presumably, the diffusion coefficient reported here is the average of a number of single-molecule tracks, which should result in error bars. It is unclear why these have not been reported. Next, the authors assessed the ability of Grb2 to stimulate a mutant of Btk that is impaired in its ability to dimerize on PIP3-containing membranes. In contrast to wild-type Btk, autophosphorylation of dimerization-deficient Btk is not enhanced by Grb2. Whilst the data are consistent with this conclusion, again, the experiment has only been repeated once and the western blot presented in Figure 5 Supplement 2 is unreadable. It is also puzzling why Grb2 gets phosphorylated in this experiment, but not in the same experiment reported in Figure 3 Supplement 2.
Finally, the authors argue that Grb2 facilitates the recruitment of Btk to molecular condensates of adaptor and scaffold proteins immobilized on a supported lipid bilayer (SLB) (Figure 6). This is a highly complex series of experiments in which various components are added to supported lipid bilayers and the diffusion of labelled Btk is measured. When Btk is added to SLBs containing the LAT adaptor protein (phosphorylated in situ by Hck immobilized on the membrane via its His tag), it exhibits similar mobility to LAT alone, and its mobility is decreased by the addition of Grb2. The addition of the proline-rich region (PRR) of SOS further decreases this mobility. In this final condition, the authors incubate the reactions for 1 h until LAT undergoes a phase transition, forming gel-like, protein-rich domains on the membrane, shown in Figure 6B. The authors' conclusion that Btk is recruited into these phase-separated domains based on a slow-down in its diffusion is not well supported by the data, which rather indicates that Btk is excluded from these domains (Figure 6B - Btk punctae (green) are almost exclusively found in between the LAT condensates (red)). As such, the restricted mobility of Btk that the authors report may simply reflect the influence of barriers to diffusion on the membrane that result from LAT condensation into phase-separated domains. The authors also present data in Figure 6 Supplement 1 indicating that Grb2 recruitment to Btk is out-competed by SOS-PRR and that Btk does not support the co-recruitment of Grb2 and SOS-PRR to the membrane. These data would appear to suggest that the authors' interpretation of the decreased mobility of Btk on the membrane may not be correct.
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Reviewer #3 (Public Review):
The study of Nocka and colleagues examines the role of membrane scaffolding in Btk kinase activation by the Grb2 adaptor protein. The studies appear to make a case for a reinterpretation of the "Saraste dimer" of Btk as a signaling entity and assigns roles to the component domains in the Src module in Btk activation. The point of distinction from earlier studies is that this work ascribes a function to an adaptor protein as promoting the kinase activation, rather than vice versa, and also illustrates why Btk can be activated via modes distinct from its close relative, such as Itk. Importantly, these studies address these key questions through membrane tethering of Btk, which is a successful, reductionist way to mimic cellular scenarios. The writing could be improved and can absolutely be more economical in …
Reviewer #3 (Public Review):
The study of Nocka and colleagues examines the role of membrane scaffolding in Btk kinase activation by the Grb2 adaptor protein. The studies appear to make a case for a reinterpretation of the "Saraste dimer" of Btk as a signaling entity and assigns roles to the component domains in the Src module in Btk activation. The point of distinction from earlier studies is that this work ascribes a function to an adaptor protein as promoting the kinase activation, rather than vice versa, and also illustrates why Btk can be activated via modes distinct from its close relative, such as Itk. Importantly, these studies address these key questions through membrane tethering of Btk, which is a successful, reductionist way to mimic cellular scenarios. The writing could be improved and can absolutely be more economical in word choice and use; currently, there is a good deal of background to each section that is not always comprehensive or crucial to contextualise the findings, while key information is often omitted. The results are currently not described in a detailed manner so there is an imbalance between the findings, which should be the focus, relative to background and interpretations or models.
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