Phosphorylation of tyrosine 90 in SH3 domain is a new regulatory switch controlling Src kinase

  1. Lenka Koudelková
  2. Markéta Pelantová
  3. Zuzana Brůhová
  4. Martin Sztacho
  5. Vojtěch Pavlík
  6. Dalibor Pánek
  7. Jakub Gemperle
  8. Pavel Talacko
  9. Jan Brábek
  10. Daniel Rösel

  • Curated by eLife

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    This manuscript explores the potential regulatory role of a previously unstudied phosphorylation site in the Src kinase SH3 domain. A mutant intended to mimic the phosphorylation of this site, Y90E, shows enhanced activity and transforming capacity, reduced mobility in the lipid bilayer, and a more open catalytic structure. In general, these findings are supported by compelling evidence. The paper will be of interest to biochemists and structural biologists studying new mechanisms that are capable of modulating the allosteric regulation of multi-domain protein kinases.

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Abstract

The activation of Src kinase in cells is strictly controlled by intramolecular inhibitory interactions mediated by SH3 and SH2 domains. They impose structural constraints on the kinase domain holding it in a catalytically non-permissive state. The transition between inactive and active conformation is known to be largely regulated by the phosphorylation state of key tyrosines 416 and 527. Here, we identified that phosphorylation of tyrosine 90 reduces binding affinity of the SH3 domain to its interacting partners, opens the Src structure, and renders Src catalytically active. This is accompanied by an increased affinity to the plasma membrane, decreased membrane motility, and slower diffusion from focal adhesions. Phosphorylation of tyrosine 90 controlling SH3-medited intramolecular inhibitory interaction, analogical to tyrosine 527 regulating SH2-C-terminus bond, enables SH3 and SH2 domains to serve as cooperative but independent regulatory elements. This mechanism allows Src to adopt several distinct conformations of varying catalytic activities and interacting properties, enabling it to operate not as a simple switch but as a tunable regulator functioning as a signalling hub in a variety of cellular processes.

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  1. Version published to 10.7554/elife.82428 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    This paper explores the potential regulatory role of a previously unstudied phosphorylation site in the Src kinase SH3 domain. The data presented conclusively demonstrate that a phosphomimetic mutation of this site, src90E, causes an elevation in Src kinase activity, changes the structure of the Src catalytic domain as determined with a FRET sensor, disrupts certain SH3 domain interactions, causes changes in kinase intracellular dynamicity, and promotes cell invasiveness. Based on the behavior of the phosphomimetic mutant, the idea that constitutive phosphorylation of Y90 could have all of these effects is well-supported by the data. However, in wild-type cells or cells transformed by activated forms of Src, there is no constitutive phosphorylation of this site. Therefore, the question remains whether Y90 phosphorylation occurs to any significant extent in cells, and the data suggesting that it could do so is limited. It also remains to be conclusively established whether Y90 phosphorylation occurs via autophosphorylation.

    Major comments:

    1. Y90 was identified as a site of phosphorylation in Luo et al. It would be helpful if more information were provided about its significance relative to other sites identified in that study. Was it detected in non-transformed cells? Was it a major site? How does it relate to Y416 in abundance? The reference to the identification of the site in a different study from the White lab is made in the discussion but not in the introduction (this should be corrected). How abundant was it that study? A fuller description of its detection would strengthen the rationale for this study. Any additional phosphoproteomics studies that identified it should also be included.

    As indicated in the manuscript (Figure 3C and new 3D), the amount of Y90 phosphorylation increases with the level of Src activation. Standard proteomic/phosphoproteomic data cannot be quantified in absolute values for technical reasons, only relative quantification is possible to some extent. To overcome this issue and address the question of the amount of Y90 phosphorylation, we newly prepared the corresponding stable isotope-labeled phosphopeptides and used them as internal standards. To the best of our knowledge, this allowed us to quantify for the first time the amount of specific tyrosine phosphorylation of Src kinase in cells. We found that in case of WT Src, the major phosphorylation site localized in the activation loop of the kinase domain, Y416, is phosphorylated in 22 % of molecules. In activated Src, this pool of Y416-phosphorylated molecules increases 2,5 times to 57 %. Y90 is phosphorylated in approximately 1 % of WT Src molecules but becomes 5 times more abundant in case of the activated kinase (5,3 % of phosphorylated molecules). This newly added data of absolute Src tyrosine phosphorylation (Figure 3D) is consistent with values we obtained from relative MS quantification of Src variants differing in catalytic activity (Figure 3C). Although the enrichment of Y90 phosphorylation in the catalytically active kinase is lower compared to Y416 phosphorylation in terms of percentage of phosphorylated molecules, it’s increment with respect to the basal state is significantly higher. We believe that this broader dynamic range of Y90 phosphorylation is in agreement with the demonstrated regulatory function of Y90 phosphorylation. We incorporated these new results and methodological approach into the revised manuscript. We also extended the original description of the MS protocol to include a description of relative quantification, which was included in the original manuscript.

    Phosphorylation of Y90 was only detected in Luo et al. and Johnson et al. phosphoproteomic screens. However, phosphorylation of tyrosines homologous to Src Y90 was described in a vast number of proteins. Some of them are mentioned in the discussion e.g., Btk, Abl, p130Cas or Src family kinases Yes and Fyn. The presence of phosphorylation on homologous tyrosines and the evolutionary conserved nature of Y90 in SH3 domains supports relevance of Src Y90 phosphorylation despite the small number of studies that were able to identify it. In our opinion, this can be attributed to its low abundance in the basal state and the technical difficulties of its detection, as discussed below in response to point 2.

    We emphasize the Luo et al. study in the introduction because it was the only study reporting Y90 phosphorylation at the time of the project’s initiation and led us to study Y90 further. Both studies are then mentioned in the discussion, which we believe is appropriate and sufficient.

    1. Related to point 1, is there evidence from the literature indicating a significant site of phosphorylation in Src has been overlooked? Or, was this site only identified because of the recent advances in MS technology and increased sensitivity of this methodology? An introduction to these points would also enhance the rationale for the study.

    In the manuscript discussion, we mention an early study (Erpel et al., 1995) which mapped conserved residues within the binding surface of the Src SH3 domain. It showed that mutation of Y90 to alanine led to partially deregulated Src and reduced affinity of the SH3 domain. Although they acknowledged the importance of Y90 for SH3 domain binding ability, they did not probe or discuss the effect of Y90 phosphorylation status. Furthermore, the level of Src Y90 phosphorylation in untransformed cells is relatively low (20-fold lower than Y416 phosphorylation). It is therefore not surprising that it has not been identified in most general phosphoproteomic studies performed on untransformed cells. In fact, in many of these studies, Y416 phosphorylation was not detected either. The detection of Y90 phosphorylation by Luo et al. likely reflects the fact that it was performed in Src527F-transformed cells, similarly Johnson et al. used HGF-activated cells. Last, we also cannot exclude that the tryptic peptides with Y90/pY90 are less detectable in MS depending on the experimental conditions. In fact, the "heavy" Y90 peptide was consistently much less (10-80 times less) detectable in our hands than the Y416 peptide. This could be because of its worse ionizability, stability in vacuum or some other technical reasons.

    In our approach, we used immunoprecipitated Src molecules to maximize the amount of Src in the sample and targeted MS, which allowed us to specifically detect even low abundant ions/peptides. This represented the critical technical approach that allowed us to consistently detect Y90 phosphorylation in untransformed cells.

    1. The explanation of the MS experiment designed to show that Y90 phosphorylation happens in cells is insufficient in the text. It is not clear why the SYF cells were not used and not clear why the FRET sensor constructs were used. It is also not clear whether or how the proteins were purified before MS analysis. Also, rather than showing the MS data as a relative level, it would be preferable to provide the number of spectra obtained for each peptide/phosphopeptide and compare this also to Y416. A fuller comparison between the phosphorylation of Y90 to that of Y416 is necessary in order to place the potential Y90-mediated phosphoregulation in context.

    We are sorry for the confusing description. With the new quantification data, we have rewritten this section and hopefully made it clearer. We kept the original relative quantification data as they nicely show that abundance of Y90 phosphorylation increases with enhanced activity of Src. However, we added new MS analysis of Src tyrosine phosphorylation performed with labeled peptides as internal standards that provides absolute numbers of Y416 and Y90 phosphorylation in cells. The new
    measurements confirm the original data showing increased Y90 phosphorylation in activated Src variants and suggest that Y90 phosphorylation is not a rare event but represents an important regulatory element in Src activation. Our approach of MS quantification of phosphorylation events using labeled peptides as standards, allowed us, to the best of our knowledge, for the first time, to measure absolute quantities of Y416 and Y90 phosphorylation and therefore also the amount of activated Src molecules in cells.

    For technical reasons, the SrcFRET biosensor was used in all these experiments. We attempted to analyze endogenous Src in several cell lines to assess its Y90 phosphorylation. However, in our hands, the amount of Src efficiently precipitated was never sufficient to detect the "very elusive" phosphopeptide containing Y90. We believe this was not caused by low amounts of Src in the cells,
    but rather because the anti-Src antibody performed much worse than the anti-GFP antibody used for SrcFRET biosensor (two high affinity epitopes) immunoprecipitation. We have previously shown that the SrcFRET biosensor functions in the same way as endogenous Src (Koudelková et al., 2019), and therefore we presume that it is phosphorylated in a similar manner and rate as endogenous Src.

    1. I would like to see conclusive evidence that Y90 phosphorylation is due to autophosphorylation. This would involve relatively simple experiments. As one possibility, an IP kinase assay followed by immunoblotting with a site-specific antibody or MS or other types of phosphopeptide visualization/identification.

    We further addressed the question of Y90 autophosphorylation using a kinase dead version of Src527F bearing K295M substitution. To quantify the amount of phosphorylated Src we applied the identical approach with labeled standards and measured phosphorylation levels of Y416 and Y90. Compared to Src WT and Src527F, phosphorylation of both tyrosines in the kinase dead variant was negligible despite the presence of endogenous Src and other SFKs in the U2OS cells we used for the experiments. These results suggest that phosphorylation of Y90 does indeed depend on the intrinsic kinase activity of Src and is therefore very likely autophosphorylation.

    We have tried to address the question of Src autophosphorylation on Y90 by analyzing the level of Y90 phosphorylation in cells expressing a kinase-inactive SrcFRET construct with open conformation (527FKD) by quantitative MS. Despite the open conformation, SrcFRET527F-KD did not display any significant phosphorylation of neither Y90 nor Y416, even though we used U2OS cells which express endogenous Src and other SFKs. These results suggest that phosphorylation of Y90 depends on catalytic activity of the kinase rather than on compactness of its conformation and is therefore very likely autophosphorylation.

    1. A few other mutations would be interesting to examine in both kinase and transformation assays for comparison to the mutants that were: Y527F Y416F; Y527F Y416F Y90E. The first is a low activity control and the second is for understanding whether Y90E could overcome the lack of Y416 phosphorylation.

    Due to lack of time, we did not perform these experiments. However, we analyzed our new kinasedead 527F mutant for FRET and found that despite its inactive kinase domain and lack of Y416 phosphorylation, it still retains an open conformation. We believe that this is a strong indication that the Y90E kinase-dead mutant would behave the same way, maintaining an open conformation albeit the kinase domain is inactive.

    1. I recommend that the results are discussed in a more circumspect manner. The results presented in Figure 7 on the double mutant, Y527F Y90F, suggest that phosphorylation of Y90 is not a very significant component of Src kinase regulation, at least in these biological contexts. That Y90 phosphorylation isn't a major regulatory factor does not diminish the value of the work describing Y90 phosphorylation. However, it does alter the interpretations. I encourage a more conservative discussion of its importance and a broader discussion of why it isn't a major site of Src phosphorylation, particularly if it is due to autophosphorylation.

    We believe that given our new quantifications showing that Y90 phosphorylation is indeed considerably present and utilized in cells, the original discussion is consistent with the new data and does not need to be changed.

    Reviewer #2 (Public Review):

    The manuscript "Phosphorylation of tyrosine 90 in SH3 domain is a new regulatory switch controlling Src kinase" describes efforts to understand how phosphorylation of tyrosine (Y90) in the SH3 domain of Src affects the activity and function of this multi-domain kinase. The authors find that an Src variant containing a phospho-mimetic mutation (Glu) at position 90 demonstrates elevated activation levels in lysates and cells (Figure 1) and adopts a less compact autoinhibited conformation within the context of a SrcFRET biosensor in lysates (Figures 3A, 3B). A series of pulldown experiments with an isolated SH3 domain (Figure 2A, 2B) or full-length Src (Figure 2C, 2D) that contain the phospho-mimetic Y90E mutation demonstrates that phosphorylation of Tyr90 would likely disrupt the interaction of Src's SH3 domain with intermolecular binding partners and the linker that couples SH2 domain/C-tail binding to autoinhibition, which provides a mechanistic basis for the observed elevated kinase activity of Src Y90E. By performing a series of imaging experiments with a SrcFRET biosensor, the authors show that the Y90E mutation does not show enhanced localization at focal adhesions like a hyperactivated Src mutant (Y527F) that contains a non-phosphorylatable C-tail (Figure 4A). However, using ImFCS combined with TIRF microscopy (Figure 4B), the authors demonstrate that Src Y90E shows similarly reduced mobility (relative to the WT SrcFRET biosensor) at the plasma membrane (especially at focal adhesions) as Src Y527F. Consistent with the elevated kinase activity of Src Y90E, the authors go on to demonstrate that the Src Y90E variant shows an ability to transform fibroblasts-at levels that are intermediate between wild-type Src and the hyperactive Src mutant Y527F (Figure 5). Similarly, Src Y90E confers an intermediate level (between wild-type Src and Src Y527F) of invasiveness and ability to form spheroids. Together, these comprehensive experiments with a Y90 phospho-mimetic strongly support a model where phosphorylation of Src's SH3 domain at Tyr90 would lead to a more intramolecularly disengaged SH3 regulatory domain and enhanced kinase activity in cells.

    Most of the conclusions in this paper are well supported by solid data, but confidence in several assays would be higher if additional technical detail or controls were provided and the biological significance of these findings would be higher if the role that Y90 phosphorylation plays in Src regulation and function were better delineated.

    1. The kinase activity assays in Figures 1C,1D, and 7A need to be scaled to the Src variant levels present in the lysate (quantification of relative Src levels is not provided).

    For kinase activity measurements, we used lysates of equal protein concentrations prepared from cell lines stably expressing Src variants. These cell lines were sorted and repeatedly tested for equal expression of Src constructs using immunodetection of Src on Western blots. We corrected the
    methods section and added this information to the description of kinase assays experimental setup.

    1. More details are required for the experiments quantifying Y90 phosphorylation levels in Figure 3C. The experimental states that equal amounts of IP'd proteins were used for these analyses but there are no details on how this was confirmed. In addition, the experimental states that normalized intensities were used for your quantifying the Y90 phospho-peptide but no details are provided on how normalization was performed (the legend states that a base peptide was used but it is unclear what this means).

    The paragraph on mass spectrometry analysis in the Materials and Methods section has been updated with the required information.

    1. A key question is whether Y90 phosphorylation serves a regulatory role in Src's cellular activity and, if so, what is the regulatory network that mediates this phospho-event. Using a mass spectrometry readout with three Src variants (wild type vs. Y527F vs. E381G) that possess differing kinase activities, the authors demonstrate that Y90 phosphorylation levels correlate to Src's kinase activity (Figure 3C), which they suggest is an indication that this residue is an autophosphorylation site (or phosphorylated by another Src family kinase). However, as Src's kinase activity correlates with SH3 domain disengagement (which leads to a more accessible Y90), it is also entirely possible that another tyrosine kinase is responsible for this phosphorylation event. More importantly, it is unclear under which signaling regime Y90 phosphorylation would play a significant regulatory role. This phospho-event was observed in a previous phospho-proteomic study but it is unclear whether the phosphorylation levels of this site occur high enough stoichiometry to modulate the intracellular function of Src and whether there is a regulatory signaling network that influences Y90 phosphorylation levels.

    We have tried to address the question of Src autophosphorylation on Y90 by analyzing the level of Y90 phosphorylation in cells expressing a kinase-inactive SrcFRET construct with open conformation (527F-KD) by quantitative MS. Despite the open conformation, SrcFRET527F-KD did not display any significant phosphorylation of neither Y90 nor Y416, even though we used U2OS cells which express endogenous Src and other SFKs. These results suggest that phosphorylation of Y90 depends on catalytic activity of the kinase rather than on compactness of its conformation and is therefore very likely autophosphorylation.

    To further support our data on relevance of Y90 phosphorylation in cells, we performed a new MS analysis of Y90 and Y416 phosphorylation in WT and activated Src. This time we used corresponding stable isotope-labeled peptides and phosphopeptides as internal standards for MS quantification. This allowed us to measure absolute amounts of phosphorylated molecules and changes in their numbers, which is information that cannot be acquired by standard biochemical or proteomic approaches and is usually lacking for the majority of known phosphorylation sites. We found that in case of WT Src, the major phosphorylation site localized in the activation loop of the kinase domain, Y416, is phosphorylated in 22,6 % of molecules. In activated Src, this pool of Y416-phosphorylated molecules increases 2,5 times to 57 %. Y90 is phosphorylated in approximately 1 % of WT Src molecules but becomes 5,1 times more abundant in case of the activated kinase (5,3 % of phosphorylated molecules). This newly added data of absolute Src tyrosine phosphorylation (Figure 3D) is consistent with values we obtained from relative MS quantification of Src variants differing in catalytic activity (Figure 3C). Although the enrichment of Y90 phosphorylation in the catalytically active kinase is lower compared to Y416 phosphorylation in terms of percentage of phosphorylated molecules, it’s increment with respect to the basal state is significantly higher. We believe that this broader dynamic range of Y90 phosphorylation is consistent with and reflects the demonstrated regulatory function of Y90 phosphorylation. We incorporated these new results and methodological approach into the revised manuscript.

  3. eLife

    eLife assessment

    This manuscript explores the potential regulatory role of a previously unstudied phosphorylation site in the Src kinase SH3 domain. A mutant intended to mimic the phosphorylation of this site, Y90E, shows enhanced activity and transforming capacity, reduced mobility in the lipid bilayer, and a more open catalytic structure. In general, these findings are supported by compelling evidence. The paper will be of interest to biochemists and structural biologists studying new mechanisms that are capable of modulating the allosteric regulation of multi-domain protein kinases.

  4. eLife

    Reviewer #1 (Public Review):

    This paper explores the potential regulatory role of a previously unstudied phosphorylation site in the Src kinase SH3 domain. The data presented conclusively demonstrate that a phosphomimetic mutation of this site, src90E, causes an elevation in Src kinase activity, changes the structure of the Src catalytic domain as determined with a FRET sensor, disrupts certain SH3 domain interactions, causes changes in kinase intracellular dynamicity, and promotes cell invasiveness. Based on the behavior of the phosphomimetic mutant, the idea that constitutive phosphorylation of Y90 could have all of these effects is well-supported by the data. However, in wild-type cells or cells transformed by activated forms of Src, there is no constitutive phosphorylation of this site. Therefore, the question remains whether Y90 phosphorylation occurs to any significant extent in cells, and the data suggesting that it could do so is limited. It also remains to be conclusively established whether Y90 phosphorylation occurs via autophosphorylation.

    Major comments:

    1. Y90 was identified as a site of phosphorylation in Luo et al. It would be helpful if more information were provided about its significance relative to other sites identified in that study. Was it detected in non-transformed cells? Was it a major site? How does it relate to Y416 in abundance? The reference to the identification of the site in a different study from the White lab is made in the discussion but not in the introduction (this should be corrected). How abundant was it that study? A fuller description of its detection would strengthen the rationale for this study. Any additional phosphoproteomics studies that identified it should also be included.

    2. Related to point 1, is there evidence from the literature indicating a significant site of phosphorylation in Src has been overlooked? Or, was this site only identified because of the recent advances in MS technology and increased sensitivity of this methodology? An introduction to these points would also enhance the rationale for the study.

    3. The explanation of the MS experiment designed to show that Y90 phosphorylation happens in cells is insufficient in the text. It is not clear why the SYF cells were not used and not clear why the FRET sensor constructs were used. It is also not clear whether or how the proteins were purified before MS analysis. Also, rather than showing the MS data as a relative level, it would be preferable to provide the number of spectra obtained for each peptide/phosphopeptide and compare this also to Y416. A fuller comparison between the phosphorylation of Y90 to that of Y416 is necessary in order to place the potential Y90-mediated phosphoregulation in context.

    4. I would like to see conclusive evidence that Y90 phosphorylation is due to autophosphorylation. This would involve relatively simple experiments. As one possibility, an IP kinase assay followed by immunoblotting with a site-specific antibody or MS or other types of phosphopeptide visualization/identification.

    5. A few other mutations would be interesting to examine in both kinase and transformation assays for comparison to the mutants that were: Y527F Y416F; Y527F Y416F Y90E. The first is a low activity control and the second is for understanding whether Y90E could overcome the lack of Y416 phosphorylation.

    5. I recommend that the results are discussed in a more circumspect manner. The results presented in Figure 7 on the double mutant, Y527F Y90F, suggest that phosphorylation of Y90 is not a very significant component of Src kinase regulation, at least in these biological contexts. That Y90 phosphorylation isn't a major regulatory factor does not diminish the value of the work describing Y90 phosphorylation. However, it does alter the interpretations. I encourage a more conservative discussion of its importance and a broader discussion of why it isn't a major site of Src phosphorylation, particularly if it is due to autophosphorylation.

  5. eLife

    Reviewer #2 (Public Review):

    The manuscript "Phosphorylation of tyrosine 90 in SH3 domain is a new regulatory switch controlling Src kinase" describes efforts to understand how phosphorylation of tyrosine (Y90) in the SH3 domain of Src affects the activity and function of this multi-domain kinase. The authors find that an Src variant containing a phospho-mimetic mutation (Glu) at position 90 demonstrates elevated activation levels in lysates and cells (Figure 1) and adopts a less compact autoinhibited conformation within the context of a SrcFRET biosensor in lysates (Figures 3A, 3B). A series of pulldown experiments with an isolated SH3 domain (Figure 2A, 2B) or full-length Src (Figure 2C, 2D) that contain the phospho-mimetic Y90E mutation demonstrates that phosphorylation of Tyr90 would likely disrupt the interaction of Src's SH3 domain with intermolecular binding partners and the linker that couples SH2 domain/C-tail binding to autoinhibition, which provides a mechanistic basis for the observed elevated kinase activity of Src Y90E. By performing a series of imaging experiments with a SrcFRET biosensor, the authors show that the Y90E mutation does not show enhanced localization at focal adhesions like a hyperactivated Src mutant (Y527F) that contains a non-phosphorylatable C-tail (Figure 4A). However, using ImFCS combined with TIRF microscopy (Figure 4B), the authors demonstrate that Src Y90E shows similarly reduced mobility (relative to the WT SrcFRET biosensor) at the plasma membrane (especially at focal adhesions) as Src Y527F. Consistent with the elevated kinase activity of Src Y90E, the authors go on to demonstrate that the Src Y90E variant shows an ability to transform fibroblasts-at levels that are intermediate between wild-type Src and the hyperactive Src mutant Y527F (Figure 5). Similarly, Src Y90E confers an intermediate level (between wild-type Src and Src Y527F) of invasiveness and ability to form spheroids. Together, these comprehensive experiments with a Y90 phospho-mimetic strongly support a model where phosphorylation of Src's SH3 domain at Tyr90 would lead to a more intramolecularly disengaged SH3 regulatory domain and enhanced kinase activity in cells.

    Most of the conclusions in this paper are well supported by solid data, but confidence in several assays would be higher if additional technical detail or controls were provided and the biological significance of these findings would be higher if the role that Y90 phosphorylation plays in Src regulation and function were better delineated.

    1. The kinase activity assays in Figures 1C,1D, and 7A need to be scaled to the Src variant levels present in the lysate (quantification of relative Src levels is not provided).

    2. More details are required for the experiments quantifying Y90 phosphorylation levels in Figure 3C. The experimental states that equal amounts of IP'd proteins were used for these analyses but there are no details on how this was confirmed. In addition, the experimental states that normalized intensities were used for your quantifying the Y90 phospho-peptide but no details are provided on how normalization was performed (the legend states that a base peptide was used but it is unclear what this means).

    3. A key question is whether Y90 phosphorylation serves a regulatory role in Src's cellular activity and, if so, what is the regulatory network that mediates this phospho-event. Using a mass spectrometry readout with three Src variants (wild type vs. Y527F vs. E381G) that possess differing kinase activities, the authors demonstrate that Y90 phosphorylation levels correlate to Src's kinase activity (Figure 3C), which they suggest is an indication that this residue is an autophosphorylation site (or phosphorylated by another Src family kinase). However, as Src's kinase activity correlates with SH3 domain disengagement (which leads to a more accessible Y90), it is also entirely possible that another tyrosine kinase is responsible for this phosphorylation event. More importantly, it is unclear under which signaling regime Y90 phosphorylation would play a significant regulatory role. This phospho-event was observed in a previous phospho-proteomic study but it is unclear whether the phosphorylation levels of this site occur high enough stoichiometry to modulate the intracellular function of Src and whether there is a regulatory signaling network that influences Y90 phosphorylation levels.

  6. eLife

    Reviewer #3 (Public Review):

    The relevance of Y90 phosphorylation as a regulatory mechanism is shown by the comparison of Src kinase activity, transforming potential, cell invasiveness, and lateral diffusion in membranes. Mechanistically, Y90E mutation affects the opening of the structure, estimated from FRET experiments, and the phosphorylation status of the three main downstream signaling pathways.

    The effect of the Y90E mutation is very clear, although its description as "phosphomimicking" is, in my opinion, not accurate. Glutamic acid has a negative charge but is significantly different from phosphotyrosine. Maybe other polar mutants (lysine, glutamine...) would have a similar destabilizing effect on hydrophobic interactions. Erpel 1995 showed some effects of the Y90A mutants.

    The effect of tyrosine phosphorylation on the SH3 domain of proteins having the conserved ALYDY motif supports the proposed role, although the evidence for in vivo Y90 phosphorylation in c-Src is scarce. The possible autophosphorylation of Y90 is suggested but the evidence is not very strong and does not rule out other kinases, especially some downstream of Src itself -as already suggested by the authors.

    The authors suggest that the perturbation of Y90 reduces the interaction with the connector domain. This is a reasonable explanation, supported by the opening of the structure, but additional effects may exist: The SH3 hydrophobic region including Y90 is also the binding site for the myristoyl group (Le Roux et al. iScience, 12, 194-203) and mutations in the SH3 RT loop significantly affected lipid binding. This could contribute to the observed reduced diffusion in the lipid bilayer.

  7. Version published to 10.1101/2022.08.23.504940v1 on bioRxiv