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SH2-catalytic domain linker heterogeneity influences allosteric coupling across the SFK family.

Register AC, Leonard SE, Maly DJ - Biochemistry (2014)

Bottom Line: Biochemical and structural studies indicate that the SH2-catalytic domain (SH2-CD) linker, the intramolecular binding epitope for SFK SH3 domains, is responsible for allosterically coupling SH3 domain engagement to autoinhibition of the ATP-binding site through the conformation of the αC helix.Analyses of Fyn1 and Fyn2, isoforms that are identical but for a 50-residue sequence spanning the SH2-CD linker, demonstrate that SH2-CD linker sequence differences can have profound effects on allosteric coupling between otherwise identical kinases.Most notably, a dampened allosteric connection between the SH3 domain and αC helix leads to greater autoinhibitory phosphorylation by Csk, illustrating the complex effects of SH2-CD linker sequence on cellular function.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Washington , Seattle, Washington 98195, United States.

ABSTRACT
Src-family kinases (SFKs) make up a family of nine homologous multidomain tyrosine kinases whose misregulation is responsible for human disease (cancer, diabetes, inflammation, etc.). Despite overall sequence homology and identical domain architecture, differences in SH3 and SH2 regulatory domain accessibility and ability to allosterically autoinhibit the ATP-binding site have been observed for the prototypical SFKs Src and Hck. Biochemical and structural studies indicate that the SH2-catalytic domain (SH2-CD) linker, the intramolecular binding epitope for SFK SH3 domains, is responsible for allosterically coupling SH3 domain engagement to autoinhibition of the ATP-binding site through the conformation of the αC helix. As a relatively unconserved region between SFK family members, SH2-CD linker sequence variability across the SFK family is likely a source of nonredundant cellular functions between individual SFKs via its effect on the availability of SH3 and SH2 domains for intermolecular interactions and post-translational modification. Using a combination of SFKs engineered with enhanced or weakened regulatory domain intramolecular interactions and conformation-selective inhibitors that report αC helix conformation, this study explores how SH2-CD sequence heterogeneity affects allosteric coupling across the SFK family by examining Lyn, Fyn1, and Fyn2. Analyses of Fyn1 and Fyn2, isoforms that are identical but for a 50-residue sequence spanning the SH2-CD linker, demonstrate that SH2-CD linker sequence differences can have profound effects on allosteric coupling between otherwise identical kinases. Most notably, a dampened allosteric connection between the SH3 domain and αC helix leads to greater autoinhibitory phosphorylation by Csk, illustrating the complex effects of SH2-CD linker sequence on cellular function.

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The Fyn1 andFyn2 SH3eng constructs have similarly inaccessibleSH3 domains. (A) Schematic of the SH3 pull-down assay. SFK constructsare exposed to beads displaying an SH3-binding peptide. After beingwashed, the retained kinase is eluted, subjected to SDS–PAGE,and quantified by Western blotting or Coomassie staining. (B) Pull-downcomparing the percent SFK retained on SH3-binding resin. Fyn1Y527F possesses an SH3 domain that is relatively accessiblecompared to those of Fyn1SH3eng and Fyn2SH3eng, which are similarly inaccessible (mean ± SEM; n = 3). (C) SDS–PAGE quantification of unbound Fyn1SH3eng and Fyn2SH3eng after incubation with 1.5 mM (1×),7.5 mM (5×), and 15 mM (10×) SH3-binding peptide resin (mean± SEM; n = 3). (D) SDS–PAGE quantificationof Fyn1SH3eng and Fyn2SH3eng eluted from resinafter incubation with 1×, 5×, and 10× loading resin(mean ± standard deviation; n = 2).
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fig3: The Fyn1 andFyn2 SH3eng constructs have similarly inaccessibleSH3 domains. (A) Schematic of the SH3 pull-down assay. SFK constructsare exposed to beads displaying an SH3-binding peptide. After beingwashed, the retained kinase is eluted, subjected to SDS–PAGE,and quantified by Western blotting or Coomassie staining. (B) Pull-downcomparing the percent SFK retained on SH3-binding resin. Fyn1Y527F possesses an SH3 domain that is relatively accessiblecompared to those of Fyn1SH3eng and Fyn2SH3eng, which are similarly inaccessible (mean ± SEM; n = 3). (C) SDS–PAGE quantification of unbound Fyn1SH3eng and Fyn2SH3eng after incubation with 1.5 mM (1×),7.5 mM (5×), and 15 mM (10×) SH3-binding peptide resin (mean± SEM; n = 3). (D) SDS–PAGE quantificationof Fyn1SH3eng and Fyn2SH3eng eluted from resinafter incubation with 1×, 5×, and 10× loading resin(mean ± standard deviation; n = 2).

Mentions: Consistent with the introduced regulatory mutations strengtheningautoinhibitory interactions, SFKSH3eng and SFKSH2eng constructs are less active than their SFKAct counterparts(Figure 2B). However, in contrast to SFKAct constructs, there is greater diversity in catalytic activityamong autoinhibited constructs. For example, Fyn1SH3eng is notably more active than Fyn2SH3eng or LynSH3eng (Figure 2B, right panel). This Fyn1 mutantexhibits an activity more than 10-fold greater than that of Fyn1SH2eng [being only ∼3-fold less active than Fyn1Act (Figure 2C, right panel)]. However,Fyn2SH2eng is much more active than either Fyn1SH2eng or LynSH2eng (Figure 2B, middlepanel). To confirm that Fyn1SH3eng’s relativelyhigh catalytic activity compared to that of Fyn2SH3eng isnot a result of differences in occupancy between the introduced high-affinitySH2-CD linker and the Fyn SH3 domain, a series of pull-down experimentsto determine SH3 domain accessibility were performed (Figure 3A). Fyn constructs of interest were incubated withresin displaying an SH3-binding peptide. After being washed, the boundkinase was eluted and quantified. The amount of SFK retained on thebeads is a reflection of the relative accessibility of their SH3 domainsto engage in intermolecular binding interactions. Comparing the relativeamounts of retained Fyn1SH3eng and Fyn2SH3eng provides a measure of how tightly each construct’s high-affinitylinker engages the Fyn SH3 domain. Both Fyn1SH3eng andFyn2SH3eng possess relatively inaccessible SH3 domains,presumably because of linker-SH3 domain engagement, relative to Fyn1Y527F (Figure 3B). Pull-downs usingresin loaded with 5- and 10-fold more SH3 ligand than in the previousexperiment were performed to test if intramolecular engagement couldbe outcompeted by higher concentrations of the immobilized SH3 domainligand. The amount of Fyn1SH3eng and Fyn2SH3eng captured (and unbound) by each resin loading is almost identical,demonstrating that both Fyn1 and Fyn2 SH3eng constructs have similarlyengaged SH3 domains (Figure 3C,D). Thus, theability of regulatory interactions to transmit autoinhibition to theATP-binding site varies dramatically among Fyn1, Fyn2, and Lyn. Thefact that Fyn1 and Fyn2 have identical SH3 and SH2 domains providesevidence that SH2-CD linker variability strongly contributes to therelative ability of an SH2 or SH3 domain to autoinhibit the kinasedomain. The functional consequences of SH2-CD linker variability,modulating the degree of allosteric coupling between intramolecularregulatory domain engagement and the ATP-binding site, may point toa source of nonredundancy between SFK family members in cells.


SH2-catalytic domain linker heterogeneity influences allosteric coupling across the SFK family.

Register AC, Leonard SE, Maly DJ - Biochemistry (2014)

The Fyn1 andFyn2 SH3eng constructs have similarly inaccessibleSH3 domains. (A) Schematic of the SH3 pull-down assay. SFK constructsare exposed to beads displaying an SH3-binding peptide. After beingwashed, the retained kinase is eluted, subjected to SDS–PAGE,and quantified by Western blotting or Coomassie staining. (B) Pull-downcomparing the percent SFK retained on SH3-binding resin. Fyn1Y527F possesses an SH3 domain that is relatively accessiblecompared to those of Fyn1SH3eng and Fyn2SH3eng, which are similarly inaccessible (mean ± SEM; n = 3). (C) SDS–PAGE quantification of unbound Fyn1SH3eng and Fyn2SH3eng after incubation with 1.5 mM (1×),7.5 mM (5×), and 15 mM (10×) SH3-binding peptide resin (mean± SEM; n = 3). (D) SDS–PAGE quantificationof Fyn1SH3eng and Fyn2SH3eng eluted from resinafter incubation with 1×, 5×, and 10× loading resin(mean ± standard deviation; n = 2).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4230323&req=5

fig3: The Fyn1 andFyn2 SH3eng constructs have similarly inaccessibleSH3 domains. (A) Schematic of the SH3 pull-down assay. SFK constructsare exposed to beads displaying an SH3-binding peptide. After beingwashed, the retained kinase is eluted, subjected to SDS–PAGE,and quantified by Western blotting or Coomassie staining. (B) Pull-downcomparing the percent SFK retained on SH3-binding resin. Fyn1Y527F possesses an SH3 domain that is relatively accessiblecompared to those of Fyn1SH3eng and Fyn2SH3eng, which are similarly inaccessible (mean ± SEM; n = 3). (C) SDS–PAGE quantification of unbound Fyn1SH3eng and Fyn2SH3eng after incubation with 1.5 mM (1×),7.5 mM (5×), and 15 mM (10×) SH3-binding peptide resin (mean± SEM; n = 3). (D) SDS–PAGE quantificationof Fyn1SH3eng and Fyn2SH3eng eluted from resinafter incubation with 1×, 5×, and 10× loading resin(mean ± standard deviation; n = 2).
Mentions: Consistent with the introduced regulatory mutations strengtheningautoinhibitory interactions, SFKSH3eng and SFKSH2eng constructs are less active than their SFKAct counterparts(Figure 2B). However, in contrast to SFKAct constructs, there is greater diversity in catalytic activityamong autoinhibited constructs. For example, Fyn1SH3eng is notably more active than Fyn2SH3eng or LynSH3eng (Figure 2B, right panel). This Fyn1 mutantexhibits an activity more than 10-fold greater than that of Fyn1SH2eng [being only ∼3-fold less active than Fyn1Act (Figure 2C, right panel)]. However,Fyn2SH2eng is much more active than either Fyn1SH2eng or LynSH2eng (Figure 2B, middlepanel). To confirm that Fyn1SH3eng’s relativelyhigh catalytic activity compared to that of Fyn2SH3eng isnot a result of differences in occupancy between the introduced high-affinitySH2-CD linker and the Fyn SH3 domain, a series of pull-down experimentsto determine SH3 domain accessibility were performed (Figure 3A). Fyn constructs of interest were incubated withresin displaying an SH3-binding peptide. After being washed, the boundkinase was eluted and quantified. The amount of SFK retained on thebeads is a reflection of the relative accessibility of their SH3 domainsto engage in intermolecular binding interactions. Comparing the relativeamounts of retained Fyn1SH3eng and Fyn2SH3eng provides a measure of how tightly each construct’s high-affinitylinker engages the Fyn SH3 domain. Both Fyn1SH3eng andFyn2SH3eng possess relatively inaccessible SH3 domains,presumably because of linker-SH3 domain engagement, relative to Fyn1Y527F (Figure 3B). Pull-downs usingresin loaded with 5- and 10-fold more SH3 ligand than in the previousexperiment were performed to test if intramolecular engagement couldbe outcompeted by higher concentrations of the immobilized SH3 domainligand. The amount of Fyn1SH3eng and Fyn2SH3eng captured (and unbound) by each resin loading is almost identical,demonstrating that both Fyn1 and Fyn2 SH3eng constructs have similarlyengaged SH3 domains (Figure 3C,D). Thus, theability of regulatory interactions to transmit autoinhibition to theATP-binding site varies dramatically among Fyn1, Fyn2, and Lyn. Thefact that Fyn1 and Fyn2 have identical SH3 and SH2 domains providesevidence that SH2-CD linker variability strongly contributes to therelative ability of an SH2 or SH3 domain to autoinhibit the kinasedomain. The functional consequences of SH2-CD linker variability,modulating the degree of allosteric coupling between intramolecularregulatory domain engagement and the ATP-binding site, may point toa source of nonredundancy between SFK family members in cells.

Bottom Line: Biochemical and structural studies indicate that the SH2-catalytic domain (SH2-CD) linker, the intramolecular binding epitope for SFK SH3 domains, is responsible for allosterically coupling SH3 domain engagement to autoinhibition of the ATP-binding site through the conformation of the αC helix.Analyses of Fyn1 and Fyn2, isoforms that are identical but for a 50-residue sequence spanning the SH2-CD linker, demonstrate that SH2-CD linker sequence differences can have profound effects on allosteric coupling between otherwise identical kinases.Most notably, a dampened allosteric connection between the SH3 domain and αC helix leads to greater autoinhibitory phosphorylation by Csk, illustrating the complex effects of SH2-CD linker sequence on cellular function.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Washington , Seattle, Washington 98195, United States.

ABSTRACT
Src-family kinases (SFKs) make up a family of nine homologous multidomain tyrosine kinases whose misregulation is responsible for human disease (cancer, diabetes, inflammation, etc.). Despite overall sequence homology and identical domain architecture, differences in SH3 and SH2 regulatory domain accessibility and ability to allosterically autoinhibit the ATP-binding site have been observed for the prototypical SFKs Src and Hck. Biochemical and structural studies indicate that the SH2-catalytic domain (SH2-CD) linker, the intramolecular binding epitope for SFK SH3 domains, is responsible for allosterically coupling SH3 domain engagement to autoinhibition of the ATP-binding site through the conformation of the αC helix. As a relatively unconserved region between SFK family members, SH2-CD linker sequence variability across the SFK family is likely a source of nonredundant cellular functions between individual SFKs via its effect on the availability of SH3 and SH2 domains for intermolecular interactions and post-translational modification. Using a combination of SFKs engineered with enhanced or weakened regulatory domain intramolecular interactions and conformation-selective inhibitors that report αC helix conformation, this study explores how SH2-CD sequence heterogeneity affects allosteric coupling across the SFK family by examining Lyn, Fyn1, and Fyn2. Analyses of Fyn1 and Fyn2, isoforms that are identical but for a 50-residue sequence spanning the SH2-CD linker, demonstrate that SH2-CD linker sequence differences can have profound effects on allosteric coupling between otherwise identical kinases. Most notably, a dampened allosteric connection between the SH3 domain and αC helix leads to greater autoinhibitory phosphorylation by Csk, illustrating the complex effects of SH2-CD linker sequence on cellular function.

Show MeSH
Related in: MedlinePlus