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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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Stabilizing an activeATP-binding site conformation overcomes regulatoryinteractions in Lyn and Fyn1. (A) SH3 pull-downs were performed usingSFKSH2eng constructs. SFKSH2eng constructs wereincubated with control ligand 1 or active-preferringligand 2 and the amounts of kinase retained on the resincompared. (B) Quantification of the SFKSH2eng SH3 pull-downexperiment (mean ± SEM; n = 3). All data arenormalized to the SFKY527F·1 complex.
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fig8: Stabilizing an activeATP-binding site conformation overcomes regulatoryinteractions in Lyn and Fyn1. (A) SH3 pull-downs were performed usingSFKSH2eng constructs. SFKSH2eng constructs wereincubated with control ligand 1 or active-preferringligand 2 and the amounts of kinase retained on the resincompared. (B) Quantification of the SFKSH2eng SH3 pull-downexperiment (mean ± SEM; n = 3). All data arenormalized to the SFKY527F·1 complex.

Mentions: Inhibitorbinding-mediated changes in SH3 domain accessibilitywere also explored using the SFKSH2eng construct (Figure 8A). Both LynSH2eng·1 and Fyn1SH2eng·1 complexes displaya relatively inaccessible SH3 domain compared with that of the SFKY527F·1 complex, consistent with enhancedSH2–C-terminal tail intramolecular engagement promoting SH3domain–SH2-CD linker interaction in both kinases. When theyare bound to ligand 2, the SH3 domain accessibility ofFyn1SH2eng and LynSH2eng returns to a levelon par with those of the Fyn1Y527F·1 andLynY527F·1 complexes, respectively (Figure 8B). These data show that adopting an active ATP-bindingsite conformation can overcome engineered regulatory domain engagement.It also indicates that although Fyn1 displays a reduced level of couplingbetween the SH3 domain and the ATP-binding site, enhancing the interactionbetween the SH2 domain and C-terminal tail is able to engage the SH3domain and stabilize an αC helix-out inactive ATP-binding site.These effects can be reversed by stabilizing an αC helix-in,active ATP-binding site with a ligand.


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

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

Stabilizing an activeATP-binding site conformation overcomes regulatoryinteractions in Lyn and Fyn1. (A) SH3 pull-downs were performed usingSFKSH2eng constructs. SFKSH2eng constructs wereincubated with control ligand 1 or active-preferringligand 2 and the amounts of kinase retained on the resincompared. (B) Quantification of the SFKSH2eng SH3 pull-downexperiment (mean ± SEM; n = 3). All data arenormalized to the SFKY527F·1 complex.
© Copyright Policy
Related In: Results  -  Collection

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

fig8: Stabilizing an activeATP-binding site conformation overcomes regulatoryinteractions in Lyn and Fyn1. (A) SH3 pull-downs were performed usingSFKSH2eng constructs. SFKSH2eng constructs wereincubated with control ligand 1 or active-preferringligand 2 and the amounts of kinase retained on the resincompared. (B) Quantification of the SFKSH2eng SH3 pull-downexperiment (mean ± SEM; n = 3). All data arenormalized to the SFKY527F·1 complex.
Mentions: Inhibitorbinding-mediated changes in SH3 domain accessibilitywere also explored using the SFKSH2eng construct (Figure 8A). Both LynSH2eng·1 and Fyn1SH2eng·1 complexes displaya relatively inaccessible SH3 domain compared with that of the SFKY527F·1 complex, consistent with enhancedSH2–C-terminal tail intramolecular engagement promoting SH3domain–SH2-CD linker interaction in both kinases. When theyare bound to ligand 2, the SH3 domain accessibility ofFyn1SH2eng and LynSH2eng returns to a levelon par with those of the Fyn1Y527F·1 andLynY527F·1 complexes, respectively (Figure 8B). These data show that adopting an active ATP-bindingsite conformation can overcome engineered regulatory domain engagement.It also indicates that although Fyn1 displays a reduced level of couplingbetween the SH3 domain and the ATP-binding site, enhancing the interactionbetween the SH2 domain and C-terminal tail is able to engage the SH3domain and stabilize an αC helix-out inactive ATP-binding site.These effects can be reversed by stabilizing an αC helix-in,active ATP-binding site with a ligand.

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