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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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Effects ofactivation loop phosphorylation and regulatory domainengagement on the ATP-binding sites of SFKs. (A) Schematic of theanalysis that was performed. Ki valuesfor SFKSH2eng and SFKAct are compared to thosefor SFKY527F to determine whether activation loop phosphorylationor SH2 domain engagement influences the affinity of 2–4. (B) Quantitative comparison of the differences in Ki values between SFKSH2eng and SFKY527F for ligands 2–4. All values are the averageof assays performed in triplicate; the SEM for each value is lessthan 20% of the average Ki value. (C)Quantitative comparison of the fold difference in Ki values for SFKY527F vs SFKAct.All values are the average of assays performed in triplicate; theSEM for each value is less than 20% of the average Ki value. Ki values are listedin Figure S4 of the Supporting Information.
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fig5: Effects ofactivation loop phosphorylation and regulatory domainengagement on the ATP-binding sites of SFKs. (A) Schematic of theanalysis that was performed. Ki valuesfor SFKSH2eng and SFKAct are compared to thosefor SFKY527F to determine whether activation loop phosphorylationor SH2 domain engagement influences the affinity of 2–4. (B) Quantitative comparison of the differences in Ki values between SFKSH2eng and SFKY527F for ligands 2–4. All values are the averageof assays performed in triplicate; the SEM for each value is lessthan 20% of the average Ki value. (C)Quantitative comparison of the fold difference in Ki values for SFKY527F vs SFKAct.All values are the average of assays performed in triplicate; theSEM for each value is less than 20% of the average Ki value. Ki values are listedin Figure S4 of the Supporting Information.

Mentions: SFK catalytic activity is predominantly governed by twofactors: activation loop phosphorylation (activating) and regulatorydomain engagement (autoinhibiting). We were interested in seeing ifwe could use our panel of inhibitors and regulatory state mutantsto determine which factor, activation loop phosphorylation or regulatorydomain engagement, governs the changes in ATP-binding site conformationdriving the extreme difference in affinity observed between Fyn1Act and Fyn1SH2eng for ligands 2–4. To do this, Ki values for ligands 2–4 were determined for each SFKY527F constructand compared to those for SFKAct, to probe activation loopphosphorylation, or SFKSH2eng, to probe SH2 domain engagement(Figure 5 and Figure S4 of the Supporting Information). The Y527F constructwas chosen as a basis for comparison because it is neither activationloop-phosphorylated nor engineered to favor regulatory domain engagement.This analysis shows that SH2 domain engagement determines the affinityof ligands 2–4 for the ATP-binding sites of allSFKs tested, because a larger difference in affinity is observed betweenSFKSH2eng and SFKY527F than between SFKY527F and SFKAct. This is particularly true forFyn1, providing evidence that the ATP-binding site conformation equilibriumfor Fyn1Y527F favors the active, αC helix-in conformationto a greater extent than all other SFKY527F constructstested, even in the absence of activation loop phosphorylation.


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

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

Effects ofactivation loop phosphorylation and regulatory domainengagement on the ATP-binding sites of SFKs. (A) Schematic of theanalysis that was performed. Ki valuesfor SFKSH2eng and SFKAct are compared to thosefor SFKY527F to determine whether activation loop phosphorylationor SH2 domain engagement influences the affinity of 2–4. (B) Quantitative comparison of the differences in Ki values between SFKSH2eng and SFKY527F for ligands 2–4. All values are the averageof assays performed in triplicate; the SEM for each value is lessthan 20% of the average Ki value. (C)Quantitative comparison of the fold difference in Ki values for SFKY527F vs SFKAct.All values are the average of assays performed in triplicate; theSEM for each value is less than 20% of the average Ki value. Ki values are listedin Figure S4 of the Supporting Information.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Effects ofactivation loop phosphorylation and regulatory domainengagement on the ATP-binding sites of SFKs. (A) Schematic of theanalysis that was performed. Ki valuesfor SFKSH2eng and SFKAct are compared to thosefor SFKY527F to determine whether activation loop phosphorylationor SH2 domain engagement influences the affinity of 2–4. (B) Quantitative comparison of the differences in Ki values between SFKSH2eng and SFKY527F for ligands 2–4. All values are the averageof assays performed in triplicate; the SEM for each value is lessthan 20% of the average Ki value. (C)Quantitative comparison of the fold difference in Ki values for SFKY527F vs SFKAct.All values are the average of assays performed in triplicate; theSEM for each value is less than 20% of the average Ki value. Ki values are listedin Figure S4 of the Supporting Information.
Mentions: SFK catalytic activity is predominantly governed by twofactors: activation loop phosphorylation (activating) and regulatorydomain engagement (autoinhibiting). We were interested in seeing ifwe could use our panel of inhibitors and regulatory state mutantsto determine which factor, activation loop phosphorylation or regulatorydomain engagement, governs the changes in ATP-binding site conformationdriving the extreme difference in affinity observed between Fyn1Act and Fyn1SH2eng for ligands 2–4. To do this, Ki values for ligands 2–4 were determined for each SFKY527F constructand compared to those for SFKAct, to probe activation loopphosphorylation, or SFKSH2eng, to probe SH2 domain engagement(Figure 5 and Figure S4 of the Supporting Information). The Y527F constructwas chosen as a basis for comparison because it is neither activationloop-phosphorylated nor engineered to favor regulatory domain engagement.This analysis shows that SH2 domain engagement determines the affinityof ligands 2–4 for the ATP-binding sites of allSFKs tested, because a larger difference in affinity is observed betweenSFKSH2eng and SFKY527F than between SFKY527F and SFKAct. This is particularly true forFyn1, providing evidence that the ATP-binding site conformation equilibriumfor Fyn1Y527F favors the active, αC helix-in conformationto a greater extent than all other SFKY527F constructstested, even in the absence of activation loop phosphorylation.

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