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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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Intramolecular engagement of Fyn1’sSH3 domain minimallyinfluences the conformation of its αC helix. (A) Schematic ofthe analysis that was performed. Ki valuesfor SFKSH2eng and SFKSH3eng were measured andcompared for each SFK. Differences in affinity between SFKSH2eng and SFKSH3eng indicate differential effects of SH3 andSH2 binding on the ATP-binding site. (B) Quantitative comparison ofthe fold difference in Ki values for SFKSH2eng and SFKSH3eng, illustrating the effects ofSH2 domain or SH3 domain binding on ATP-binding site conformation.Compounds 2–4 greatly prefer Fyn1SH3eng over Fyn1SH2eng, showing that despite enhanced SH3 domainengagement Fyn1 maintains an active, αC helix-in binding site. Ki values are listed in Figure S5 of the Supporting Information.
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fig6: Intramolecular engagement of Fyn1’sSH3 domain minimallyinfluences the conformation of its αC helix. (A) Schematic ofthe analysis that was performed. Ki valuesfor SFKSH2eng and SFKSH3eng were measured andcompared for each SFK. Differences in affinity between SFKSH2eng and SFKSH3eng indicate differential effects of SH3 andSH2 binding on the ATP-binding site. (B) Quantitative comparison ofthe fold difference in Ki values for SFKSH2eng and SFKSH3eng, illustrating the effects ofSH2 domain or SH3 domain binding on ATP-binding site conformation.Compounds 2–4 greatly prefer Fyn1SH3eng over Fyn1SH2eng, showing that despite enhanced SH3 domainengagement Fyn1 maintains an active, αC helix-in binding site. Ki values are listed in Figure S5 of the Supporting Information.

Mentions: Because of the disparate activities of the autoinhibited SH2eng andSH3eng Fyn1 constructs (Figure 2B), we investigatedwhether this difference in activity is manifested in the observedaffinities of inhibitors 2–4 for Fyn1SH3eng versus Fyn1SH2eng using a similar strategy as describedin Figure 5 (Figure 6A). SH3 domain binding is predicted to align a network of residuesin the SH2-CD linker that stabilizes the αC helix in an inactiveconformation (Figure 1C). Therefore, it isodd that enhancing the affinity of Fyn1’s SH2-CD linker forits SH3 domain would not autoinhibit the enzyme to an extent similarto that observed for Lyn and Fyn2 (Figures 2 and 3). On the basis of the relatively largedifferences in catalytic activities between the Fyn1SH3eng and Fyn1SH2eng constructs, we predicted that Fyn1SH3eng would possess a more active ATP-binding site conformation.To test this, the Ki values of ligandsthat prefer an active conformation (2–4) weredetermined for Fyn1SH3eng and Fyn1SH2eng (FigureS5 of the Supporting Information). As predicted,ligands that prefer an active ATP-binding site conformation possessa higher affinity for Fyn1SH3eng than for Fyn1SH2eng (Figure 6B). In contrast, the SH3eng andSH2eng constructs of Lyn and Fyn2 possess similar affinities (∼1–10SH3eng/SH2eng ratios) for these ligands. The fact that ligands 2–4 do not show an equally strong preference for Fyn2SH3eng over Fyn2SH2eng is strong evidence that thedecoupling of Fyn1’s SH3 domain from the αC helix isdependent on the SH2-CD linker. Unlike most SFKs, in which SH3 domainengagement directly results in autoinhibition, Fyn1’s SH2-CDlinker requires SH2 domain engagement to allosterically couple SH3domain binding to the αC helix.


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

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

Intramolecular engagement of Fyn1’sSH3 domain minimallyinfluences the conformation of its αC helix. (A) Schematic ofthe analysis that was performed. Ki valuesfor SFKSH2eng and SFKSH3eng were measured andcompared for each SFK. Differences in affinity between SFKSH2eng and SFKSH3eng indicate differential effects of SH3 andSH2 binding on the ATP-binding site. (B) Quantitative comparison ofthe fold difference in Ki values for SFKSH2eng and SFKSH3eng, illustrating the effects ofSH2 domain or SH3 domain binding on ATP-binding site conformation.Compounds 2–4 greatly prefer Fyn1SH3eng over Fyn1SH2eng, showing that despite enhanced SH3 domainengagement Fyn1 maintains an active, αC helix-in binding site. Ki values are listed in Figure S5 of the Supporting Information.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Intramolecular engagement of Fyn1’sSH3 domain minimallyinfluences the conformation of its αC helix. (A) Schematic ofthe analysis that was performed. Ki valuesfor SFKSH2eng and SFKSH3eng were measured andcompared for each SFK. Differences in affinity between SFKSH2eng and SFKSH3eng indicate differential effects of SH3 andSH2 binding on the ATP-binding site. (B) Quantitative comparison ofthe fold difference in Ki values for SFKSH2eng and SFKSH3eng, illustrating the effects ofSH2 domain or SH3 domain binding on ATP-binding site conformation.Compounds 2–4 greatly prefer Fyn1SH3eng over Fyn1SH2eng, showing that despite enhanced SH3 domainengagement Fyn1 maintains an active, αC helix-in binding site. Ki values are listed in Figure S5 of the Supporting Information.
Mentions: Because of the disparate activities of the autoinhibited SH2eng andSH3eng Fyn1 constructs (Figure 2B), we investigatedwhether this difference in activity is manifested in the observedaffinities of inhibitors 2–4 for Fyn1SH3eng versus Fyn1SH2eng using a similar strategy as describedin Figure 5 (Figure 6A). SH3 domain binding is predicted to align a network of residuesin the SH2-CD linker that stabilizes the αC helix in an inactiveconformation (Figure 1C). Therefore, it isodd that enhancing the affinity of Fyn1’s SH2-CD linker forits SH3 domain would not autoinhibit the enzyme to an extent similarto that observed for Lyn and Fyn2 (Figures 2 and 3). On the basis of the relatively largedifferences in catalytic activities between the Fyn1SH3eng and Fyn1SH2eng constructs, we predicted that Fyn1SH3eng would possess a more active ATP-binding site conformation.To test this, the Ki values of ligandsthat prefer an active conformation (2–4) weredetermined for Fyn1SH3eng and Fyn1SH2eng (FigureS5 of the Supporting Information). As predicted,ligands that prefer an active ATP-binding site conformation possessa higher affinity for Fyn1SH3eng than for Fyn1SH2eng (Figure 6B). In contrast, the SH3eng andSH2eng constructs of Lyn and Fyn2 possess similar affinities (∼1–10SH3eng/SH2eng ratios) for these ligands. The fact that ligands 2–4 do not show an equally strong preference for Fyn2SH3eng over Fyn2SH2eng is strong evidence that thedecoupling of Fyn1’s SH3 domain from the αC helix isdependent on the SH2-CD linker. Unlike most SFKs, in which SH3 domainengagement directly results in autoinhibition, Fyn1’s SH2-CDlinker requires SH2 domain engagement to allosterically couple SH3domain binding to the αC helix.

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