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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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SH2-CDlinker affects availability of the C-terminal tail to post-translationalmodification by Csk. (A) Crystal structure of regulatory domain-disengagedSrc (blue, PDB entry 1Y57) superimposed on a cocrystal structure of the SrcCD–Csk complex(green, PDB entry 3D7T). The interface between Csk and Src is boxed, while the variableregion between Fyn1 and Fyn2 is colored red. Regions colored blueare identical between Fyn1 and Fyn2. (B) Scheme for measuring pTyr527phosphorylation of inhibitor-bound Fyn1 and Fyn2 complexes by Csk.(C) Structure of a Michael acceptor analogue of ligand 1. (D) Graph displaying the results of the pTyr527 experiment. Theradioactive phosphate signal is plotted vs Fyn concentration (mean± SEM; n = 3).
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fig9: SH2-CDlinker affects availability of the C-terminal tail to post-translationalmodification by Csk. (A) Crystal structure of regulatory domain-disengagedSrc (blue, PDB entry 1Y57) superimposed on a cocrystal structure of the SrcCD–Csk complex(green, PDB entry 3D7T). The interface between Csk and Src is boxed, while the variableregion between Fyn1 and Fyn2 is colored red. Regions colored blueare identical between Fyn1 and Fyn2. (B) Scheme for measuring pTyr527phosphorylation of inhibitor-bound Fyn1 and Fyn2 complexes by Csk.(C) Structure of a Michael acceptor analogue of ligand 1. (D) Graph displaying the results of the pTyr527 experiment. Theradioactive phosphate signal is plotted vs Fyn concentration (mean± SEM; n = 3).

Mentions: Fyn1 and Fyn2’s SH2-CD linker variabilityresults in surprising differences in the degree of allosteric couplingbetween the SH2 and SH3 domains and the ATP-binding site, but whatare the biological consequences of more versus less coupling for aparticular SFK? Csk is the primary kinase responsible for autoinhibitionof SFKs in most cells.39,40 It phosphorylates Tyr527 on theC-terminal tail of all SFK family members, resulting in enhanced intramolecularengagement of the SH2 domain and autoinhibition. The biochemical studiesdescribed so far suggest that Fyn1’s reduced ATP-binding site–regulatorydomain coupling results in greater overall regulatory domain accessibilityregardless of ATP-binding site conformation compared to Fyn2. Thus,one would predict that Fyn1’s C-terminal tail would be morevulnerable to phosphorylation by Csk than Fyn2’s tail. Cskhas been shown crystallographically to interact only with the C-terminusof Src.41 Fyn1 and Fyn2 possess identicalC-termini; thus, comparing the rate of phosphorylation is directlyprobing how SH2-CD linker variation between the two isoforms affectspost-translational modification (Figure 9A).


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

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

SH2-CDlinker affects availability of the C-terminal tail to post-translationalmodification by Csk. (A) Crystal structure of regulatory domain-disengagedSrc (blue, PDB entry 1Y57) superimposed on a cocrystal structure of the SrcCD–Csk complex(green, PDB entry 3D7T). The interface between Csk and Src is boxed, while the variableregion between Fyn1 and Fyn2 is colored red. Regions colored blueare identical between Fyn1 and Fyn2. (B) Scheme for measuring pTyr527phosphorylation of inhibitor-bound Fyn1 and Fyn2 complexes by Csk.(C) Structure of a Michael acceptor analogue of ligand 1. (D) Graph displaying the results of the pTyr527 experiment. Theradioactive phosphate signal is plotted vs Fyn concentration (mean± SEM; n = 3).
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fig9: SH2-CDlinker affects availability of the C-terminal tail to post-translationalmodification by Csk. (A) Crystal structure of regulatory domain-disengagedSrc (blue, PDB entry 1Y57) superimposed on a cocrystal structure of the SrcCD–Csk complex(green, PDB entry 3D7T). The interface between Csk and Src is boxed, while the variableregion between Fyn1 and Fyn2 is colored red. Regions colored blueare identical between Fyn1 and Fyn2. (B) Scheme for measuring pTyr527phosphorylation of inhibitor-bound Fyn1 and Fyn2 complexes by Csk.(C) Structure of a Michael acceptor analogue of ligand 1. (D) Graph displaying the results of the pTyr527 experiment. Theradioactive phosphate signal is plotted vs Fyn concentration (mean± SEM; n = 3).
Mentions: Fyn1 and Fyn2’s SH2-CD linker variabilityresults in surprising differences in the degree of allosteric couplingbetween the SH2 and SH3 domains and the ATP-binding site, but whatare the biological consequences of more versus less coupling for aparticular SFK? Csk is the primary kinase responsible for autoinhibitionof SFKs in most cells.39,40 It phosphorylates Tyr527 on theC-terminal tail of all SFK family members, resulting in enhanced intramolecularengagement of the SH2 domain and autoinhibition. The biochemical studiesdescribed so far suggest that Fyn1’s reduced ATP-binding site–regulatorydomain coupling results in greater overall regulatory domain accessibilityregardless of ATP-binding site conformation compared to Fyn2. Thus,one would predict that Fyn1’s C-terminal tail would be morevulnerable to phosphorylation by Csk than Fyn2’s tail. Cskhas been shown crystallographically to interact only with the C-terminusof Src.41 Fyn1 and Fyn2 possess identicalC-termini; thus, comparing the rate of phosphorylation is directlyprobing how SH2-CD linker variation between the two isoforms affectspost-translational modification (Figure 9A).

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