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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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Intramolecularregulatory domain interactions affect the catalyticactivities of Lyn, Fyn1, and Fyn2. (A) Cartoon representations ofthe SFK regulatory state mutants used in this study (SFKAct, SFKSH2eng, and SFKSH3eng). Disruptive mutationsare illustrated as red X’s, while mutations that lead to greaterintramolecular engagement are illustrated as red dots. (B) Activityof Lyn, Fyn1, and Fyn2 activated (left) and autoinhibited [SH2eng(middle) and SH3eng (right)] regulatory state mutants obtained viaa radioactive phosphate transfer assay and plotted as signal vs enzymeconcentration (mean ± SEM; n = 3). (C) Folddifference in activity between SFKAct and SFKSH2eng (left), SFKAct and SFKSH3eng (middle), andSFKSH2eng and SFKSH3eng (right) (mean ±SEM; n = 3).
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fig2: Intramolecularregulatory domain interactions affect the catalyticactivities of Lyn, Fyn1, and Fyn2. (A) Cartoon representations ofthe SFK regulatory state mutants used in this study (SFKAct, SFKSH2eng, and SFKSH3eng). Disruptive mutationsare illustrated as red X’s, while mutations that lead to greaterintramolecular engagement are illustrated as red dots. (B) Activityof Lyn, Fyn1, and Fyn2 activated (left) and autoinhibited [SH2eng(middle) and SH3eng (right)] regulatory state mutants obtained viaa radioactive phosphate transfer assay and plotted as signal vs enzymeconcentration (mean ± SEM; n = 3). (C) Folddifference in activity between SFKAct and SFKSH2eng (left), SFKAct and SFKSH3eng (middle), andSFKSH2eng and SFKSH3eng (right) (mean ±SEM; n = 3).

Mentions: Autoinhibited and activated Src and Hck constructs have previouslybeen generated by introducing mutations that enhance or weaken intramolecularSH2 and SH3 regulatory domain interactions. To explore the effectsof SH2 and SH3 domain engagement on catalytic activity within thecontext of Lyn, Fyn1, and Fyn2, analogous regulatory state mutantsof each SFK were generated (Figure 2A). AllSFKs discussed in this work are three-domain (3D) constructs, meaningthat they are full length except the unique domain has been excluded.Activated Lyn and Fyn (SFKAct) constructs were generatedby activation loop autophosphorylation of Y527F mutants, which cannotundergo C-terminal tail autoinhibitory phosphorylation.28−30 Quantitative activation loop phosphorylation was confirmed via immunoblottingwith antibodies that selectively recognize either phosphorylated ornonphosphorylated SFK activation loops (Figure S1 of the Supporting Information). Mutations that strengthenintramolecular SH2 and SH3 regulatory domain interactions were usedto create autoinhibited Lyn and Fyn constructs. Constructs with anincreased level of engagement between the C-terminal regulatory tailand the SH2 domain (SFKSH2eng) were generated by changingthe three residues following Tyr527 to a high-affinity SH2 domain-bindingepitope (Glu-Glu-Ile).31,32 Constructs with an increasedlevel of engagement between the SH2-CD linker and the SH3 domain [SFKSH3eng (Figure S2 of the Supporting Information)] were generated by introducing two high-affinity PXXP motifs (PPXPP)into the SH2-CD linker (Figure S2 of the SupportingInformation).33 This epitope hasbeen shown to possess a similarly high affinity for the SH3 domainsof SFKs.21,34−38 Adding PXXP sequences to the linkers of Fyn1, Fyn2,and Lyn enhances intramolecular SH3 domain engagement within the contextof the native SFK linker, which has been shown in chimera studiesto be tuned specifically for each SFK.24,25


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

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

Intramolecularregulatory domain interactions affect the catalyticactivities of Lyn, Fyn1, and Fyn2. (A) Cartoon representations ofthe SFK regulatory state mutants used in this study (SFKAct, SFKSH2eng, and SFKSH3eng). Disruptive mutationsare illustrated as red X’s, while mutations that lead to greaterintramolecular engagement are illustrated as red dots. (B) Activityof Lyn, Fyn1, and Fyn2 activated (left) and autoinhibited [SH2eng(middle) and SH3eng (right)] regulatory state mutants obtained viaa radioactive phosphate transfer assay and plotted as signal vs enzymeconcentration (mean ± SEM; n = 3). (C) Folddifference in activity between SFKAct and SFKSH2eng (left), SFKAct and SFKSH3eng (middle), andSFKSH2eng and SFKSH3eng (right) (mean ±SEM; n = 3).
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fig2: Intramolecularregulatory domain interactions affect the catalyticactivities of Lyn, Fyn1, and Fyn2. (A) Cartoon representations ofthe SFK regulatory state mutants used in this study (SFKAct, SFKSH2eng, and SFKSH3eng). Disruptive mutationsare illustrated as red X’s, while mutations that lead to greaterintramolecular engagement are illustrated as red dots. (B) Activityof Lyn, Fyn1, and Fyn2 activated (left) and autoinhibited [SH2eng(middle) and SH3eng (right)] regulatory state mutants obtained viaa radioactive phosphate transfer assay and plotted as signal vs enzymeconcentration (mean ± SEM; n = 3). (C) Folddifference in activity between SFKAct and SFKSH2eng (left), SFKAct and SFKSH3eng (middle), andSFKSH2eng and SFKSH3eng (right) (mean ±SEM; n = 3).
Mentions: Autoinhibited and activated Src and Hck constructs have previouslybeen generated by introducing mutations that enhance or weaken intramolecularSH2 and SH3 regulatory domain interactions. To explore the effectsof SH2 and SH3 domain engagement on catalytic activity within thecontext of Lyn, Fyn1, and Fyn2, analogous regulatory state mutantsof each SFK were generated (Figure 2A). AllSFKs discussed in this work are three-domain (3D) constructs, meaningthat they are full length except the unique domain has been excluded.Activated Lyn and Fyn (SFKAct) constructs were generatedby activation loop autophosphorylation of Y527F mutants, which cannotundergo C-terminal tail autoinhibitory phosphorylation.28−30 Quantitative activation loop phosphorylation was confirmed via immunoblottingwith antibodies that selectively recognize either phosphorylated ornonphosphorylated SFK activation loops (Figure S1 of the Supporting Information). Mutations that strengthenintramolecular SH2 and SH3 regulatory domain interactions were usedto create autoinhibited Lyn and Fyn constructs. Constructs with anincreased level of engagement between the C-terminal regulatory tailand the SH2 domain (SFKSH2eng) were generated by changingthe three residues following Tyr527 to a high-affinity SH2 domain-bindingepitope (Glu-Glu-Ile).31,32 Constructs with an increasedlevel of engagement between the SH2-CD linker and the SH3 domain [SFKSH3eng (Figure S2 of the Supporting Information)] were generated by introducing two high-affinity PXXP motifs (PPXPP)into the SH2-CD linker (Figure S2 of the SupportingInformation).33 This epitope hasbeen shown to possess a similarly high affinity for the SH3 domainsof SFKs.21,34−38 Adding PXXP sequences to the linkers of Fyn1, Fyn2,and Lyn enhances intramolecular SH3 domain engagement within the contextof the native SFK linker, which has been shown in chimera studiesto be tuned specifically for each SFK.24,25

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