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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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Allostericrelationships in the Src-family kinases (SFKs). (A)Dendrogram showing the evolutionary relationship of the Src-familykinases (SFKs). (B) Conserved domain architecture of SFKs. SH3 andSH2 regulatory domains are connected to the catalytic domain (CD)by the SH2-CD linker and C-terminal tail. The SH3 domain-binding epitopesin the linkers of Src, Fyn1, Fyn2, Hck, and Lyn are boxed, and keyresidues thought to allosterically connect the αC helix (ATP-bindingsite) and the SH3 domain are boxed and labeled (Src numbering). Notethat Fyn1 has a linker longer than those of Fyn2 and Src. (C) Cartoonrepresentation of the three-dimensional structure of an autoinhibitedSFK. The crystal structure (PDB entry 2SRC) shows a portion of the CD (yellow),αC helix (red), SH2-CD linker (green), and SH3 domain (blue),known to be important for mediating allosteric connection of the ATP-bindingsite and regulatory domains. Key residues highlighted in panel B areshown as sticks. Of particular interest are the proximity of helixαC to Trp260 and the hydrophobic contacts made by Leu255. (D)Schematic illustrating the goal of this study, to probe the degreeof bidirectional allosteric coupling between the ATP-binding site(helix αC) and the regulatory domains among SFK family membersvia the SH3–linker interaction.
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fig1: Allostericrelationships in the Src-family kinases (SFKs). (A)Dendrogram showing the evolutionary relationship of the Src-familykinases (SFKs). (B) Conserved domain architecture of SFKs. SH3 andSH2 regulatory domains are connected to the catalytic domain (CD)by the SH2-CD linker and C-terminal tail. The SH3 domain-binding epitopesin the linkers of Src, Fyn1, Fyn2, Hck, and Lyn are boxed, and keyresidues thought to allosterically connect the αC helix (ATP-bindingsite) and the SH3 domain are boxed and labeled (Src numbering). Notethat Fyn1 has a linker longer than those of Fyn2 and Src. (C) Cartoonrepresentation of the three-dimensional structure of an autoinhibitedSFK. The crystal structure (PDB entry 2SRC) shows a portion of the CD (yellow),αC helix (red), SH2-CD linker (green), and SH3 domain (blue),known to be important for mediating allosteric connection of the ATP-bindingsite and regulatory domains. Key residues highlighted in panel B areshown as sticks. Of particular interest are the proximity of helixαC to Trp260 and the hydrophobic contacts made by Leu255. (D)Schematic illustrating the goal of this study, to probe the degreeof bidirectional allosteric coupling between the ATP-binding site(helix αC) and the regulatory domains among SFK family membersvia the SH3–linker interaction.

Mentions: Src-familykinases (SFKs) makeup a family of nine non-receptor tyrosine kinases (Src, Hck, Fyn,Lyn, Lck, Yes, Fgr, Blk, and Frk) that play a variety of importantbiological functions through both catalysis and intermolecular protein–proteininteractions (Figure 1A).1,2 Largelybecause of the potential roles that they play in human disease, SFKshave become popular subjects of study, with most biochemical and structuralresearch focusing on Src and Hck.2−4 All SFKs consist of anN-terminal unique domain, regulatory SH3 and SH2 domains, a catalyticdomain (CD), and a C-terminal tail (Figure 1B). Catalytic activity in SFKs is regulated by a combination of post-translationalmodification (phosphorylation) and intramolecular protein–proteininteractions.2,4 In the autoinhibited form, SFKsadopt a closed global conformation stabilized by intramolecular interactionsbetween the SH3 domain and the SH2-CD linker [polyproline type II(PPII) helix] and between the SH2 domain and the C-terminal tail,which is enhanced by phosphorylation of Tyr527 on the C-terminal tail.In the active, open conformation, these intramolecular interactionsare weakened and the regulatory domains are freed to interact withother binding partners in the cell. The active form is further stabilizedby phosphorylation of the activation loop at Tyr416.5−10


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

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

Allostericrelationships in the Src-family kinases (SFKs). (A)Dendrogram showing the evolutionary relationship of the Src-familykinases (SFKs). (B) Conserved domain architecture of SFKs. SH3 andSH2 regulatory domains are connected to the catalytic domain (CD)by the SH2-CD linker and C-terminal tail. The SH3 domain-binding epitopesin the linkers of Src, Fyn1, Fyn2, Hck, and Lyn are boxed, and keyresidues thought to allosterically connect the αC helix (ATP-bindingsite) and the SH3 domain are boxed and labeled (Src numbering). Notethat Fyn1 has a linker longer than those of Fyn2 and Src. (C) Cartoonrepresentation of the three-dimensional structure of an autoinhibitedSFK. The crystal structure (PDB entry 2SRC) shows a portion of the CD (yellow),αC helix (red), SH2-CD linker (green), and SH3 domain (blue),known to be important for mediating allosteric connection of the ATP-bindingsite and regulatory domains. Key residues highlighted in panel B areshown as sticks. Of particular interest are the proximity of helixαC to Trp260 and the hydrophobic contacts made by Leu255. (D)Schematic illustrating the goal of this study, to probe the degreeof bidirectional allosteric coupling between the ATP-binding site(helix αC) and the regulatory domains among SFK family membersvia the SH3–linker interaction.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Allostericrelationships in the Src-family kinases (SFKs). (A)Dendrogram showing the evolutionary relationship of the Src-familykinases (SFKs). (B) Conserved domain architecture of SFKs. SH3 andSH2 regulatory domains are connected to the catalytic domain (CD)by the SH2-CD linker and C-terminal tail. The SH3 domain-binding epitopesin the linkers of Src, Fyn1, Fyn2, Hck, and Lyn are boxed, and keyresidues thought to allosterically connect the αC helix (ATP-bindingsite) and the SH3 domain are boxed and labeled (Src numbering). Notethat Fyn1 has a linker longer than those of Fyn2 and Src. (C) Cartoonrepresentation of the three-dimensional structure of an autoinhibitedSFK. The crystal structure (PDB entry 2SRC) shows a portion of the CD (yellow),αC helix (red), SH2-CD linker (green), and SH3 domain (blue),known to be important for mediating allosteric connection of the ATP-bindingsite and regulatory domains. Key residues highlighted in panel B areshown as sticks. Of particular interest are the proximity of helixαC to Trp260 and the hydrophobic contacts made by Leu255. (D)Schematic illustrating the goal of this study, to probe the degreeof bidirectional allosteric coupling between the ATP-binding site(helix αC) and the regulatory domains among SFK family membersvia the SH3–linker interaction.
Mentions: Src-familykinases (SFKs) makeup a family of nine non-receptor tyrosine kinases (Src, Hck, Fyn,Lyn, Lck, Yes, Fgr, Blk, and Frk) that play a variety of importantbiological functions through both catalysis and intermolecular protein–proteininteractions (Figure 1A).1,2 Largelybecause of the potential roles that they play in human disease, SFKshave become popular subjects of study, with most biochemical and structuralresearch focusing on Src and Hck.2−4 All SFKs consist of anN-terminal unique domain, regulatory SH3 and SH2 domains, a catalyticdomain (CD), and a C-terminal tail (Figure 1B). Catalytic activity in SFKs is regulated by a combination of post-translationalmodification (phosphorylation) and intramolecular protein–proteininteractions.2,4 In the autoinhibited form, SFKsadopt a closed global conformation stabilized by intramolecular interactionsbetween the SH3 domain and the SH2-CD linker [polyproline type II(PPII) helix] and between the SH2 domain and the C-terminal tail,which is enhanced by phosphorylation of Tyr527 on the C-terminal tail.In the active, open conformation, these intramolecular interactionsare weakened and the regulatory domains are freed to interact withother binding partners in the cell. The active form is further stabilizedby phosphorylation of the activation loop at Tyr416.5−10

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