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Structural coupling of SH2-kinase domains links Fes and Abl substrate recognition and kinase activation.

Filippakopoulos P, Kofler M, Hantschel O, Gish GD, Grebien F, Salah E, Neudecker P, Kay LE, Turk BE, Superti-Furga G, Pawson T, Knapp S - Cell (2008)

Bottom Line: Our data indicate that Fes kinase activation is closely coupled to substrate recognition through cooperative SH2-kinase-substrate interactions.Similarly, we find that the SH2 domain of the active Abl kinase stimulates catalytic activity and substrate phosphorylation through a distinct SH2-kinase interface.Thus, the SH2 and catalytic domains of active Fes and Abl pro-oncogenic kinases form integrated structures essential for effective tyrosine kinase signaling.

View Article: PubMed Central - PubMed

Affiliation: Structural Genomics Consortium, University of Oxford, Old Road Campus, Roosevelt Drive, Oxford OX3 7DQ, UK.

ABSTRACT
The SH2 domain of cytoplasmic tyrosine kinases can enhance catalytic activity and substrate recognition, but the molecular mechanisms by which this is achieved are poorly understood. We have solved the structure of the prototypic SH2-kinase unit of the human Fes tyrosine kinase, which appears specialized for positive signaling. In its active conformation, the SH2 domain tightly interacts with the kinase N-terminal lobe and positions the kinase alphaC helix in an active configuration through essential packing and electrostatic interactions. This interaction is stabilized by ligand binding to the SH2 domain. Our data indicate that Fes kinase activation is closely coupled to substrate recognition through cooperative SH2-kinase-substrate interactions. Similarly, we find that the SH2 domain of the active Abl kinase stimulates catalytic activity and substrate phosphorylation through a distinct SH2-kinase interface. Thus, the SH2 and catalytic domains of active Fes and Abl pro-oncogenic kinases form integrated structures essential for effective tyrosine kinase signaling.

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Activation of Fes Kinase Activity by SH2 Domain Interactions(A) The effect of mutations in the SH2 domain on Fes kinase activity: HEK293T cells were cotransfected with Flag-tagged Fes and GST-cortactin. Cell lysates were immunoprecipitated with anti-Flag and blotted with anti-pTyr to reveal autophosphorylated (p) Flag-Fes (top panel) or with anti-Flag (lower panel) as a control. WT indicates wild-type Fes, and mutants are described in the text.(B) GST-cortactin was affinity purified from cells coexpressing WT or mutant Fes proteins and blotted either with anti-pTyr antibodies to measure Fes-induced phosphorylation (p) (top panel) or with anti-GST (bottom panel).(C) Comparison of the N-terminal SH2 domain sequence of Fps/Fes orthologs. Insertions originally used to identify the SH2 domain in v-Fps are indicated (RX15m and AX9m) (Sadowski et al., 1986; Stone et al., 1984). Conserved residues are highlighted in red and similar residues in yellow.(D) Details of the Fes SH2-kinase interface. Residues important for the SH2 domain-kinase interaction are conserved in Fps/Fes family members but not in other tyrosine kinases. Residues mutated in this study are indicated by a green asterisk. Interactions of interface residues and the involved secondary structure elements are shown in the structure of active Fes. The alignment of human sequences is colored as in (C).
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fig2: Activation of Fes Kinase Activity by SH2 Domain Interactions(A) The effect of mutations in the SH2 domain on Fes kinase activity: HEK293T cells were cotransfected with Flag-tagged Fes and GST-cortactin. Cell lysates were immunoprecipitated with anti-Flag and blotted with anti-pTyr to reveal autophosphorylated (p) Flag-Fes (top panel) or with anti-Flag (lower panel) as a control. WT indicates wild-type Fes, and mutants are described in the text.(B) GST-cortactin was affinity purified from cells coexpressing WT or mutant Fes proteins and blotted either with anti-pTyr antibodies to measure Fes-induced phosphorylation (p) (top panel) or with anti-GST (bottom panel).(C) Comparison of the N-terminal SH2 domain sequence of Fps/Fes orthologs. Insertions originally used to identify the SH2 domain in v-Fps are indicated (RX15m and AX9m) (Sadowski et al., 1986; Stone et al., 1984). Conserved residues are highlighted in red and similar residues in yellow.(D) Details of the Fes SH2-kinase interface. Residues important for the SH2 domain-kinase interaction are conserved in Fps/Fes family members but not in other tyrosine kinases. Residues mutated in this study are indicated by a green asterisk. Interactions of interface residues and the involved secondary structure elements are shown in the structure of active Fes. The alignment of human sequences is colored as in (C).

Mentions: An overview of unphosphorylated Fes SH2-kinase in its catalytically active conformation is shown in Figure 1B. In this active configuration, the SH2 domain is stabilized by a sulphate ion, which mimics the phosphate of a pTyr ligand by coordinating a conserved SH2 domain arginine (R483), as well as R467 and S485 (see Figure 5A). In addition, a sulphate ion is coordinated by Y713 and R706 in the activation segment, thereby mimicking autophosphorylation (see Figure 3B). The SH2 and kinase domains form a stable unit linked by the N-terminal region of the SH2 domain and polar interactions between the SH2 domain helix αA and the catalytically important helix αC (Figures 1C, 2D, and S2). In Fes/Fer family members, the linker region between the SH2 and kinase domains is about 8 residues shorter when compared to other cytoplasmic tyrosine kinases, constraining the packing of the SH2 domain. The N terminus of the SH2 domain (462HGAI) intercalates between the central SH2 β sheet and the loop region between strands β4 and β5 in the kinase domain (Figures 2D and S3). This tight packing does not leave room for bulky side chains, and the central glycine residue is present in all Fes family members, suggesting that this domain packing is conserved.


Structural coupling of SH2-kinase domains links Fes and Abl substrate recognition and kinase activation.

Filippakopoulos P, Kofler M, Hantschel O, Gish GD, Grebien F, Salah E, Neudecker P, Kay LE, Turk BE, Superti-Furga G, Pawson T, Knapp S - Cell (2008)

Activation of Fes Kinase Activity by SH2 Domain Interactions(A) The effect of mutations in the SH2 domain on Fes kinase activity: HEK293T cells were cotransfected with Flag-tagged Fes and GST-cortactin. Cell lysates were immunoprecipitated with anti-Flag and blotted with anti-pTyr to reveal autophosphorylated (p) Flag-Fes (top panel) or with anti-Flag (lower panel) as a control. WT indicates wild-type Fes, and mutants are described in the text.(B) GST-cortactin was affinity purified from cells coexpressing WT or mutant Fes proteins and blotted either with anti-pTyr antibodies to measure Fes-induced phosphorylation (p) (top panel) or with anti-GST (bottom panel).(C) Comparison of the N-terminal SH2 domain sequence of Fps/Fes orthologs. Insertions originally used to identify the SH2 domain in v-Fps are indicated (RX15m and AX9m) (Sadowski et al., 1986; Stone et al., 1984). Conserved residues are highlighted in red and similar residues in yellow.(D) Details of the Fes SH2-kinase interface. Residues important for the SH2 domain-kinase interaction are conserved in Fps/Fes family members but not in other tyrosine kinases. Residues mutated in this study are indicated by a green asterisk. Interactions of interface residues and the involved secondary structure elements are shown in the structure of active Fes. The alignment of human sequences is colored as in (C).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2572732&req=5

fig2: Activation of Fes Kinase Activity by SH2 Domain Interactions(A) The effect of mutations in the SH2 domain on Fes kinase activity: HEK293T cells were cotransfected with Flag-tagged Fes and GST-cortactin. Cell lysates were immunoprecipitated with anti-Flag and blotted with anti-pTyr to reveal autophosphorylated (p) Flag-Fes (top panel) or with anti-Flag (lower panel) as a control. WT indicates wild-type Fes, and mutants are described in the text.(B) GST-cortactin was affinity purified from cells coexpressing WT or mutant Fes proteins and blotted either with anti-pTyr antibodies to measure Fes-induced phosphorylation (p) (top panel) or with anti-GST (bottom panel).(C) Comparison of the N-terminal SH2 domain sequence of Fps/Fes orthologs. Insertions originally used to identify the SH2 domain in v-Fps are indicated (RX15m and AX9m) (Sadowski et al., 1986; Stone et al., 1984). Conserved residues are highlighted in red and similar residues in yellow.(D) Details of the Fes SH2-kinase interface. Residues important for the SH2 domain-kinase interaction are conserved in Fps/Fes family members but not in other tyrosine kinases. Residues mutated in this study are indicated by a green asterisk. Interactions of interface residues and the involved secondary structure elements are shown in the structure of active Fes. The alignment of human sequences is colored as in (C).
Mentions: An overview of unphosphorylated Fes SH2-kinase in its catalytically active conformation is shown in Figure 1B. In this active configuration, the SH2 domain is stabilized by a sulphate ion, which mimics the phosphate of a pTyr ligand by coordinating a conserved SH2 domain arginine (R483), as well as R467 and S485 (see Figure 5A). In addition, a sulphate ion is coordinated by Y713 and R706 in the activation segment, thereby mimicking autophosphorylation (see Figure 3B). The SH2 and kinase domains form a stable unit linked by the N-terminal region of the SH2 domain and polar interactions between the SH2 domain helix αA and the catalytically important helix αC (Figures 1C, 2D, and S2). In Fes/Fer family members, the linker region between the SH2 and kinase domains is about 8 residues shorter when compared to other cytoplasmic tyrosine kinases, constraining the packing of the SH2 domain. The N terminus of the SH2 domain (462HGAI) intercalates between the central SH2 β sheet and the loop region between strands β4 and β5 in the kinase domain (Figures 2D and S3). This tight packing does not leave room for bulky side chains, and the central glycine residue is present in all Fes family members, suggesting that this domain packing is conserved.

Bottom Line: Our data indicate that Fes kinase activation is closely coupled to substrate recognition through cooperative SH2-kinase-substrate interactions.Similarly, we find that the SH2 domain of the active Abl kinase stimulates catalytic activity and substrate phosphorylation through a distinct SH2-kinase interface.Thus, the SH2 and catalytic domains of active Fes and Abl pro-oncogenic kinases form integrated structures essential for effective tyrosine kinase signaling.

View Article: PubMed Central - PubMed

Affiliation: Structural Genomics Consortium, University of Oxford, Old Road Campus, Roosevelt Drive, Oxford OX3 7DQ, UK.

ABSTRACT
The SH2 domain of cytoplasmic tyrosine kinases can enhance catalytic activity and substrate recognition, but the molecular mechanisms by which this is achieved are poorly understood. We have solved the structure of the prototypic SH2-kinase unit of the human Fes tyrosine kinase, which appears specialized for positive signaling. In its active conformation, the SH2 domain tightly interacts with the kinase N-terminal lobe and positions the kinase alphaC helix in an active configuration through essential packing and electrostatic interactions. This interaction is stabilized by ligand binding to the SH2 domain. Our data indicate that Fes kinase activation is closely coupled to substrate recognition through cooperative SH2-kinase-substrate interactions. Similarly, we find that the SH2 domain of the active Abl kinase stimulates catalytic activity and substrate phosphorylation through a distinct SH2-kinase interface. Thus, the SH2 and catalytic domains of active Fes and Abl pro-oncogenic kinases form integrated structures essential for effective tyrosine kinase signaling.

Show MeSH