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Sequence requirements for binding of Src family tyrosine kinases to activated growth factor receptors
G Alonso1, M Koegl, N Mazurenko
1Differentiation Programme, European Molecular Biology Laboratory, Heidelberg, Germany.
Abstract:
Activation of growth factor receptor protein tyrosine kinases frequently results in the binding of numerous proteins to their tyrosine-phosphorylated cytoplasmic domains. These interactions involve the SH2 domains of the binding proteins and phosphorylated tyrosines on the receptor molecules, with the specificity of interaction dictated by the amino acid composition surrounding the phosphorylated tyrosine. In the case of the platelet-derived growth factor (PDGF) receptor, the major binding site for Src family tyrosine kinases is in the juxtamembrane domain and includes tyrosine 579 (Mori, S., Rönnstrand, L., Yokote, K., Engström, A., Courtneidge, S. A., Claesson-Welsh, L., and Heldin, C-H. (1993) EMBO J. 12, 2257-2264). To analyze in more detail which amino acids surrounding the phosphorylated tyrosine at position 579 were important for high affinity interaction with Src family kinases, we synthesized a series of phosphopeptides corresponding to this binding site in which single amino acids were individually changed and tested their ability to compete with the PDGF receptor for binding of Fyn. We found that not only the three residues carboxyl-terminal to the phosphorylated tyrosine were important but that also residues at positions -1 and +4 relative to the tyrosine were required. Phosphorylation of both tyrosines 579 and 581 significantly increased competition efficiency. The activated colony stimulating factor-1 (CSF-1) receptor, which is known to associate with Src family kinases, has a sequence in its juxtamembrane region similar to that surrounding Tyr-579 of the PDGF receptor, and a phosphopeptide modeled on this sequence competed the association of Fyn with the receptor in vitro. Furthermore, mutational analysis demonstrated that these sequences were required for the efficient association of Src family kinases with the activated CSF-1 receptor in vivo. Phosphopeptides corresponding to the Src family binding sites of both PDGF and CSF-1 receptors activated Src kinase activity in vitro. These observations support a model in which the enzymatic activity of Src family tyrosine kinases is controlled by intra- and intermolecular interactions of tyrosine-phosphorylated peptides with the SH2 domain of the kinases.
Insights
Specific amino acids surrounding phosphorylated tyrosines on growth factor receptors are crucial for binding Src family kinases. This interaction regulates Src kinase activity, impacting cellular signaling pathways.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Growth factor receptor protein tyrosine kinases activate signaling pathways upon ligand binding.
- SH2 domains of binding proteins mediate interactions with tyrosine-phosphorylated receptor cytoplasmic domains.
- Specific amino acid sequences surrounding phosphorylated tyrosines dictate binding specificity.
Purpose of the Study:
- To identify critical amino acids surrounding tyrosine 579 of the platelet-derived growth factor (PDGF) receptor involved in high-affinity binding of Src family kinases.
- To investigate the role of specific juxtamembrane sequences in the association of Src family kinases with activated colony stimulating factor-1 (CSF-1) receptor.
- To explore the impact of these interactions on Src kinase activity.
Main Methods:
- Synthesis and testing of phosphopeptides with single amino acid mutations corresponding to the PDGF receptor binding site.
- In vitro competition assays to measure binding affinity of Fyn to PDGF receptor phosphopeptides.
- In vitro and in vivo mutational analysis of the CSF-1 receptor juxtamembrane region for Src family kinase association.
- In vitro kinase assays using phosphopeptides to assess Src kinase activity.
Main Results:
- Amino acids at positions -1 and +4 relative to phosphorylated tyrosine 579 of the PDGF receptor are critical for high-affinity Src family kinase binding.
- Phosphorylation of both tyrosines 579 and 581 significantly enhances competition efficiency for Fyn binding.
- A phosphopeptide mimicking the CSF-1 receptor juxtamembrane sequence competed Fyn association with the receptor in vitro and was required for in vivo association.
- Phosphopeptides from both PDGF and CSF-1 receptors activated Src kinase activity in vitro.
Conclusions:
- Specific amino acid residues surrounding phosphorylated tyrosines in growth factor receptors are essential for SH2 domain-mediated binding of Src family kinases.
- These interactions play a role in regulating Src family kinase activity, potentially through intra- and intermolecular peptide-SH2 domain interactions.
- The findings support a model where Src kinase activity is controlled by tyrosine-phosphorylated peptides binding to the SH2 domain.