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Tyrosine phosphorylation of Sprouty2 enhances its interaction with c-Cbl and is crucial for its function
Chee Wai Fong1, Hwei Fen Leong, Esther Sook Miin Wong
1Signal Transduction Laboratory, Institute of Molecular and Cell Biology, National University of Singapore, Singapore, 117609 Singapore.
Abstract:
Mammalian Sprouty (Spry) proteins are now established as receptor tyrosine kinase-induced modulators of the Ras/mitogen-activated protein kinase pathway. Specifically, hSpry2 inhibits the fibroblast growth factor receptor (FGFR)-induced mitogen-activated protein kinase pathway but conversely prolongs activity of the same pathway following epidermal growth factor (EGF) stimulation, where activated EGF receptors are retained on the cell surface. In this study it is demonstrated that hSpry2 is tyrosine-phosphorylated upon stimulation by either FGFR or EGF and subsequently binds endogenous c-Cbl with high affinity. A conserved motif on hSpry2, together with phosphorylation on tyrosine 55, is required for its enhanced interaction with the SH2-like domain of c-Cbl. A hSpry2 mutant (Y55F) that did not exhibit an enhanced binding with c-Cbl failed to retain EGF receptors on the cell surface. Furthermore, individually mutating hSpry2 residues 52-59 to alanine indicated a tight correlation between their affinity for c-Cbl binding and their inhibition of ERK2 activity in the FGFR pathway. We postulate that tyrosine phosphorylation "activates" hSpry2 by enhancing its interaction with c-Cbl and that this interaction is critical for its physiological function in a signal-specific context.
Insights
Mammalian Sprouty 2 (hSpry2) protein phosphorylation enhances its binding to c-Cbl, a key interaction for regulating cell signaling pathways like the Ras/mitogen-activated protein kinase pathway.
Area of Science:
- Cell Biology
- Molecular Biology
- Signal Transduction
Background:
- Sprouty (Spry) proteins modulate receptor tyrosine kinase (RTK) signaling.
- hSpry2 differentially regulates fibroblast growth factor receptor (FGFR) and epidermal growth factor (EGF) signaling pathways.
- hSpry2's role in RTK pathway regulation requires further elucidation.
Purpose of the Study:
- To investigate the mechanism of hSpry2-mediated regulation of RTK signaling.
- To determine the role of hSpry2 phosphorylation and c-Cbl interaction in signal transduction.
- To elucidate the signal-specific functions of hSpry2.
Main Methods:
- Tyrosine phosphorylation assays upon FGFR and EGF stimulation.
- Co-immunoprecipitation to assess hSpry2 and c-Cbl binding.
- Site-directed mutagenesis of hSpry2 (Y55F and residues 52-59).
- Analysis of EGF receptor retention and ERK2 activity.
Main Results:
- hSpry2 undergoes tyrosine phosphorylation upon stimulation by FGFR or EGF.
- Phosphorylated hSpry2 binds with high affinity to endogenous c-Cbl.
- Tyrosine 55 phosphorylation is critical for hSpry2-c-Cbl interaction.
- Mutations affecting c-Cbl binding impair EGF receptor retention and alter ERK2 activity in the FGFR pathway.
Conclusions:
- Tyrosine phosphorylation of hSpry2 enhances its interaction with c-Cbl, acting as an activation mechanism.
- The hSpry2-c-Cbl interaction is crucial for hSpry2's physiological function in a signal-specific manner.
- This study provides insights into the context-dependent regulation of RTK signaling by hSpry2.