Related Experiment Videos
The PDGF receptor phosphorylates Tyr 138 in the c-Src SH3 domain in vivo reducing peptide ligand binding
1Molecular Biology and Virology Laboratory, The Salk Institute, La Jolla, California 92037, USA.
Abstract:
Treatment of quiescent NIH3T3 cells with PDGF BB results in the transient activation and hyperphosphorylation of the protein-tyrosine kinase, c-Src. These effects correlate with novel serine and tyrosine phosphorylations in the N-terminal non-catalytic region of the molecule, which contains an SH3 and SH2 domain. In this study, a site of PDGF-induced tyrosine phosphorylation was mapped to Tyr 138 in the SH3 domain; Tyr 138 is exposed on the SH3 peptide binding surface. This same site is phosphorylated in vitro by the PDGF receptor when purified baculovirus-expressed c-Src is complexed with the activated receptor. Phosphorylation of Tyr 138 required association of c-Src with the PDGF receptor via its SH2 domain. When a c-Src Phe 138 mutant was stably expressed in Src- mouse fibroblasts, it was activated to the same extent as wild type c-Src following PDGF stimulation, indicating that phosphorylation of this site is not required for PDGF-mediated activation. However, Tyr 138 phosphorylation was found to diminish SH3 domain peptide ligand binding ability in vitro.
Insights
Platelet-derived growth factor (PDGF) BB induces phosphorylation of c-Src protein-tyrosine kinase at Tyr 138 within its SH3 domain. This phosphorylation does not affect c-Src activation but reduces its SH3 domain
Area of Science:
- Cellular signaling
- Protein phosphorylation
- Kinase regulation
Background:
- Platelet-derived growth factor (PDGF) BB stimulates quiescent NIH3T3 cells.
- This stimulation leads to transient activation and hyperphosphorylation of c-Src protein-tyrosine kinase.
- Novel serine and tyrosine phosphorylations occur in c-Src's N-terminal non-catalytic region, containing SH3 and SH2 domains.
Purpose of the Study:
- To map the specific site of PDGF-induced tyrosine phosphorylation in c-Src.
- To investigate the role of this phosphorylation site in c-Src activation and function.
- To understand the mechanism of c-Src phosphorylation by the PDGF receptor.
Main Methods:
- Site-directed mutagenesis to create a c-Src Phe 138 mutant.
- Stable expression of the mutant in Src- mouse fibroblasts.
- Stimulation with PDGF BB and analysis of c-Src activation.
- In vitro phosphorylation assays using purified c-Src and PDGF receptor.
- Assessment of SH3 domain peptide ligand binding.
Main Results:
- PDGF-induced tyrosine phosphorylation was mapped to Tyr 138 in the c-Src SH3 domain.
- Phosphorylation of Tyr 138 required association of c-Src with the activated PDGF receptor via its SH2 domain.
- The Tyr 138 phosphorylation site is exposed on the SH3 peptide binding surface.
- A c-Src Phe 138 mutant showed normal PDGF-mediated activation, indicating Tyr 138 phosphorylation is not required for activation.
- Tyr 138 phosphorylation diminished SH3 domain peptide ligand binding in vitro.
Conclusions:
- PDGF receptor directly phosphorylates c-Src at Tyr 138 within the SH3 domain.
- This phosphorylation event is regulated by SH2 domain-mediated association with the PDGF receptor.
- While not essential for PDGF-induced c-Src activation, Tyr 138 phosphorylation modulates c-Src's SH3 domain binding capabilities.
- This finding provides insights into the intricate regulation of c-Src activity by growth factor signaling pathways.