Related Experiment Videos
Ligand-induced phosphorylation of the colony-stimulating factor 1 receptor can occur through an intermolecular
M Ohtsuka1, M F Roussel, C J Sherr
1Department of Tumor Cell Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105.
Abstract:
Ligand-induced tyrosine phosphorylation of the human colony-stimulating factor 1 receptor (CSF-1R) could involve either an intra- or intermolecular mechanism. We therefore examined the ability of a CSF-1R carboxy-terminal truncation mutant to phosphorylate a kinase-defective receptor, CSF-1R[met 616], that contains a methionine-for-lysine substitution at its ATP-binding site. By using an antipeptide serum that specifically reacts with epitopes deleted from the enzymatically competent truncation mutant, cross-phosphorylation of CSF-1R[met 616] on tyrosine was demonstrated, both in immune-complex kinase reactions and in intact cells stimulated with CSF-1. Both in vitro and in vivo, CSF-1R[met 616] was phosphorylated on tryptic peptides identical to those derived from wild-type CSF-1R, suggesting that receptor phosphorylation on tyrosine can proceed via an intermolecular interaction between receptor monomers. When expressed alone, CSF-1R[met 616] did not undergo ligand-induced down modulation, but its phosphorylation in cells coexpressing the kinase-active truncation mutant accelerated its degradation.
Insights
Tyrosine phosphorylation of the colony-stimulating factor 1 receptor (CSF-1R) can occur through intermolecular interactions between receptor monomers. This intermolecular mechanism facilitates the phosphorylation and subsequent degradation of kinase-defective CSF-1R mutants.
Area of Science:
- Cellular signaling
- Molecular biology
- Biochemistry
Background:
- The colony-stimulating factor 1 receptor (CSF-1R) is a key regulator of cell proliferation and differentiation.
- Ligand-induced tyrosine phosphorylation of CSF-1R is crucial for its signaling but the precise mechanism (intra- or intermolecular) remains debated.
Purpose of the Study:
- To investigate whether CSF-1R tyrosine phosphorylation occurs via an intermolecular mechanism.
- To characterize the phosphorylation of a kinase-defective CSF-1R mutant.
Main Methods:
- Utilized a carboxy-terminal truncation mutant of CSF-1R and a kinase-defective mutant (CSF-1R[met 616]) with a specific ATP-binding site mutation.
- Employed an antipeptide serum to detect phosphorylation of the mutant receptor.
- Performed immune-complex kinase reactions and experiments in intact cells stimulated with CSF-1.
Main Results:
- Demonstrated cross-phosphorylation of the kinase-defective CSF-1R[met 616] on tyrosine residues, both in vitro and in vivo.
- Identified tryptic peptides from phosphorylated CSF-1R[met 616] identical to those from wild-type CSF-1R.
- Showed that coexpression with a kinase-active mutant accelerated the degradation of CSF-1R[met 616] upon ligand stimulation.
Conclusions:
- Receptor phosphorylation on tyrosine can occur through intermolecular interactions between CSF-1R monomers.
- This intermolecular mechanism plays a role in CSF-1R signaling and regulation, including ligand-induced degradation.