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Tyrosine 569 in the c-Fms juxtamembrane domain is essential for kinase activity and macrophage colony-stimulating
G M Myles1, C S Brandt, K Carlberg
1Cell Biology Department, Fred Hutchinson Cancer Research Center, Seattle, Washington 98104.
Abstract:
The receptor (Fms) for macrophage colony-stimulating factor (M-CSF) is a member of the tyrosine kinase class of growth factor receptors. It maintains survival, stimulates growth, and drives differentiation of the macrophage lineage of hematopoietic cells. Fms accumulates on the cell surface and becomes activated for signal transduction after M-CSF binding and is then internalized via endocytosis for eventual degradation in lysosomes. We have investigated the mechanism of endocytosis as part of the overall signaling process of this receptor and have identified an amino acid segment near the cytoplasmic juxtamembrane region surrounding tyrosine 569 that is important for internalization. Mutation of tyrosine 569 to alanine (Y569A) eliminates ligand-induced rapid endocytosis of receptor molecules. The mutant Fms Y569A also lacks tyrosine kinase activity; however, tyrosine kinase activity is not essential for endocytosis because the kinase inactive receptor Fms K614A does undergo ligand-induced endocytosis, albeit at a reduced rate. Mutation of tyrosine 569 to phenylalanine had no effect on the M-CSF-induced endocytosis of Fms, and a four-amino-acid sequence containing Y-569 could support endocytosis when transferred into the cytoplasmic juxtamembrane region of a glycophorin A construct. These results indicate that tyrosine 569 within the juxtamembrane region of Fms is part of a signal recognition sequence for endocytosis that does not require tyrosine phosphorylation at this site and that this domain also influences the kinase activity of the receptor. These results are consistent with a ligand-dependent step in recognition of the potential cryptic internalization signal.
Insights
A specific tyrosine (Y569) in the Fms receptor is crucial for its internalization after binding macrophage colony-stimulating factor (M-CSF). This internalization signal does not require tyrosine phosphorylation and influences receptor kinase activity.
Area of Science:
- Cell biology
- Molecular signaling
- Hematopoiesis
Background:
- The Fms receptor, a tyrosine kinase, mediates macrophage colony-stimulating factor (M-CSF) signaling.
- M-CSF binding triggers Fms receptor activation, cell surface accumulation, and subsequent endocytosis for degradation.
- Understanding Fms receptor endocytosis is key to its overall signaling mechanism.
Purpose of the Study:
- To investigate the mechanism of Fms receptor endocytosis.
- To identify specific amino acid sequences involved in Fms receptor internalization.
- To determine the role of tyrosine kinase activity in Fms receptor endocytosis.
Main Methods:
- Site-directed mutagenesis of the Fms receptor, specifically targeting tyrosine 569 (Y569A) and a kinase-inactive mutant (K614A).
- Assessing ligand-induced endocytosis rates for wild-type and mutant Fms receptors.
- Functional transfer of the identified juxtamembrane sequence into a heterologous protein (glycophorin A).
Main Results:
- Mutation of tyrosine 569 to alanine (Y569A) abolished M-CSF-induced rapid endocytosis of Fms.
- The Y569A mutation also eliminated Fms tyrosine kinase activity.
- A kinase-inactive Fms mutant (K614A) still underwent ligand-induced endocytosis, indicating kinase activity is not essential for internalization.
- Mutation of tyrosine 569 to phenylalanine did not affect endocytosis.
- A four-amino-acid sequence containing Y569 conferred endocytosis when transferred to glycophorin A.
Conclusions:
- Tyrosine 569 in the Fms juxtamembrane region is a critical component of a signal recognition sequence for endocytosis.
- This internalization signal does not require tyrosine phosphorylation at Y569.
- The juxtamembrane domain containing Y569 influences the receptor's tyrosine kinase activity.
- These findings suggest a ligand-dependent recognition of a cryptic internalization signal within the Fms receptor.