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Src-dependent tyrosine phosphorylation regulates dynamin self-assembly and ligand-induced endocytosis of the
Seungkirl Ahn1, Jihee Kim, Carmen L Lucaveche
1Howard Hughes Medical Institute, Department of Pharmacology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Abstract:
Endocytosis of ligand-activated receptors requires dynamin-mediated GTP hydrolysis, which is regulated by dynamin self-assembly. Here, we demonstrate that phosphorylation of dynamin I by c-Src induces its self-assembly and increases its GTPase activity. Electron microscopic analyses reveal that tyrosine-phosphorylated dynamin I spontaneously self-assembles into large stacks of rings. Tyrosine 597 was identified as being phosphorylated both in vitro and in cultured cells following epidermal growth factor receptor stimulation. The replacement of tyrosine 597 with phenylalanine impairs Src kinase-induced dynamin I self-assembly and GTPase activity in vitro. Expression of Y597F dynamin I in cells attenuates agonist-driven epidermal growth factor receptor internalization. Thus, c-Src-mediated tyrosine phosphorylation is required for the function of dynamin in ligand-induced signaling receptor internalization.
Insights
Phosphorylation of dynamin I by c-Src kinase triggers its self-assembly and enhances GTPase activity, crucial for receptor internalization. This tyrosine phosphorylation at residue 597 is essential for the endocytosis process.
Area of Science:
- Cell biology
- Molecular biology
- Biochemistry
Background:
- Endocytosis of activated receptors relies on dynamin-mediated GTP hydrolysis.
- Dynamin self-assembly is a key regulatory mechanism for this process.
Purpose of the Study:
- To investigate the role of c-Src kinase in regulating dynamin I function.
- To elucidate the mechanism by which c-Src influences dynamin I self-assembly and GTPase activity.
Main Methods:
- In vitro phosphorylation assays using c-Src kinase.
- Electron microscopy to visualize dynamin I self-assembly.
- Site-directed mutagenesis (Y597F) to assess the role of tyrosine 597.
- Cellular expression studies to evaluate receptor internalization.
Main Results:
- Phosphorylation of dynamin I by c-Src induces its self-assembly into ring stacks and increases GTPase activity.
- Tyrosine 597 was identified as the specific phosphorylation site by c-Src.
- Mutation of tyrosine 597 to phenylalanine abrogated c-Src-induced self-assembly and GTPase activity.
- Expression of the Y597F mutant dynamin I impaired epidermal growth factor receptor internalization.
Conclusions:
- c-Src-mediated tyrosine phosphorylation of dynamin I at residue 597 is essential for its self-assembly and GTPase activity.
- This phosphorylation event is critical for the proper function of dynamin in ligand-induced receptor internalization.