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Dynamin is required for the activation of mitogen-activated protein (MAP) kinase by MAP kinase kinase
O Kranenburg1, I Verlaan, W H Moolenaar
1Division of Cellular Biochemistry, The Netherlands Cancer Institute and Centre for Biomedical Genetics, Plesmanlaan 121, 1066CX Amsterdam, The Netherlands.
Abstract:
Internalization of activated receptors from the plasma membrane has been implicated in the activation of mitogen-activated protein (MAP) kinase. However, the mechanism whereby membrane trafficking may regulate mitogenic signaling remains unclear. Here we report that dominant-negative dynamin (K44A), an inhibitor of endocytic vesicle formation, abrogates MAP kinase activation in response to epidermal growth factor, lysophosphatidic acid, and protein kinase C-activating phorbol ester. In contrast, dynamin-K44A does not affect the activation of Ras, Raf, and MAP kinase kinase (MEK) by either agonist. Through immunofluorescence and subcellular fractionation studies, we find that activated MEK is present both at the plasma membrane and in intracellular vesicles but not in the cytosol. Our findings suggest that dynamin-regulated endocytosis of activated MEK, rather than activated receptors, is a critical event in the MAP kinase activation cascade.
Insights
Endocytosis regulates mitogen-activated protein (MAP) kinase activation. Dynamin inhibition blocks MAP kinase activation by preventing the internalization of activated MAP kinase kinase (MEK), suggesting MEK endocytosis is critical for signaling.
Area of Science:
- Cell Biology
- Molecular Biology
- Signal Transduction
Background:
- Receptor internalization is linked to mitogen-activated protein (MAP) kinase activation.
- The precise role of membrane trafficking in regulating mitogenic signaling remains elusive.
Purpose of the Study:
- To investigate the mechanism by which membrane trafficking regulates MAP kinase activation.
- To determine if dynamin-mediated endocytosis is essential for MAP kinase signaling.
Main Methods:
- Utilized dominant-negative dynamin (K44A) as an inhibitor of endocytic vesicle formation.
- Employed immunofluorescence and subcellular fractionation to track protein localization.
- Assessed MAP kinase activation in response to various agonists.
Main Results:
- Dominant-negative dynamin (K44A) inhibited MAP kinase activation by epidermal growth factor, lysophosphatidic acid, and phorbol ester.
- Dynamin inhibition did not affect the activation of Ras, Raf, or MAP kinase kinase (MEK).
- Activated MEK was localized to the plasma membrane and intracellular vesicles, but not the cytosol.
Conclusions:
- Dynamin-regulated endocytosis of activated MEK is a critical step in the MAP kinase activation cascade.
- This process is essential for propagating the mitogenic signal downstream of receptor activation.