Mixed lineage kinase 2 interacts with clathrin and influences clathrin-coated vesicle trafficking.
Mixed lineage kinase 2 (MLK2) is a protein kinase that signals in the stress-activated Jun N-terminal kinase signal transduction pathway. We used immunoprecipitation and mass spectrometric analysis to identify MLK2-binding proteins in cell lines with inducible expression of green fluorescent protein-tagged MLK2. Here we report the identification of clathrin as ... a binding partner for MLK2 in both cultured cells and mammalian brain. We demonstrate that clathrin binding requires a motif (LLDMD) located near the MLK2 C terminus, which is similar to "clathrin box" motifs important for binding of clathrin coat assembly and accessory proteins to the clathrin heavy chain. A C-terminal fragment of MLK2 containing this motif binds strongly to clathrin, and mutation of the LLDMD sequence to LAAAD completely abrogates clathrin binding. We isolated clathrin-coated vesicles from green fluorescent protein-MLK2-expressing cells and from mouse brain lysates and found that MLK2 is enriched along with clathrin in these vesicles. In addition, we demonstrated that endogenous MLK2 co-immunoprecipitates with clathrin heavy chain from the vesicle-enriched fraction of mouse brain lysate. Furthermore, overexpression of MLK2 in cultured cells inhibits accumulation of labeled transferrin in recycling endosomes during receptor-mediated endocytosis. These findings suggest a role for MLK2 and the stress-signaling pathway at sites of clathrin activity in vesicle formation or trafficking.
Mesh Terms:
Amino Acid Motifs, Amino Acid Sequence, Animals, Biological Transport, Brain, Clathrin, Clathrin-Coated Vesicles, Electrophoresis, Polyacrylamide Gel, Endocytosis, Endosomes, Green Fluorescent Proteins, Humans, Immunoblotting, Luminescent Proteins, MAP Kinase Kinase Kinases, Mass Spectrometry, Mice, Microscopy, Fluorescence, Models, Genetic, Molecular Sequence Data, Peptides, Plasmids, Precipitin Tests, Protein Binding, Protein Structure, Tertiary, Sequence Homology, Amino Acid, Signal Transduction, Transfection, Transferrin
Amino Acid Motifs, Amino Acid Sequence, Animals, Biological Transport, Brain, Clathrin, Clathrin-Coated Vesicles, Electrophoresis, Polyacrylamide Gel, Endocytosis, Endosomes, Green Fluorescent Proteins, Humans, Immunoblotting, Luminescent Proteins, MAP Kinase Kinase Kinases, Mass Spectrometry, Mice, Microscopy, Fluorescence, Models, Genetic, Molecular Sequence Data, Peptides, Plasmids, Precipitin Tests, Protein Binding, Protein Structure, Tertiary, Sequence Homology, Amino Acid, Signal Transduction, Transfection, Transferrin
J. Biol. Chem.
Date: Sep. 27, 2002
PubMed ID: 12105200
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