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.
Akbarzadeh
Shiva
S
Trescowthick Research Laboratories, Peter MacCallum Cancer Institute, Melbourne 8006, Victoria, Australia.
Ji
Hong
H
Frecklington
David
D
Marmy-Conus
Nelly
N
Mok
Yee-Foong
YF
Bowes
Leanne
L
Devereux
Lisa
L
Linsenmeyer
Martha
M
Simpson
Richard J
RJ
Dorow
Donna S
DS
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D001921:Brain / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism*; D022163:Clathrin-Coated Vesicles / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D015151:Immunoblotting; D008164:Luminescent Proteins / Q000378:metabolism; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism*; D013058:Mass Spectrometry; D051379:Mice; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010455:Peptides; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D014162:Transfection; D014168:Transferrin / Q000378:metabolism
D002966:Clathrin; D008164:Luminescent Proteins; D010455:Peptides; D014168:Transferrin; D049452:Green Fluorescent Proteins; D020930:MAP Kinase Kinase Kinases; C093290:MAP3K10 protein, human; C490541:Map3k10 protein, mouse