CLIP-170 interacts with dynactin complex and the APC-binding protein EB1 by different mechanisms.
CLIP-170 is a "cytoplasmic linker protein" implicated in endosome-microtubule interactions and in control of microtubule dynamics. CLIP-170 localizes dynamically to growing microtubule plus ends, colocalizing with the dynein activator dynactin and the APC-binding protein EB1. This shared "plus-end tracking" behavior suggests that CLIP-170 might interact with dynactin and/or EB1. We have used site-specific mutagenesis of CLIP-170 and a transfection/colocalization assay to address this question in mammalian tissue culture cells. Our results indicate that CLIP-170 interacts, directly or indirectly, with both dynactin and EB1. We find that the CLIP-170/dynactin interaction is mediated by the second metal binding motif of the CLIP-170 tail. In contrast, the CLIP-170/EB1 interaction requires neither metal binding motif. In addition, our experiments suggest that the CLIP-170/dynactin interaction occurs via the shoulder/sidearm subcomplex of dynactin and can occur in the cytosol (i.e., it does not require microtubule binding). These results have implications for the targeting of both dynactin and EB1 to microtubule plus ends. Our data suggest that the CLIP-170/dynactin interaction can target dynactin complex to microtubule plus ends, although dynactin likely also targets MT plus ends directly via the microtubule binding motif of the p150(Glued) subunit. We find that CLIP-170 mutants alter p150(Glued) localization without affecting EB1, indicating that EB1 can target microtubule plus ends independently of dynactin.
Goodson
Holly V
HV
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA. hgoodson@nd.edu
Skube
Susan B
SB
Stalder
Romaine
R
Valetti
Caterina
C
Kreis
Thomas E
TE
Morrison
Ewan E
EE
Schroer
Trina A
TA
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000072159:Dynactin Complex; D006367:HeLa Cells; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009363:Neoplasm Proteins; D017434:Protein Structure, Tertiary
C000606360:DCTN1 protein, human; D000072159:Dynactin Complex; C405804:EB1 microtubule binding proteins; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; C076744:cytoplasmic linker protein 170