Conformational changes in CLIP-170 regulate its binding to microtubules and dynactin localization

doi:10.1083/jcb.200402082

Published online 4 October 2004. doi:10.1083/jcb.200402082
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Article

Conformational changes in CLIP-170 regulate its binding to microtubules and dynactin localization

Gideon Lansbergen¹, Yulia Komarova³^,4, Mauro Modesti¹, Claire Wyman¹^,2, Casper C. Hoogenraad¹, Holly V. Goodson⁵, Régis P. Lemaitre⁶, David N. Drechsel⁶, Erik van Munster⁷, Theodorus W.J. Gadella, Jr.⁷, Frank Grosveld¹, Niels Galjart¹, Gary G. Borisy³, and Anna Akhmanova¹

¹ MGC Department of Cell Biology and Genetics and ² Department of Radiation Oncology, Erasmus Medical Center, 3000 DR Rotterdam, Netherlands
³ Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, IL 60611
⁴ Laboratory of Cell Motility, A.N. Belozersky Institute, Moscow State University, Moscow, 119992, Russia
⁵ Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556
⁶ Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany
⁷ Section of Molecular Cytology and Centre for Advanced Microscopy, Swammerdam Institute for Life Science, University of Amsterdam, 1098 SM Amsterdam, Netherlands

Address correspondence to Anna Akhmanova, MGC Dept. of Cell Biology and of Genetics, Erasmus Medical Center, P.O. Box 1738, 3000 DR Rotterdam, Netherlands. Tel.: 31-10-4087166. Fax: 31-10-4089468. email: anna.akhmanova{at}chello.nl

Cytoplasmic linker protein (CLIP)-170, CLIP-115, and the dynactinsubunit p150^Glued are structurally related proteins, which associatespecifically with the ends of growing microtubules (MTs). Here,we show that down-regulation of CLIP-170 by RNA interferenceresults in a strongly reduced accumulation of dynactin at theMT tips. The NH₂ terminus of p150^Glued binds directly to theCOOH terminus of CLIP-170 through its second metal-binding motif.p150^Glued and LIS1, a dynein-associating protein, compete forthe interaction with the CLIP-170 COOH terminus, suggestingthat LIS1 can act to release dynactin from the MT tips. We alsoshow that the NH₂-terminal part of CLIP-170 itself associateswith the CLIP-170 COOH terminus through its first metal-bindingmotif. By using scanning force microscopy and fluorescence resonanceenergy transfer-based experiments we provide evidence for anintramolecular interaction between the NH₂ and COOH terminiof CLIP-170. This interaction interferes with the binding ofthe CLIP-170 to MTs. We propose that conformational changesin CLIP-170 are important for binding to dynactin, LIS1, andthe MT tips.

Key Words: plus end–tracking proteins; motor protein; cytoplasmic dynein; LIS1; CLIP-115

C.C. Hoogenraad's present address is The Picower Center forLearning and Memory, Massachusetts Institute of Technology,Cambridge, MA 02139.

Abbreviations used in this paper: CLIP, cytoplasmic linker protein;FRET, fluorescence resonance energy transfer; HIS, 6X histidine;IP, immunoprecipitation; MT, microtubule; RNAi, RNA interference;siRNA, small interfering RNA; SFM, scanning force microscopy;+TIP, plus end–tracking protein.

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