CLIPR-59 Is a Lipid Raft-associated Protein Containing a Cytoskeleton-associated Protein Glycine-rich Domain (CAP-Gly) That Perturbs Microtubule Dynamics

doi:10.1074/jbc.M406482200

CLIPR-59 Is a Lipid Raft-associated Protein Containing a Cytoskeleton-associated Protein Glycine-rich Domain (CAP-Gly) That Perturbs Microtubule Dynamics*

^‡CNRS Unité Mixte de Recherche 144, Institut Curie Section Recherche, 75248 Paris Cedex 05, and ^¶Equipe d'Accueil Doctorale 1595, Faculté de Pharmacie, Université Paris-Sud, 92296 Châtenay-Malabry, France

∥ To whom correspondence should be addressed. Tel.: 33-1-42-34-63-88; Fax: 33-1-42-34-63-82; E-mail: franck.perez{at}curie.fr.

Abstract

We recently have identified a new cytoplasmic linker protein (CLIP), CLIPR-59, which is involved in the regulation of early endosome/trans-Golgi network dynamics. In contrast with CLIP-170, CLIPR-59 is not localized to microtubules at steady state but is associated with the trans-Golgi network and the plasma membrane. Here we show that the last 30 amino acids (C30) are sufficient for membrane targeting and that two cysteines in the C30 domain are palmitoylated. We demonstrate that CLIPR-59 is associated with lipid rafts via its C-terminal palmitoylated domain. In vitro experiments suggest that CLIPR-59 and its microtubule-binding domain alone have a better affinity for unpolymerized tubulin or small oligomers than for microtubules. In contrast with the CLIP-170 microtubule-binding domain, the CLIPR-59 microtubule-binding domain diminishes microtubule regrowth after nocodazole washout in vivo, showing that this domain can prevent microtubule polymerization. In contrast with the role of linker between membranes and microtubules that was proposed for CLIP function, CLIPR-59 thus may have an “anti-CLIP” function by preventing microtubule-raft interactions.

Footnotes

↵1 The abbreviations used are: CLIP, cytoplasmic linker protein; CLIPR-59, CLIP-170-related protein of 59 kDa; CAP-Gly, cytoskeleton-associated protein glycine-rich domain; MTB, microtubule-binding domain; C59MTB, the MTB of CLIPR-59; TGN, trans-Golgi network; MEF, mouse embryonic fibroblast; GFP, green fluorescent protein; GFP-C59, GFP-labeled CLIPR-59; GFP-ΔC60, GFP-labeled CLIPR-59 with the last 60 amino acids deleted; GFP-C30, enhanced GFP cDNA fused with the sequence encoding the last 30 amino acids of CLIPR-59; HA, hemagglutinin; MBP, maltose-binding protein; TfR, transferrin receptor; MβCD, methyl-β-cyclodextrin; PIPES, 1,4-piperazinedi-ethanesulfonic acid; PNS, postnuclear supernatant; NZ, nocodazole; DRM, detergent-resistant membrane(s); GPI, glycosylphosphatidyl inositol; PIC, protease inhibitor mixture.
↵* This work was supported by the Centre National de la Recherche Scientifique and the Institut Curie. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ Supported by a postdoctoral fellowship from the Association pour la Recherche contre le Cancer. Present address: CNRS UPR 9051, Centre Hayem-Hôpital Saint-Louis, Paris, France.
- Received June 10, 2004.
The American Society for Biochemistry and Molecular Biology, Inc.