title: Targeting of CRMP-2 to the primary cilium is modulated by GSK-3β.
abstract: CRMP-2 plays a pivotal role in promoting axon formation, neurite outgrowth and elongation in neuronal cells. CRMP-2's role in other cells is unknown. Our preliminary results showed CRMP-2 expression in cilia of fibroblasts. To localize CRMP-2, define its role and study the regulation of CRMP-2's expression in cilia we carried out the following experiments. We find that in fibroblasts CRMP-2 localizes to the centrosome and is associated with the basal body and -at a low level- is present in primary cilia. Phosphorylated pCRMP-2 can only be detected at the basal body. RNAi knockdown of CRMP-2 interfered with primary cilium assembly demonstrating a critical requirement for CRMP-2. Deletion analysis of CRMP-2 identified a 51 amino acid sequence in the C-terminus that is required for targeting to the basal body and primary cilium. This domain contains GSK-3β phosphorylation sites as well as two repeats of the VxPx motif, previously identified as a cilium targeting signal in other primary cilium proteins. To our surprise, mutation of the CRMP-2 VxPx motifs did not eliminate primary cilium targeting. Instead, mutation of the GSK-3β phosphorylation sites abolished CRMP-2 targeting to the primary cilium without affecting basal body localization. Treatment of cells with lithium, a potent GSK-3β inhibitor, or with two specific GSK-3β inhibitors (the L803-mts peptide inhibitor and CHIR99021) resulted in cilium elongation and decreased basal body levels of pCRMP-2 as well as increased levels of total CRMP-2 at the primary cilium. In summary, we identified CRMP-2 as a protein critically involved in primary cilia formation. To our knowledge this is the first demonstration of modulation of primary cilium targeting by GSK-3β.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Canada.
authors.forename: Young
authors.initials: Y
authors.lastname: Ou
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
authors.forename: Min
authors.initials: M
authors.lastname: Cheng
authors.forename: Jerome B
authors.initials: JB
authors.lastname: Rattner
authors.forename: Ina
authors.initials: I
authors.lastname: Dobrinski
authors.forename: Frans A
authors.initials: FA
authors.lastname: van der Hoorn
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D018385:Centrosome / Q000378:metabolism; D002923:Cilia / Q000378:metabolism*; D004252:DNA Mutational Analysis; D005347:Fibroblasts / Q000378:metabolism; D052816:Foreskin / Q000166:cytology; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008297:Male; D008954:Models, Biological; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism
chemical_list: D036341:Intercellular Signaling Peptides and Proteins; D050505:Mutant Proteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; C095114:collapsin response mediator protein-2; D049452:Green Fluorescent Proteins; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3