Journal
PLOS ONE
Volume 7, Issue 11, Pages -Publisher
PUBLIC LIBRARY SCIENCE
DOI: 10.1371/journal.pone.0048773
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Funding
- Canadian Institutes of Health Research [MOP-111008]
- National Center for Research Resources
- Office of Research Infrastructure Programs (ORIP) of the National Institutes of Health [2 R01 RR17359-10]
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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-3 beta 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-3 beta phosphorylation sites abolished CRMP-2 targeting to the primary cilium without affecting basal body localization. Treatment of cells with lithium, a potent GSK-3 beta inhibitor, or with two specific GSK-3 beta 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-3 beta.
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