Date of Award
Master of Science (MS)
School of Biological Sciences
Martin F Engelke
Cilia and flagella are microtubule-based organelles that protrude from the plasma membrane of most cells in the body and play a critical role in cell signaling, motility, and development. Numerous diseases, collectively known as ciliopathies, with phenotypes like developmental abnormalities, cystic kidney disease, and obesity, have been linked to ciliary dysfunction. Intraflagellar transport (IFT) shuttles cellular cargoes along axonemal microtubules and into and out of cilia. IFT is essential for the assembly of cilia and flagella and is powered by heterodimeric kinesin-2 (KIF3A/KIF3B) and dynein-2. While the essential role of kinesin-2 in mammalian ciliogenesis is well established, little is known about how its activity is regulated for IFT. Chaya and colleagues generated a construct in which eight potential phosphosites in the KIF3A tail, where cargo binds, were mutated (Chaya et al., 2014, EMBO J). This construct could not rescue ciliogenesis in Kif3a RNAi cells, suggesting that phosphorylation might regulate this motor for IFT.To determine which site mediates phospho-regulation, we created a library of constructs in which all potential phosphorylation sites in the KIF3A tail were mutated to dephosphomimetic or phosphomimetic residues. When expressed in knockout cells three phosphomimetic constructs resulted in ciliation rate that were significantly different from rescue with wild-type motor. However, replacing the phosphomimetic residues with structurally resembling residues that are not negatively charged phenocopied the phosphomimetic constructs. Thus, we conclude that the identified sites are important for KIF3A to function, but that phosphorylation of the KIF3A tail does not regulate kinesin-2 for ciliogenesis.
Fasawe, Ayoola, "Kif3 a Tail Domain Phosphorylation Is Not Required for Ciliogenesis in Mouse Embryonic Fibroblasts" (2023). Theses and Dissertations. 1663.
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