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K. L. M. Coene, D. A. Mans, S. J. F. Letteboer, K. Boldt, C. J. Gloeckner, F. P. M. Cremers, M. Ueffing, R. Roepman; The Retinal Ciliopathy-Associated Protein Homologs RPGRIP1 and RPGRIP1L Are Linked to (Connecting) Cilium Dynamics Through Interaction With a Serine/Threonine Kinase. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2583.
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Recent studies have established ciliary dysfunction as the underlying cause of a broad range of multi-organ phenotypes, often including retinal pathology. Although more and more ‘ciliopathy’ genes are identified, little is known about their actual function in the (connecting) cilium. Our aim is to gain more insight in the function of two retinal ciliopathy-associated protein homologs, RPGR interacting protein 1 (RPGRIP1), and RPGRIP1-like protein (RPGRIP1L). Mutations in RPGRIP1 lead to Leber congenital amaurosis, while mutations in RPGRIP1L were identified in patients with Joubert, Meckel, and COACH syndrome. The RPGRIP1L A229T variant is also a common modifier for retinal degeneration.
RPGRIP1 and RPGRIP1L were used as bait proteins in Strep-Flag tandem affinity purification (SF-TAP) experiments in human embryonic kidney (HEK293T) cells. The associated protein complexes were identified by mass spectrometry (LC-MS), and further validated by affinity biochemistry and immunohistochemistry. Genes were silenced in ciliated retinal pigment epithelium cells by transfection of small interfering RNA (siRNA) oligos.
The SF-TAP procedure allowed the identification of functionally active protein complexes in cultured cells in vivo. We identified a serine/threonine kinase as a component of both the RPGRIP1- as well as the RPGRIP1L-associated protein complex. The interaction between this kinase and RPGRIP1/1L was confirmed in GST-pulldown and co-immunoprecipitation assays. In ciliated retinal pigment epithelium cells, the kinase localized to basal bodies. In adult mouse retinas, the kinase was predominantly detected at the ciliary rootlet. Downregulation of the gene encoding the kinase led to a significant decrease in cilium assembly.
We have identified a serine/threonine kinase as interacting partner of RPGRIP1 and RPGRIP1L. This kinase localizes to centrosomes and plays a role in cilium dynamics, possibly through regulation of cell cycle progression. Its direct association with the connecting cilium protein complex defines this kinase as a candidate gene for (retinal) ciliopathies.
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