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Jason R. Bader, Joseph C. Besharse; Phosphorylation Regulates the Function of the Kinesin-2 Motor KIF17 in Cone Photoreceptors. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2586.
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Multiple proteins are targeted to photoreceptor outer segments (OSs) where they function in the vision pathway, and intraflagellar transport (IFT), a highly conserved bidirectional transport pathway occurring along the microtubule-based ciliary axoneme which has been implicated in trafficking. The canonical anterograde motor for IFT is the heterotrimeric kinesin II or KIF3 complex. Previous work from our laboratory has demonstrated a requirement for an additional kinesin 2 family motor, the homodimeric KIF17. The mechanism used to coordinate KIF17 and the KIF3 complex is not clear. However, we have identified a role for phosphorylation in regulating KIF17-based photoreceptor IFT.
Zebrafish expressing GFP-tagged WT, phosphomimetic, or non-phosphorylatable KIF17 were analyzed by live cell imaging and immunofluorescence microscopy. These constructs were also introduced into mammalian cells and the impact was assessed by immunoflourescence. Biochemical assays including immunoprecipitation and sucrose density centrifugation were used to test the impact of KIF17 phosphorylation on the motor-cargo interaction.
Analysis utilizing non-phosphorylatable and phosphomimetic KIF17 mutants revealed that phosphorylation modulates KIF17’s translocation to the distal tip of zebrafish cone OSs. Consistent with this, phosphorylation impacts KIF17 primary cilium distal tip accumulation in cultured cells. Biochemical analysis revealed that phosphorylation of the KIF17 tail domain regulates the interaction between the motor and the IFT complex. Real time imaging revealed that KIF17 moves at rates predicted for microtubule based motility in the distal domain of the cone OS.
IFT is essential for photoreceptor development and maintenance. These results demonstrate that KIF17 translocates to the distal region of cone OSs and this activity is dependent on phosphorylation. Phosphorylation also impacts the distal tip accumulation of KIF17 in kidney epithelial cell primary cilia, suggesting a conserved mechanism of KIF17 regulation. Furthermore, phosphorylation modulates KIF17’s interaction with the IFT complex. Overall, these findings reveal a new mechanism used to coordinate KIF17 activity in photoreceptor OSs, which is essential for photoreceptor maintenance.
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