Regulation of cilium length and intraflagellar transport by the MAP kinases MAK, MRK, and MOK

摘要

Cilia are assembled and maintained by intraflagellar transport(IFT), which moves cargo bi-directionally along theaxoneme of the cilium. Regulation of IFT allows modulationof cilia length and influences signaling pathways thatinitiate in the cilium. We characterized C. elegans’ DYF-5,a MAP kinase that regulates cilia length and IFT. DYF-5has three mammalian homologues: MAK (male germ cellassociatedkinase), MRK (MAK-related kinase) and MOK(MAPK/MAK/MRK overlapping kinase). Here we analyzedthe functions of MAK, MRK and MOK. GFP fusionsof MAK, MRK and MOK showed that all three proteinslocalize along the length of the cilia of IMCD3 and RPEcells, and accumulate at the distal tip. Knockdown of MRKor MOK resulted in longer cilia, while overexpression ofMAK or MRK resulted in short and stumpy cilia. Interestingly,overexpression of MOK did not affect cilia length.These effects on cilia length are in line with our data onDYF-5 function in C. elegans (Burghoorn et al., 2007) andpublished data on MAK (Omori et al., 2010). To visualizeIFT and measure its speed we have generated IMCD3 celllines that stably express GFP-fusion constructs of differentcomponents of the IFT complex: Kinesin-II, Kif17,complex A, complex B, and the BBSome. All these GFPfusion proteins showed an average anterograde speed of~0.4 μm/s and a similar average retrograde speed. Knockdown of MOK using shRNAs resulted in increased anterogradespeeds of all constructs to ~0.6 μm/s, while knockdown of MRK had no effect. Retrograde speeds were notaffected.