The green biflagellate alga Chlamydomonas reinhardtii serves as model for studying structural and functional features of flagella. The axoneme of C. reinhardtii anchors a network of kinases and phosphatases that control motility. One of them, Casein Kinase 1 (CK1), is known to phosphorylate the Inner Dynein Arm I1 Intermediate Chain 138 (IC138), thereby regulating motility. CK1 is also involved in regulating the circadian rhythm of phototaxis and is relevant for the formation of flagella. By a comparative phosphoproteome approach, we determined phosphoproteins in the flagellum that are targets of CK1. Thereby, we applied the specific CK1 inhibitor CKI-7 that causes significant changes in the flagellum phosphoproteome and reduces the swimming velocity of the cells. In the CKI-7-treated cells, 14 phosphoproteins were missing compared to the phosphoproteome of untreated cells, including IC138, and four additional phosphoproteins had a reduced number of phosphorylation sites. Notably, inhibition of CK1 causes also novel phosphorylation events, indicating that it is part of a kinase network. Among them, Glycogen Synthase Kinase 3 is of special interest, because it is involved in the phosphorylation of key clock components in flies and mammals and in parallel plays an important role in the regulation of assembly in the flagellum. 1. Introduction Eukaryotic cilia or flagella are microtubule-based organelles that are highly conserved in protein composition and structural organization from protozoa to mammals. They are structurally characterized by nine microtubular doublets surrounding two central microtubular singlets [1]. Substructures like dynein arms and radial spokes are associated with the axoneme and important for motility in the flagellum. Matrix proteins that are not tightly associated with the flagellar membrane or the axoneme serve diverse functions in the flagellum and can be involved in intraflagellar transport [2]. Since many years, the green biflagellate alga Chlamydomonas reinhardtii, whose genome has been sequenced, is used as a model to study flagella structure, assembly, formation, and motility [3]. C. reinhardtii uses flagella for motility in aqueous environments, for attaching to surfaces and for cell-cell recognition during mating. A proteomic analysis of Chlamydomonas flagella revealed more than 600 proteins [4] that include, for example, motor and signal transduction components as well as proteins with homologues associated with human diseases (e.g., polycystic kidney disease, retinal degeneration, hydrocephalus, or changes in the
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