Application of Phosphoproteomics to Find Targets of Casein Kinase 1 in the Flagellum of Chlamydomonas
Analysis of CK1 targets in the flagellum. (a) Diagram of flagellum phosphoproteins in wild-type and CK1-inhibited cells. A cross-section of a flagellum from C. reinhardtii (left panel) and a more detailed view (red rectangle) are shown according to . Structural phosphoproteins in CKI-7-inhibited cells, and such with a reduced number of phosphorylation sites are indicated in yellow color with a red frame. Novel phosphopeptides of structural proteins or additional phosphorylation sites of known phosphopeptides from structural phosphoproteins that were identified in the CKI-7-inhibited proteome are indicated by yellow color with a blue frame. Structural proteins with previously identified phosphopeptides, whose phosphorylation sites were detected again, are indicated in yellow without frame. Abbreviations are: C1 central pair projection (C1P), C2 central pair projection (C2P), PF6 protein (PF6), Hydin (H), Radial Spoke Protein17 (RSP17); Outer Dynein Arm Docking Complex (DC); Inner Dynein Arm Intermedite Chain138 (IC138), Tectin (T) as well as an Intraflagellar Transport Protein43 (IFT43). (b) and (c) Positions of identified phosphopeptides in the predicted domains of RSP11 (b) and GSK3 (c). Identified phosphopeptides are indicted by black boxes. The amino acid positions are mentioned. “p” indicates in vivo phosphorylation sites. RIIa, regulatory subunit of cAMP-Dependent Protein Kinase A; Ser/Thr Kin, Ser/Thr protein kinase catalytic domain. (d) and (e) Hypothetical model of GSK3 de-/activation via reversible phosphorylation triggered by CK1. (d) Regulatory signaling involves an additional kinase. The noninhibited active form of CK1 inactivates another still unknown kinase by phosphorylation. This kinase is needed in its active nonphosphorylated form for activating GSK3. (e) If CK1 is inhibited by CKI-7, the unknown kinase is not phosphorylated and thus active. This active kinase phosphorylates in turn GSK3, which is then activated.