Casein Kinase I Is Anchored on Axonemal Doublet Microtubules and Regulates Flagellar Dynein Phosphorylation and Activity

Abstract

Flagellar dynein activity is regulated by phosphorylation. One critical phosphoprotein substrate in Chlamydomonas is the 138-kDa intermediate chain (IC138) of the inner arm dyneins (Habermacher, G., and Sale, W. S. (1997) J. Cell Biol. 136, 167-176). In this study, several approaches were used to determine that casein kinase I (CKI) is physically anchored in the flagellar axoneme and regulates IC138 phosphorylation and dynein activity. First, using a videomicroscopic motility assay, selective CKI inhibitors rescued dynein-driven microtubule sliding in axonemes isolated from paralyzed flagellar mutants lacking radial spokes. Rescue of dynein activity failed in axonemes isolated from these mutant cells lacking IC138. Second, CKI was unequivocally identified in salt extracts from isolated axonemes, whereas casein kinase II was excluded from the flagellar compartment. Third, Western blots indicate that within flagella, CKI is anchored exclusively to the axoneme. Analysis of multiple Chlamydomonas motility mutants suggests that the axonemal CKI is located on the outer doublet microtubules. Finally, CKI inhibitors that rescued dynein activity blocked phosphorylation of IC138. We propose that CKI is anchored on the outer doublet microtubules in position to regulate flagellar dynein.The dynein ATPase is a family of molecular motors responsible for diverse cellular functions including retrograde microtubule-based transport of organelles, assembly and function of the Golgi and mitotic apparatus, and movement of cilia and flagella (1, 2). One of the primary questions is how dynein activity is regulated. The focus of this report is based on a series of studies revealing that the flagellar central pair apparatus and radial spokes play a primary role in regulation of flagellar dynein activity (3, 4). The regulatory mechanism involves a structural network of axonemal kinases and phosphatases that control phosphorylation of key subunits within the inner arm dyneins (5-8). In particular, phosphorylation of a dynein intermediate chain subunit, IC138, 1 correlates with inhibition of flagellar dynein activity (7). This network of enzymes along with the central pair apparatus, radial spokes, and the inner arm dyneins operates to control flagellar waveform (9 -17). These conclusions are based on genetic analysis and in vitro functional studies of flagella from Chlamydomonas reinhardtii. For example, mutations that disrupt either the central pair or the radial spokes result in flagellar paralysis (2,3,18). These paralyzed axonemes undergo dynein-driven microtubule sliding in vitro (19) but at greatly reduced rates of sliding when compared with wild-type axonemes (20). Reconstitution and functional assays demonstrate that the radial spokes are required for wild-type dynein activity, and the velocity of dyneindriven microtubule sliding is mediated by posttranslational modification of the inner dynein arms (20). These results are consistent with other evidence indicating that the central pair/ radial spoke apparatus along with the dynein...

Possible Roles of Protein Kinase A in Cell Motility and Excystation of the Early Diverging Eukaryote Giardia lamblia

“…In bovine and human sperm, mAKAP82 localized PKA, via its RII subunit, to the fibrous sheath (48), between the axoneme and the surrounding mitochondrion. In contrast, PKA of a single-celled flagellated alga, Chlamydomonas, is reported to be localized to the radial spokes of the flagellar structure, where it may be involved in regulating dynein ATPase (49). The location of PKA in giardial flagellar structure appears closer to that of mammalian sperm than to that of Chlamydomonas.…”

Asymmetry of the central apparatus defines the location of active microtubule sliding in Chlamydomonas flagella

“…3). Recent evidence has revealed that inner dynein arm subform I1 may play a role in controlling flagellar bending (23,31,(35)(36)(37)(38)(39). Therefore, we analyzed central apparatus orientation in axonemes isolated from the I1 defective strain ida1.…”

Flagellar and ciliary beating: the proven and the possible

“…Inner-arm-dynein mutations of Chlamydomonas result in either complete or partial loss of coordinated beating. The 138 kD intermediate chain (IC138) is a protein that is associated with the inner-arm dyneins (Habermacher and Sale, 1997) and is actively phosphorylated by casein kinase 1 (Yang and Sale, 2000). Wirschell and colleagues state: 'The current model for regulation of I1-dynein is that chemical and/or mechanical signals from the central pair are transmitted through the radial spokes to affect IC138 phosphorylation on specific outer doublets (e.g.…”