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Abstract

Mitotic phosphatases play crucial roles in anaphase regulation and mitotic exit by annulling the kinase-mediated protein phosphorylation. In budding yeast Saccharomyces cerevisiae, Cdc14 (cell division cycle 14) phosphatase antagonizes key Cdk1 (cyclin-dependent kinase 1) functions to drive cells out of mitosis. Despite the presence of highly conserved catalytic domains, human CDC14s are dispensable for cell cycle progression. Nevertheless, to decipher the molecular mechanisms of human CDC14s functions, we have investigated the knockout cellular models aided by various imaging and proteomics approaches. Phenotypic analyses of the generated hCDC14A knockout in human retinal pigment epithelium (hTERT-RPE1) cells have exhibited the occurrence of longer primary cilia upon serum starvation. The intermediate longer cilia in haploid-insufficient cells, as well as the extended cilia observed upon siRNA-mediated acute depletion of hCDC14A, have further confirmed the phenotype. Primary cilia are microtubule-based structures that control various aspects of growth and development through sensing extracellular signals. Defects in this regulation lead to a host of pathological conditions collectively known as ciliopathies. Indirect immunofluorescence and electron microscopy have revealed that the disassembly pathways, as well as the crucial structures like axoneme and basal body, were intact in the elongated cilia. Inducible expression of hCDC14A has indicated its presence in the proximal end and subdistal appendage of the basal body. Global phosphoproteome along with proximity-based interaction proteomics approaches under conditions that favor ciliation have identified substrates for hCDC14A. Some of the identified substrates are involved in actin cytoskeleton reorganization with a function in cilia length control. The actin bundling protein drebrin (DBN1) was one of the identified hCDC14A substrates that have recently been reported to be associated with ciliogenesis. We show that the counteracting phospho-regulation of DBN1 at serine residue 142 by the proline directed kinase CDK5 and hCDC14A phosphatase regulates cilia length. However, significantly longer cilia in hCDC14A knockout cells than those from DBN1 knockout cells indicate that there are more substrates for hCDC14A that might be involved in ciliogenesis. The longer cilia phenotype in hCDC14A knockout cells could be explained by the enhanced recycling endosomes (transferrin) as well as increased ciliary vesicle docking (smoothened) in the pericentrosomal areas.