The human phosphatase CDC14A modulates primary cilium length by regulating centrosomal actin nucleation

CDC14A codes for a conserved proline‐directed phosphatase, and mutations in the gene are associated with autosomal‐recessive severe to profound deafness, due to defective kinocilia. A role of CDC14A in cilia formation has also been described in other organisms. However, how human CDC14A impacts on cilia formation remains unclear. Here, we show that human RPE1 hCDC14A PD cells, encoding a phosphatase dead version of hCDC14A, have longer cilia than wild‐type cells, while hCDC14A overexpression reduces cilia formation. Phospho‐proteome analysis of ciliated RPE1 cells identified actin‐associated and microtubule binding proteins regulating cilia length as hCDC14A substrates, including the actin‐binding protein drebrin. Indeed, we find that hCDC14A counteracts the CDK5‐dependent phosphorylation of drebrin at S142 during ciliogenesis. Further, we show that drebrin and hCDC14A regulate the recruitment of the actin organizer Arp2 to centrosomes. In addition, during ciliogenesis hCDC14A also regulates endocytosis and targeting of myosin Va vesicles to the basal body in a drebrin‐independent manner, indicating that it impacts primary cilia formation in a multilayered manner. Synopsis Human CDC14A negatively regulates primary cilia length in a multilayered manner by dephosphorylating actin‐associated proteins, including DBN1. Serine 142 of DBN1 is dephosphorylated by hCDC14A, thereby counteracting the cilia length promoting activity of CDK5. hCDC14A localizes to the basal body and the actin cytoskeleton during ciliogenesis. hCDC14A regulates the phosphorylation of DBN1, thereby contributing to cilia length control. hCDC14A and DBN1 regulate the actin nucleator Arp2 at basal bodies. Endocytosis and transport of myosin Va vesicles to the basal body is a DBN1‐independent function of hCDC14A during ciliogenesis. Graphical Abstract Human CDC14A negatively regulates primary cilia length in a multilayered manner by dephosphorylating actin‐associated proteins, including DBN1. Serine 142 of DBN1 is dephosphorylated by hCDC14A, thereby counteracting the cilia length promoting activity of CDK5.