Abstract
The Polo-like kinase homolog in Trypanosoma brucei, TbPLK, plays essential roles in basal body segregation, flagellum attachment and cytokinesis. The level of TbPLK protein is tightly controlled, but the underlying mechanism remains elusive. Here, we report a Cullin-RING ubiquitin ligase composed of Cullin4, the DNA damage-binding protein 1 homolog TbDDB1 and a WD40-repeat protein WDR1 that controls TbPLK abundance in the basal body and the bilobe. WDR1, through its C-terminal domain, interacts with the PEST motif in TbPLK and, through its N-terminal WD40 motif, binds to TbDDB1. Depletion of WDR1 inhibits bilobe duplication and basal body segregation, disrupts the assembly of the new flagellum attachment zone filament and detaches the new flagellum. Consistent with its role in TbPLK degradation, depletion of WDR1 causes excessive accumulation of TbPLK in the basal body and the bilobe, leading to continuous phosphorylation of TbCentrin2 in the bilobe at late cell cycle stages. Together, these results identify a novel WD40-repeat protein as a TbPLK receptor in the Cullin4-DDB1 ubiquitin ligase complex for degrading TbPLK in the basal body and the bilobe after the G1/S cell cycle transition, thereby promoting bilobe duplication, basal body separation and flagellum-cell body adhesion.
Highlights
Polo-like kinases (PLKs) are evolutionarily conserved serine/threonine protein kinases and play multiple essential roles in mitotic entry, centrosome maturation, spindle assembly, chromosome segregation, mitotic exit and cytokinesis in a wide variety of eukaryotes [1,2]
The destruction box (D-box) is best known to be recognized by the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase, whereas the PEST motif is often recognized by the Cullin-RING ligase (CRL)-type ubiquitin ligase
We tagged the two TbPLK mutants and wild-type TbPLK with a triple HA epitope and overexpressed them in T. brucei. These overexpressed wild-type and mutant TbPLK proteins all localized to the basal body, the bilobe and the new flagellum attachment zone (FAZ) tip, TbPLK-DBmut was detected in the cytosol (S1A Fig)
Summary
Polo-like kinases (PLKs) are evolutionarily conserved serine/threonine protein kinases and play multiple essential roles in mitotic entry, centrosome maturation, spindle assembly, chromosome segregation, mitotic exit and cytokinesis in a wide variety of eukaryotes [1,2]. Degradation of human PLK1 and the budding yeast PLK homolog Cdc is mediated by the anaphase-promoting complex/cyclosome (APC/C) in conjugation with the cofactor Cdh, and requires a destruction box (D-box) [6,7]. Degradation of human PLK4, a regulator of centriole duplication, is mediated by the Cullin-RING ligase (CRL) SCFβ-TrCP/Slimb [8,9], and requires a PEST motif that is trans-phosphorylated by PLK4 itself [10,11]. The PEST motif is a stretch of peptide rich in proline (P), glutamic acid (E), serine (S) and threonine (T), and is well known as a signal sequence for protein degradation [12]. PLK4 controls its own stability through trans-autophosphorylation of the PEST motif to create a phospho-degron that is recognized by the F-box protein β-TrCP/Slimb for ubiquitination and degradation [13,14]
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