Abstract
Tight control of centriole duplication is critical for normal chromosome segregation and the maintenance of genomic stability. Polo-like kinase 4 (Plk4) is a key regulator of centriole biogenesis. How Plk4 dynamically promotes its symmetry-breaking relocalization and achieves its procentriole-assembly state remains unknown. Here we show that Plk4 is a unique kinase that utilizes its autophosphorylated noncatalytic cryptic polo-box (CPB) to phase separate and generate a nanoscale spherical condensate. Analyses of the crystal structure of a phospho-mimicking, condensation-proficient CPB mutant reveal that a disordered loop at the CPB PB2-tip region is critically required for Plk4 to generate condensates and induce procentriole assembly. CPB phosphorylation also promotes Plk4’s dissociation from the Cep152 tether while binding to downstream STIL, thus allowing Plk4 condensate to serve as an assembling body for centriole biogenesis. This study uncovers the mechanism underlying Plk4 activation and may offer strategies for anti-Plk4 intervention against genomic instability and cancer.
Highlights
Tight control of centriole duplication is critical for normal chromosome segregation and the maintenance of genomic stability
Using three-dimensional structured illumination microscopy (3D-SIM), we observed that treatment of cells with a Plk[4] inhibitor, centrinone[26], was sufficient to prevent Polo-like kinase 4 (Plk4)’s ring-todot localization conversion, as shown previously[27], and that this event is essential for the subsequent recruitment of Sas[6] to the procentriole assembly site (Supplementary Fig. 1a)
To understand the underlying mechanism of how PC3-phosphorylated cryptic polo-box (CPB) can drive Plk[4] to coalesce into a stable dot-like condensate, we examined whether CPB CP contains a flexible region that may cause an increase in accessible hydrophobicity, a physicochemical property known to be critical for driving protein–protein interactions32,33. 1-Anilinonaphthalene-8-sulfonic acid (ANS) alters its fluorescent properties as it binds to nonpolar regions and is considered an effective probe to investigate the remodeling in the accessible hydrophobic surfaces of a protein[34,35]
Summary
Tight control of centriole duplication is critical for normal chromosome segregation and the maintenance of genomic stability. Plk[4] dimerizes through the cryptic polo box (CPB; residues 581–808)[6,8,9] of its C-terminal domain (CTD; residues 581–970) and its N-terminal kinase domain (KD)-dependent trans-autophosphorylation activity generates a phosphodegron at S285 and S289 to induce SCF-βTrCP/Slimb-mediated ubiquitination and its own proteasomal degradation[10,11,12,13,14]. This observation suggests that Plk[4] must overcome its phosphodegron-mediated selfdestruction threshold to trigger downstream events critical for centriole biogenesis. It is unclear how the dephosphorylated linker region works in concert with its Nterminal catalytic activity to form a functional Plk[4] assembly
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