The Molecular Basis for Phosphodependent Substrate Targeting and Regulation of Plks by the Polo-Box Domain

Abstract

Polo-like kinases (Plks) perform crucial functions in cell-cycle progression and multiple stages of mitosis. Plks are characterized by a C-terminal noncatalytic region containing two tandem Polo boxes, termed the Polo-box domain (PBD), which has recently been implicated in phosphodependent substrate targeting. We show that the PBDs of human, Xenopus, and yeast Plks all recognize similar phosphoserine/threonine-containing motifs. The 1.9 A X-ray structure of a human Plk1 PBD-phosphopeptide complex shows that the Polo boxes each comprise beta6alpha structures that associate to form a 12-stranded beta sandwich domain. The phosphopeptide binds along a conserved, positively charged cleft located at the edge of the Polo-box interface. Mutations that specifically disrupt phosphodependent interactions abolish cell-cycle-dependent localization and provide compelling phenotypic evidence that PBD-phospholigand binding is necessary for proper mitotic progression. In addition, phosphopeptide binding to the PBD stimulates kinase activity in full-length Plk1, suggesting a conformational switching mechanism for Plk regulation and a dual functionality for the PBD.

Phosphorylation of the cytokinesis regulator ECT2 at G2/M phase stimulates association of the mitotic kinase Plk1 and accumulation of GTP-bound RhoA

“…His-538 and Lys-540 in Plk1 are the residues within the PBD pincer grip, and make contact with the phosphate group. Consistent with these observations, mutations at His-538 and Lys-540 in Plk1 abolish the association with Cdc25 in vivo (Elia et al, 2003b). Whereas expression of Plk1 PBD induces an increase in the population of cells in G2/M phase, the mutations at His-538 and Lys-540 efficiently abolish this phenotype (Elia et al, 2003b), suggesting that the phospholigand-binding activity of Plk1 PBD is necessary for proper mitotic progression.…”

“…Consistent with these observations, mutations at His-538 and Lys-540 in Plk1 abolish the association with Cdc25 in vivo (Elia et al, 2003b). Whereas expression of Plk1 PBD induces an increase in the population of cells in G2/M phase, the mutations at His-538 and Lys-540 efficiently abolish this phenotype (Elia et al, 2003b), suggesting that the phospholigand-binding activity of Plk1 PBD is necessary for proper mitotic progression. Although the phosphospecific association of Plk1 is well documented concerning Cdc25, it is still not known whether other Plk1 substrates require priming phosphorylation by Cdk1.…”

Phosphorylation of Plk1 at S137 and T210 is Inhibited in Response to DNA Damage

“…4,5 Plk1 belongs to the family of Polo-like kinases (PLK's), which each have one or two conserved 30-amino-acid elements, called polo-boxes. These comprise the Polo-box domain (PBD), a phosphopeptide binding motif, [6][7][8] that is involved in regulation of Plk1 activity. The PBD mediates interactions of Plk1 with other proteins, 9 and is involved in the cellular localization of Plk1 to mitotic structures.…”

Polo Kinase and Cytokinesis Initiation in Mammalian Cells: Harnessing the Awesome Power of Chemical Genetics

“…Structurally, Plk1 is characterized by both an N-terminal kinase domain and a C-terminal Polo-box domain (PBD) that acts as a phosphopeptide-binding module and mediator of www.landesbioscience.comPlk1's localization and substrate preferences. 8,9 When overexpressed in vivo, the PBD causes delocalization of the endogenous pool of Plk1 and elicits a strong checkpoint-dependent prometaphase arrest. 10,11 However, the vast majority of these cells display mature centrosomes and bipolar spindles, unlike those injected with Plk1-specific antibodies or depleted of Plk1 via RNA interference (RNAi) or genetic deletion of the PLK1 locus (see below).…”