We have developed a proteomic approach for identifying phosphopeptide-binding domains that modulate kinase-dependent signaling pathways. An immobilized library of partially degenerate phosphopeptides biased toward a particular kinase phosphorylation motif is used to isolate phosphopeptide-binding domains that interact with substrates phosphorylated by that kinase. Applying this approach to cyclin-dependent kinases (Cdks), we identified the Polo-box Domain (PBD) of the mitotic kinase Polo-like kinase 1 (Plk1) as a specific phosphoserine (pSer) or phosphothreonine (pThr) binding domain and determined its optimal binding motif. This motif is present in known Plk1 substrates such as Cdc25, and an optimal phosphopeptide containing the motif disrupted substrate binding and centrosomal localization of the PBD. This finding reveals how Plk1 can localize to specific sites within cells in response to Cdk phosphorylation at those sites.; Through peptide library screening, we show that the PBDs of other polo-like kinase family members bind phosphopeptides with the same conserved motif as that of Plk1. The 1.9A X-ray structure of the human Plk1 PBD in complex with a high affinity phosphopeptide explains this selectivity and shows that the two Polo-box regions within the PBD each comprise alphabeta6 structural motifs that associate to form a novel 12-stranded beta-sandwich domain. Phosphopeptide-binding occurs at a conserved positively-charged cleft formed by the interface of the Polo-boxes. Mutations at this surface disrupt phosphodependent substrate targeting by the PBD and provide phenotypic cellular evidence that PBD phosphobinding is necessary for proper mitotic progression. Finally, phosphopeptide binding to the PBD of full-length Plk1 relieves inhibition of the kinase domain, suggesting a PBD-mediated conformational switching mechanism for Plk activation. In combination our data reveal a central role for phosphoprotein-binding that is crucial for many, if not all, cellular functions of the PBD.
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