Abstract
Polo like kinase 1 (Plk1) is a key player in orchestrating the wide variety of cell-cycle events ranging from centrosome maturation, mitotic entry, checkpoint recovery, transcriptional control, spindle assembly, mitotic progression, cytokinesis and DNA damage checkpoints recovery. Due to its versatile nature, Plk1 is considered an imperative regulator to tightly control the diverse aspects of the cell cycle network. Interactions among Plk1 polo box domain (PBD) and its putative binding proteins are crucial for the activation of Plk1 kinase domain (KD). To date, only a few substrate candidates have been characterized through the inclusion of both polo box and kinase domain-mediated interactions. Thus it became compelling to explore precise and specific Plk1 substrates through reassessment and extension of the structure-function paradigm. To narrow this apparently wide gap in knowledge, here we employed a thorough sequence search of Plk1 phosphorylation signature containing proteins and explored their structure-based features like conceptual PBD-binding capabilities and subsequent recruitment of KD directed phosphorylation to dissect novel targets of Plk1. Collectively, we identified 4,521 phosphodependent proteins sharing similarity to the consensus phosphorylation and PBD recognition motifs. Subsequent application of filters including similarity index, Gene Ontology enrichment and protein localization resulted in stringent pre-filtering of irrelevant candidates and isolated unique targets with well-defined roles in cell-cycle machinery and carcinogenesis. These candidates were further refined structurally using molecular docking and dynamic simulation assays. Overall, our screening approach enables the identification of several undefined cell-cycle associated functions of Plk1 by uncovering novel phosphorylation targets.
Highlights
During mitosis, faithful distribution of chromosomes to newly forming daughter cells necessitates careful coordination of multiple processes such as entry into mitosis, spindle assembly, chromosome segregation and cytokinesis
In an attempt to identify novel substrates for Polo like kinase 1 (Plk1), initially we assessed the adequacy of consensus phosphorylation motif [E/D]X[pS/pT][I/L/V/M]X[E] for Plk1 kinase domain (KD) [26], which was shared by a majority of Plk1 substrates
Identification of novel Plk1 specific substrates In order to identify novel substrate candidates for Plk1, we developed a filtering approach to increase the specificity of our predictions by integrating multiple types of relative data sets including percent identity, clustering analysis GO (Gene Ontology) database, subcellular localization, presence of kinase phosphorylation and polo box domain (PBD) recognition motif and role in cancer (Figure 1)
Summary
Faithful distribution of chromosomes to newly forming daughter cells necessitates careful coordination of multiple processes such as entry into mitosis, spindle assembly, chromosome segregation and cytokinesis. Polo like kinases (Plks) are emerging as key regulators of essential cell cycle events and have garnered a lot of attention [1,2,3]. Plks are highly conserved from baker’s yeasts to humans with specific and dynamic roles throughout cell-cycle process [6,7,8,9]. Plk localizes to the cytoplasm and centrosome during interphase and concentrates to kinetochores and the cytokinesis bridge during cell division. This protein has major functions in centrosome maturation, mitotic entry, and cytokinesis [10,11]. Plk has emerged as a novel modulator of DNA damage checkpoints, where it maintains genomic stability during DNA replication [11]
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