A Coupled Chemical-Genetic and Bioinformatic Approach to Polo-like Kinase Pathway Exploration

Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA 94158, USA.
Chemistry & Biology (Impact Factor: 6.65). 12/2007; 14(11):1261-72. DOI: 10.1016/j.chembiol.2007.09.011
Source: PubMed


Protein phosphorylation is a ubiquitous mechanism for cellular signal propagation, and signaling network complexity presents a challenge to protein kinase substrate identification. Few targets of Polo-like kinases are known, despite their significant role in coordinating cell-cycle progression. Here, we combine chemical-genetic, bioinformatic, and proteomic tools for Polo-like kinase substrate identification. Specific pharmacological inhibition of budding yeast Polo-like kinase, Cdc5, resulted in a misaligned preanaphase spindle and subsequently delayed anaphase nuclear migration, revealing a Cdc5 function. A cellular screen for Cdc5 substrates identified Spc72, a spindle pole body (SPB) component and microtubule anchor required for nuclear positioning. Spc72 bound to the Cdc5 PBD in a mitosis-specific manner, was phosphorylated by Cdc5 in vitro, and demonstrated a loss of mitotic phosphorylation in vivo upon Cdc5 inhibition. Finally, an examination of Cdc5 binding by SPB-localized proteins expanded our knowledge of Cdc5 function at the SPB.

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Available from: Drew M Lowery, Nov 09, 2015
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    • "Whether RSC components are also phosphorylated by Cdc5 remains an open question. However, we note that several subunits of the RSC complex were recently identified as high-probability substrates for Cdc5 and also contain a large number of PBD consensus binding sites (at least 28;Snead et al., 2007). This, together with the fact that the RSC complex contains 17 subunits (Cairns et al., 1999), makes the identification of the actual Cdc5 phosphorylation/PBD-binding sites in RSC subunits a daunting task that will be the subject of a future study. "
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    ABSTRACT: Cell-cycle checkpoints are essential feedback mechanisms that promote genome integrity. However, in the face of unrepairable DNA lesions, bypass mechanisms can suppress checkpoint activity and allow cells to resume proliferation. The molecular mechanisms underlying this biological response are currently not understood. Taking advantage of unique separation-of-function mutants, we show that the Polo-like kinase (PLK) Cdc5 uses a phosphopriming-based interaction mechanism to suppress G2/M checkpoint arrest by targeting Polo kinase activity to centrosomes. We also show that key subunits of the evolutionarily conserved RSC complex are critical downstream effectors of Cdc5 activity in checkpoint suppression. Importantly, the lethality and checkpoint defects associated with loss of Cdc5 Polo box activity can be fully rescued by artificially anchoring Cdc5 kinase domain to yeast centrosomes. Collectively, our results highlight a previously unappreciated role for centrosomes as key signaling centers for the suppression of cell-cycle arrest induced by persistent or unrepairable DNA damage.
    Full-text · Article · Jan 2016 · Cell Reports
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    • "Previous work has established that Mus81 activity is decisively up-regulated in mitosis in response to a sequential phosphorylation of Mms4 by CDK and the Polo-like kinase Cdc5 (Matos et al. 2011; Gallo-Ferná ndez et al. 2012; Saugar et al. 2013; Szakal and Branzei 2013). We therefore used two systems to interfere with Cdc5 activity: the cdc5-as1 analog-sensitive allele, which we inhibited using chloromethylketone (CMK) (Snead et al. 2007), and transcriptional shutoff of pGAL-CDC5 using glucose repression. Both types of Cdc5 inactivation resulted in a loss of the slower-migrating species of Mms4 in gels and at the same time diminished the binding of Dpb11 and Slx4 to Mms4 3Flag (Fig. 5C; Supplemental Fig. S9A). "
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    ABSTRACT: A key function of the cellular DNA damage response is to facilitate the bypass of replication fork-stalling DNA lesions. Template switch reactions allow such a bypass and involve the formation of DNA joint molecules (JMs) between sister chromatids. These JMs need to be resolved before cell division; however, the regulation of this process is only poorly understood. Here, we identify a regulatory mechanism in yeast that critically controls JM resolution by the Mus81-Mms4 endonuclease. Central to this regulation is a conserved complex comprising the scaffold proteins Dpb11 and Slx4 that is under stringent control. Cell cycle-dependent phosphorylation of Slx4 by Cdk1 promotes the Dpb11-Slx4 interaction, while in mitosis, phosphorylation of Mms4 by Polo-like kinase Cdc5 promotes the additional association of Mus81-Mms4 with the complex, thereby promoting JM resolution. Finally, the DNA damage checkpoint counteracts Mus81-Mms4 binding to the Dpb11-Slx4 complex. Thus, Dpb11-Slx4 integrates several cellular inputs and participates in the temporal program for activation of the JM-resolving nuclease Mus81.
    Full-text · Article · Jul 2014 · Genes & Development
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    • "Despite the significant and pleiotropic functions of Plk1 in coordinating cell-cycle progression, surprisingly only few specific targets are known which are uniquely phosphorylated by Plks. In order to explore Plk1-related cascade of molecular events, understanding of possible links between potential phosphorylation sites and subsequent interacting proteins is crucial [10], [18], [19]. Thus, linking a certain kinase with particular phosphorylation events remains intricate and elucidation of kinase-substrate relationships is vital in understanding the intracellular signal transduction and cellular physiology. "
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