Article

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

ABSTRACT

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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    • "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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    • "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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