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.

Download full-text


Available from: Drew M Lowery, Nov 09, 2015
  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    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.
    Genes & Development 07/2014; 28(14):1604-19. DOI:10.1101/gad.240515.114 · 10.80 Impact Factor
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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.
    PLoS ONE 08/2013; 8(8):e70843. DOI:10.1371/journal.pone.0070843 · 3.23 Impact Factor
  • Source
    • "The DNA damage response regulates crossover timing B Szakal and D Branzei resolution. We used the cdc5-as1 allele, which encodes for a protein that is specifically inactivated by chloromethylketone (CMK-Cl) (Snead et al, 2007). We monitored the efficiency of CMK-Cl-mediated Cdc5-as1 inhibition by assessing the phosphorylation status of Mms4, a recently reported target of Cdc5 (Matos et al, 2011; Gallo-Fernandez et al, 2012). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The error-free DNA damage tolerance (DDT) pathway is crucial for replication completion and genome integrity. Mechanistically, this process is driven by a switch of templates accompanied by sister chromatid junction (SCJ) formation. Here, we asked if DDT intermediate processing is temporarily regulated, and what impact such regulation may have on genome stability. We find that persistent DDT recombination intermediates are largely resolved before anaphase through a G2/M damage checkpoint-independent, but Cdk1/Cdc5-dependent pathway that proceeds via a previously described Mus81-Mms4-activating phosphorylation. The Sgs1-Top3- and Mus81-Mms4-dependent resolution pathways occupy different temporal windows in relation to replication, with the Mus81-Mms4 pathway being restricted to late G2/M. Premature activation of the Cdk1/Cdc5/Mus81 pathway, achieved here with phosphomimetic Mms4 variants as well as in S-phase checkpoint-deficient genetic backgrounds, induces crossover-associated chromosome translocations and precocious processing of damage-bypass SCJ intermediates. Taken together, our results underscore the importance of uncoupling error-free versus erroneous recombination intermediate processing pathways during replication, and establish a new paradigm for the role of the DNA damage response in regulating genome integrity by controlling crossover timing.
    The EMBO Journal 03/2013; 32(8). DOI:10.1038/emboj.2013.67 · 10.43 Impact Factor
Show more