title: A coupled chemical-genetic and bioinformatic approach to Polo-like kinase pathway exploration.
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.
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA 94158, USA.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Snead
authors.forename: Matthew
authors.initials: M
authors.lastname: Sullivan
authors.forename: Drew M
authors.initials: DM
authors.lastname: Lowery
authors.forename: Michael S
authors.initials: MS
authors.lastname: Cohen
authors.forename: Chao
authors.initials: C
authors.lastname: Zhang
authors.forename: David H
authors.initials: DH
authors.lastname: Randle
authors.forename: Jack
authors.initials: J
authors.lastname: Taunton
authors.forename: Michael B
authors.initials: MB
authors.lastname: Yaffe
authors.forename: David O
authors.initials: DO
authors.lastname: Morgan
authors.forename: Kevan M
authors.initials: KM
authors.lastname: Shokat
mesh_terms: D000483:Alleles; D018797:Cell Cycle Proteins / Q000378:metabolism; D019295:Computational Biology; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D018797:Cell Cycle Proteins; D029701:Saccharomyces cerevisiae Proteins; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; C081927:CDC5 protein, S cerevisiae