A coupled chemical-genetic and bioinformatic approach to Polo-like kinase pathway exploration

Chem Biol. 2007 Nov;14(11):1261-72. doi: 10.1016/j.chembiol.2007.09.011.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Alleles
  • Cell Cycle Proteins / metabolism
  • Computational Biology*
  • Protein Kinases / metabolism
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism*
  • Saccharomyces cerevisiae Proteins / metabolism
  • Substrate Specificity

Substances

  • Cell Cycle Proteins
  • Saccharomyces cerevisiae Proteins
  • Protein Kinases
  • Protein-Serine-Threonine Kinases
  • CDC5 protein, S cerevisiae