Coupling phosphoryl transfer and substrate interactions in protein kinases

Biochim Biophys Acta. 2005 Dec 30;1754(1-2):191-9. doi: 10.1016/j.bbapap.2005.07.024. Epub 2005 Sep 8.


Protein kinases control cell signaling events through the ATP-dependent phosphorylation of serine, threonine and tyrosine residues in protein targets. The recognition of these protein substrates by the kinases relies on two principal factors: proper subcellular co-localization and molecular interactions between the kinase and substrate. In this review, we will focus on the kinetic role of the latter in conveying favorable substrate recognition. Using rapid mixing technologies, we demonstrate that the intrinsic thermodynamic affinities of two protein substrates for their respective kinases (Csk with Src and Sky1p with Npl3) are weak compared to their apparent affinities measured in traditional steady-state kinetic assays (i.e.--Km < Kd). The source of the high apparent affinities rests in a very fast and highly favorable phosphoryl transfer step that serves as a clamp for substrate recognition. In this mechanism, both Csk and Sky1p utilize this step to draw the substrate toward product, thereby, converting a high Kd into a low Km. We propose that this one form of substrate recognition employed by protein kinases is advantageous since it simultaneously facilitates high apparent substrate affinity and fast protein turnover.

Publication types

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

MeSH terms

  • CSK Tyrosine-Protein Kinase
  • Catalysis
  • Humans
  • Kinetics
  • Models, Biological
  • Models, Chemical
  • Nuclear Proteins / chemistry
  • Nuclear Proteins / metabolism
  • Peptides / chemistry
  • Phosphorylation*
  • Protein Binding
  • Protein Kinases / chemistry*
  • Protein Kinases / metabolism
  • Protein Structure, Tertiary
  • Protein-Serine-Threonine Kinases / chemistry
  • Protein-Serine-Threonine Kinases / metabolism
  • Protein-Tyrosine Kinases / chemistry
  • Protein-Tyrosine Kinases / metabolism
  • RNA-Binding Proteins / chemistry
  • RNA-Binding Proteins / metabolism
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / metabolism
  • Substrate Specificity
  • src-Family Kinases


  • NPL3 protein, S cerevisiae
  • Nuclear Proteins
  • Peptides
  • RNA-Binding Proteins
  • Saccharomyces cerevisiae Proteins
  • Protein Kinases
  • SKY1 protein, S cerevisiae
  • Protein-Tyrosine Kinases
  • CSK Tyrosine-Protein Kinase
  • src-Family Kinases
  • CSK protein, human
  • Protein-Serine-Threonine Kinases