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Substrate specificity of CDK2-cyclin A. What is optimal?

Lisa M Stevenson-Lindert1, Paul Fowler, John Lew

  • 1Program in Molecular Biosciences and Engineering, University of California, Santa Barbara, California 93106, USA.

The Journal of Biological Chemistry
|September 25, 2003
PubMed

Insights

Cyclin-dependent kinases (CDKs) often use suboptimal sequences in cellular substrates, which are compensated by cyclin-binding motifs. This study reveals how these motifs affect substrate binding and catalysis kinetics for CDK2-cyclin A.

Area of Science:

  • Molecular Biology
  • Enzymology
  • Protein Kinase Signaling

Background:

  • The optimal substrate sequence for cyclin-dependent kinases (CDKs) is known, but physiological substrates often deviate.
  • Suboptimal phosphorylation sites in CDK substrates are frequently associated with a cyclin-binding (Cy) motif.
  • The Cy motif is thought to compensate for reduced catalytic efficiency at suboptimal phosphorylation sites.

Purpose of the Study:

  • To investigate the kinetic basis of substrate recognition by CDK2-cyclin A.
  • To understand how the Cy motif influences substrate binding and catalysis with optimal and suboptimal phosphorylation sequences.

Main Methods:

  • Kinetic analysis of CDK2-cyclin A with synthetic peptide substrates.
  • Comparison of catalytic efficiency (kcat/Km), substrate binding (Km), and turnover (kcat) for different substrate motifs.
  • Investigation of the interplay between phosphorylation site sequence and the Cy motif.

Main Results:

  • In the optimal motif, Pro+1 enhances binding and catalysis, while Lys+2 and Lys+3 primarily enhance binding.
  • The Cy motif enhances catalytic efficiency with suboptimal sequences (Lys+2 to Pro) by increasing affinity.
  • When fused to optimal sequences, the Cy motif minimally enhances efficiency due to high affinity impeding catalysis.

Conclusions:

  • Suboptimal phosphorylation site determinants in cellular substrates are kinetically compensated by the Cy motif.
  • The Cy motif's effect depends on the underlying phosphorylation site sequence, modulating binding and turnover.
  • These findings offer kinetic insights into the selection of specificity determinants for cellular substrate phosphorylation.

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