Quantitative reconstitution of mitotic CDK1 activation in somatic cell extracts

Mol Cell. 2010 Mar 26;37(6):753-67. doi: 10.1016/j.molcel.2010.02.023.

Abstract

The regulation of mitotic entry in somatic cells differs from embryonic cells, yet it is only for embryonic cells that we have a quantitative understanding of this process. To gain a similar insight into somatic cells, we developed a human cell extract system that recapitulates CDK1 activation and nuclear envelope breakdown in response to mitotic cyclins. As cyclin B concentrations increase, CDK1 activates in a three-stage nonlinear response, creating an ordering of substrate phosphorylations. This response is established by dual regulatory feedback loops involving WEE1/MYT1, which impose a cyclin B threshold, and CDC25, which allows CDK1 to escape the WEE1/MYT1 inhibition. This system also exhibits a complex response to cyclin A. Cyclin A promotes WEE1 phosphorylation to weaken the negative feedback loop and primes mitotic entry through cyclin B. This observation explains the requirement of both cyclin A and cyclin B to initiate mitosis in somatic cells.

Publication types

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

MeSH terms

  • CDC2 Protein Kinase / analysis*
  • CDC2 Protein Kinase / genetics
  • CDC2 Protein Kinase / metabolism
  • Cell Cycle Proteins / metabolism
  • Cell Extracts / chemistry*
  • Cyclin A2 / genetics
  • Cyclin A2 / metabolism
  • Cyclin B / metabolism
  • Enzyme Activation
  • HeLa Cells
  • Humans
  • Mitosis*
  • Nuclear Proteins / metabolism
  • Phosphotyrosine / metabolism
  • Protein Binding
  • Protein-Tyrosine Kinases / metabolism
  • Substrate Specificity

Substances

  • CCNA2 protein, human
  • Cell Cycle Proteins
  • Cell Extracts
  • Cyclin A2
  • Cyclin B
  • Nuclear Proteins
  • Phosphotyrosine
  • Protein-Tyrosine Kinases
  • WEE1 protein, human
  • CDC2 Protein Kinase