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Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
12:26

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Published on: May 3, 2018

Phosphorylation of cyclin dependent kinase 4 on tyrosine 17 is mediated by Src family kinases

Nicholas G Martin1, Peter C McAndrew, Paul D Eve

  • 1Centre for Cancer Therapeutics at The Institute of Cancer Research, Cancer Research UK, Haddow Laboratories, 15 Cotswold Road, Sutton, Surrey, UK.

The FEBS Journal
|May 16, 2008
PubMed

Insights

Src family kinases, not WEE1 or MYT1, phosphorylate cyclin-dependent kinase 4 at tyrosine 17. This finding clarifies regulation of cyclin-dependent kinase 4 (CDK4) activity, a key cell cycle regulator often altered in cancer.

Area of Science:

  • Molecular Biology
  • Cell Cycle Regulation
  • Cancer Biology

Background:

  • Cyclin-dependent kinase 4 (CDK4) is a critical regulator of the cell cycle.
  • Deregulation of CDK4 activity is common in various cancers.
  • The phosphorylation of CDK4 at tyrosine 17 (pY17) is a known regulatory mechanism, but the responsible kinases were unidentified.

Purpose of the Study:

  • To identify the kinases responsible for phosphorylating cyclin-dependent kinase 4 (CDK4) at tyrosine 17.
  • To elucidate the regulatory mechanisms controlling CDK4 activity through pY17 phosphorylation.

Main Methods:

  • Generation of a phosphospecific antibody for pY17-CDK4.
  • Purification of tyrosine 17 kinases from HeLa cell lysates.
  • In vitro kinase assays and cell transfection experiments.
  • Treatment with Src family kinase inhibitors.

Main Results:

  • Tyrosine 17 of CDK4 is phosphorylated at low levels in proliferating HCT116 cells.
  • The Src family kinase C-YES was identified as a major contributor to pY17-CDK4 activity in HeLa cells.
  • C-YES directly phosphorylated CDK4 at Y17 in transfected cells.
  • Inhibition of Src family kinases reduced pY17-CDK4 levels in cells.

Conclusions:

  • Src family kinases, including C-YES, are the primary kinases phosphorylating CDK4 at tyrosine 17.
  • WEE1 and MYT1 do not phosphorylate CDK4 at this site.
  • This study clarifies the regulation of CDK4 activity via Y17 phosphorylation, offering insights into cancer-related signaling pathways.

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