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Regulation of cyclin D1/Cdk4 complexes by calcium/calmodulin-dependent protein kinase I

Christina R Kahl1, Anthony R Means

  • 1Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.

The Journal of Biological Chemistry
|February 3, 2004
PubMed

Insights

The calcium/calmodulin-dependent kinase (CaMK) inhibitor KN-93 arrests cells in G1 by blocking cyclin D/cdk4 activation. This action involves CaMKI, preventing a late step in cell cycle progression.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • The cell cycle inhibitor KN-93 targets multifunctional calcium/calmodulin-dependent kinases (CaMK).
  • KN-93 causes G1 arrest in various cell types, but its precise mechanism and target in G1 remain unclear.
  • Understanding the G1 arrest point is crucial for cell cycle regulation research.

Purpose of the Study:

  • To elucidate the biochemical nature of the G1 arrest induced by KN-93.
  • To identify the specific CaMK isoform involved in the G1 arrest mechanism.
  • To investigate the effect of KN-93 on cyclin D/cdk4 complex activation.

Main Methods:

  • Cell cycle analysis of WI-38 human diploid fibroblasts treated with KN-93.
  • Biochemical assays to assess cyclin D1/cdk4 complex assembly, nuclear localization, and phosphorylation.
  • Gel filtration analysis to examine cdk4 complex migration and activity.
  • Expression of kinase-deficient CaMKI and CaMKII to determine the involvement of specific CaMK isoforms.

Main Results:

  • KN-93 induced a reversible G1 arrest in late G1, preceding detectable cyclin-dependent kinase 4 (cdk4) activation.
  • At the arrest point, cyclin D1/cdk4 complexes were assembled, nuclear, and phosphorylated but relatively inactive.
  • KN-93 inhibited cdk4 activation and a shift in cyclin D1/cdk4 complex migration; overexpression of cyclin D1/cdk4 rescued the arrest.
  • Kinase-deficient CaMKI, but not CaMKII, overexpression mimicked the KN-93 arrest, implicating CaMKI.

Conclusions:

  • KN-93 arrests cells in late G1 by inhibiting a late, uncharacterized step in cyclin D/cdk4 activation.
  • This KN-93-sensitive step involves CaMKI and occurs after complex assembly, nuclear entry, and phosphorylation.
  • The findings clarify the mechanism of KN-93-induced G1 arrest and highlight CaMKI's role in cell cycle progression.

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