6533b7d0fe1ef96bd125af00

RESEARCH PRODUCT

Translocation of cdk2 to the nucleus during G1-phase in PDGF-stimulated human fibroblasts.

subject

description

We studied the subcellular distribution of cdk2 in synchronized, PDGF-stimulated human fibroblasts (FH109). After contact inhibition and serum depletion, more than 95% of FH109 cells were arrested in G0/G1-phase. PDGF-AB led to a 16-fold increase in proliferation compared with untreated cells. Cell cycle progression was studied by flow cytometric analysis, [3H]thymidine incorporation, and phosphorylation of the retinoblastoma gene product, pRB. Using Western blot analysis after subcellular fractionation, we revealed that after PDGF stimulation the phosphorylated (Thr 160), i.e., activated, form of cdk2 (33 kDa) first appeared in the nucleus at late G1-phase and persisted throughout until to the end of S-phase. Since cdk2 was not synthesized de novo, and the amount of inactive cdk2 (35 kDa) remained constant in the nucleus, we suggested a translocation from the cytosol to the nucleus in late G1. Using immunofluorescence techniques, we detected a diffuse staining in quiescent cells. Starting at late G1-phase, cdk2 immunoreactivity was concentrated to the nucleus while immunoreactivity in the cytosol disappeared. We therefore draw the conclusion that cdk2 is translocated from the cytosol into the nucleus in late G1-phase. Since protein levels and activity of cdk7, which is the catalytic subunit of cdk-activating kinase (CAK) phosphorylating cdk2, remained constant throughout the cell cycle, CAK activity might therefore be regulated by the availability of its substrate cdk2.