TRANSLOCATION OF CDK2 TO THE NUCLEUS DURING G1-PHASE IN PDGF-STIMULATED HUMAN FIBROBLASTS

We studied the subcellular distribution of cdk2 in synchronized, PDGF- stimulated human fibroblasts (FH109). After contact inhibition and ser um depletion, more than 95% of FH109 cells were arrested in G0/G1-phas e. PDGF-AB led to a le-fold increase in proliferation compared with un treated cells. Cell cycle progression was studied by how cytometric an alysis, [H-3]thymidine incorporation, and phosphorylation of the retin oblastoma gene product, pRB. Using Western blot analysis after subcell ular fractionation, we revealed that after PDGF stimulation the phosph orylated (Thr 160), i.e., activated, form of cdk2 (33 kDa) first appea red 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 amo unt of inactive cdk2 (35 kDa) remained constant in the nucleus, we sug gested a translocation from the cytosol to the nucleus in late G1. Usi ng immunofluorescence techniques, we detected a diffuse staining in qu iescent cells. Starting at late G1-phase, cdk2 immunoreactivity was co ncentrated to the nucleus while immunoreactivity in the cytosol disapp eared. We therefore draw the conclusion that cdk2 is translocated from the cytosol into the nucleus in late G1-phase. Since protein levels a nd activity of cdk7, which is the catalytic subunit of cdk-activating kinase (CAK) phosphorylating cdk2, remained constant throughout the ce ll cycle, CAK activity might therefore be regulated by the availabilit y of its substrate cdk2. (C) 1997 Academic Press.