EXPRESSION AND SUBCELLULAR-LOCALIZATION OF CDK2 AND CDC2 KINASES AND THEIR COMMON PARTNER CYCLIN-A IN THYROID EPITHELIAL-CELLS - COMPARISONOF CYCLIC-AMP-DEPENDENT AND CYCLIC-AMP-INDEPENDENT CELL-CYCLES

Dog thyroid epithelial cells in primary culture constitute a model of positive control of DNA synthesis initiation and CO-S prereplicative p hase progression by cyclic AMP as a second messenger for TSH. In its e arly steps, this mitogenic control is quite distinct from cyclic AMP-i ndependent mitogenic cascades elicited by growth factors. We demonstra te here that TSH (cyclic AMP) and EGF + serum (cyclic AMP-independent) stimulations cooperate and finally converge on proteins that control the cell cycle machinery. This convergence included a common induction of the expression of cyclin A and p34(cdc2), and to a lesser extent o f p33/38(cdk2), which was already expressed in quiescent thyroid cells , and common changes of cdc2 and CDK2 phosphorylations as evidenced by electrophoretic mobility shifts. Kinetic differences in these process es after stimulation by TSH or EGF + serum or by these factors in comb ination correlated with differences in cell cycle kinetics. Moreover, an immunofluorescence analysis of these proteins using the double labe ling of PCNA as a marker of each cell cycle phase shows: (1) a previou sly undescribed nuclear translocation of CDK2 before S phase initiatio n; (2) a sudden increase of cdc2 nuclear immunoreactivity at G2/mitosi s transition. These data support the roles of CDK2 and cdc2 at G1/S an d G2/mitosis transitions, respectively. (3) We were unable to demonstr ate in individual cells a strict association between the nuclear appea rance of cyclin A and G1/S transition, and an association of cyclin A and CDK2 with PCNA-stained DNA replication sites. On the other hand, t he lengthening of G2 phase in the TSH/cyclic AMP-dependent thyroid cel l cycle was associated with a stabilization of Tyr15 inhibitory phosph orylation of cdc2 and an especially high nuclear concentration of cycl in A and CDK2. We hypothesize that high nuclear accumulation of cyclin A and CDK2 during G2 phase could be causative in the cyclic AMP-depen dent delay of mitosis onset. (C) 1996 Wiley-Liss, Inc.