Nature of the critical labile event that controls RB phosphorylation in the cyclic AMP-dependent cell cycle of thyrocytes in primary culture

This study addresses the nature of the critical labile event that couples a t restriction point mitogenic cascades with the autonomous part of the cell cycle. In primary cultures of dog thyroid epithelial cells, kinetic experi ments indicate that a labile cAMP-dependent event positively controls a lat e G1 commitment to DNA replication and RE phosphorylation. As previously sh own in this system, the cAMP-dependent mitogenic pathway differs from the m ost generally envisaged growth factor cascades as it stimulates the accumul ation of p27(kip1) but not of cyclins D. Nevertheless, cyclin D3 and CDK4 a ctivity are essential in the cAMP-dependent mitogenesis, and cAMP unmasks t he DCS-22 epitope of cyclin D3 and induces the nuclear translocations and a ssembly of cyclin D3 and CDK4 in a complex that also contains p27(kip1), Un expectedly, the washing out of forskolin rapidly arrested S phase entry and the accumulation of hyperphosphorylated RE, but did not reverse any of the above events associated with cyclin D3-CDK4 activation. This implies that even after induction of stable nuclear cyclin D3-CDK4 complexes, dog thyroc ytes still depend on cAMP for RE phosphorylation and commitment to DNA synt hesis, which suggests that a key labile event responsible for a last contro l of restriction point passage remains to be uncovered, in the cAMP-depende nt cell cycle of dog thyrocytes and possibly other systems, (C) 1999 Academ ic Press.