ADHESION-DEPENDENT CONTROL OF CYCLIN-E CDK2 ACTIVITY AND CELL-CYCLE PROGRESSION IN NORMAL-CELLS BUT NOT IN HA-RAS TRANSFORMED NRK CELLS

Loss of adhesion of NRK fibroblasts to an appropriate surface leads to cell cycle arrest in late G(1) and failure to produce cyclin A. Previ ously, we showed that adhesion-dependent expression of cyclin A is tra nscriptionally regulated. In an effort to identify elements of the adh esion-mediated signal transduction cascade upstream of cyclin A activa tion, we investigated the expression of cyclin E and its associated ki nase activity in adherent and suspended NRK cells. Expression of cycli n E was found to be unaffected by suspension. However, cyclin E comple xes immunoprecipitated from extracts prepared from NRK cells 12 h afte r release from G(0) arrest were found to be catalytically inactive in suspended but not in adherent cells. This suspension-induced inhibitio n of cyclin E-associated kinase activity was not observed in NRK cells transformed by a c-Ha-ras oncogene containing a G12V mutation. When G (0)-synchronized NRK cells were transfected with a cyclin A promoter:l uciferase reporter construct along with expression vectors for either wild-type cdk2 or a dominant-negative cdk2 mutant, transcriptional act ivation of cyclin A was found to be dependent on catalytically active cdk2. Inhibition of cyclin E/cdk2 complexes has frequently been attrib uted to association of the cdk inhibitors p21(Cip1) and p27(Kip1). How ever, no differences between adherent and suspended cells could be obs erved for either expression or cdk2 association of p21(Cip1) or p27(Ki p1) nor were any proteins specifically associated with cdk2 or cyclin E in immunoprecipitates from metabolically labeled cell extracts. Thes e results dedfine a pathway through which an adhesion-generated signal controls cyclin A expression by modulating cyclin E/cdk2 activity. (C ) 1996 Academic Press, Inc.