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 G1 and failure to produce cyclin A. Previously, we showed that adhesion-dependent expression of cyclin A is transcriptionally regulated. In an effort to identify elements of the adhesion-mediated signal transduction cascade upstream of cyclin A activation, we investigated the expression of cyclin E and its associated kinase activity in adherent and suspended NRK cells. Expression of cyclin E was found to be unaffected by suspension. However, cyclin E complexes immunoprecipitated from extracts prepared from NRK cells 12 h after release from G0 arrest were found to be catalytically inactive in suspended but not in adherent cells. This suspension-induced inhibition of cyclin E-associated kinase activity was not observed in NRK cells transformed by a c-Ha-ras oncogene containing a G12V mutation. When G0-synchronized NRK cells were transfected with a cyclin A promoter:luciferase reporter construct along with expression vectors for either wild-type cdk2 or a dominant-negative cdk2 mutant, transcriptional activation of cyclin A was found to be dependent on catalytically active cdk2. Inhibition of cyclin E/cdk2 complexes has frequently been attributed to association of the cdk inhibitors p21(Cip1) and p27(Kip1). However, no differences between adherent and suspended cells could be observed for either expression or cdk2 association of p21(Cip1) or p27(Kip1), nor were any proteins specifically associated with cdk2 or cyclin E in immunoprecipitates from metabolically labeled cell extracts. These results define a pathway through which an adhesion-generated signal controls cyclin A expression by modulating cyclin E/cdk2 activity.
Carstens
C P
CP
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706, USA.
Krämer
A
A
Fahl
W E
WE
D000818:Animals; D042846:CDC2-CDC28 Kinases; D002448:Cell Adhesion / Q000502:physiology*; D002453:Cell Cycle / Q000502:physiology*; D018797:Cell Cycle Proteins; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D051357:Cyclin-Dependent Kinase 2; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000032:analysis / Q000096:biosynthesis*; D016213:Cyclins / Q000032:analysis / Q000096:biosynthesis* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000032:analysis / Q000378:metabolism; D011905:Genes, ras; D015151:Immunoblotting; D007668:Kidney; D008156:Luciferases / Q000096:biosynthesis; D008869:Microtubule-Associated Proteins / Q000032:analysis / Q000378:metabolism; D006656:Protamine Kinase / Q000032:analysis / Q000096:biosynthesis; D017346:Protein Serine-Threonine Kinases / Q000032:analysis / Q000096:biosynthesis*; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D014162:Transfection; D025521:Tumor Suppressor Proteins
C493958:Cdkn1a protein, rat; C493962:Cdkn1b protein, rat; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004791:Enzyme Inhibitors; D008869:Microtubule-Associated Proteins; D011994:Recombinant Proteins; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D008156:Luciferases; D006656:Protamine Kinase; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495892:Cdk2 protein, rat; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases