Contact inhibition-induced inactivation of the cyclin D-dependent kinase in rat fibroblast cell line, 3Y1.
Inhibitory proteins for Cdks (CKIs) are involved in cell cycle arrest induced by anti-mitotic factors, chemicals, or DNA damage in mammalian cells. High cell density also induces cell cycle arrest with unreplicated genomic DNA even in the presence of mitotic dose of the growth factors, termed contact inhibition. Although rat fibroblast cell line, 3Y1, arrested in quiescence by contact inhibition, Cdk4 bound its regulatory subunit, cyclin D1 or D3. However, these complexes were enzymatically inactive. Phosphorylation of the cyclin D1-bound Cdk4 by Cdk4-activating kinase, composed of cyclin H and MO15 (alias Cdk7), which was reconstituted in Spodoptera frugiperda cells (Sf9) could convert inactive cyclin D1-Cdk4 complex into active form in vitro, suggesting that threonine 172 in the Cdk4, whose phosphorylation is required for its activation, was in part unphosphorylated in the contact-inhibited 3Y1. Although MO15 was active in cell extracts prepared from the contact-inhibited 3Y1, activation of bacterially produced Cdk4 in the cell extracts was inhibited. Removing p27kip1 from the cell extracts allowed MO15 holoenzyme to phosphorylate the Cdk4 and to activate it, indicating that an access of MO15 to Cdk4 was inhibited by p27kip1 in the contact-inhibited 3Y1.
Kato
A
A
Department of Biology, Nippon Roche Research Institute, Kanagawa, Japan.
Takahashi
H
H
Takahashi
Y
Y
Matsushime
H
H
D000818:Animals; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D002460:Cell Line; D003260:Contact Inhibition; D019938:Cyclin D1; D056748:Cyclin H; D051358:Cyclin-Dependent Kinase 4; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism; D005347:Fibroblasts; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D015513:Oncogene Proteins / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera; D013912:Threonine; D014162:Transfection; D025521:Tumor Suppressor Proteins; D000095043:Cyclin-Dependent Kinase-Activating Kinase
C534753:Ccnh protein, rat; C493962:Cdkn1b protein, rat; D018797:Cell Cycle Proteins; D056748:Cyclin H; D016213:Cyclins; D004791:Enzyme Inhibitors; D008869:Microtubule-Associated Proteins; D015513:Oncogene Proteins; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D025521:Tumor Suppressor Proteins; D019938:Cyclin D1; D050760:Cyclin-Dependent Kinase Inhibitor p27; D013912:Threonine; D017346:Protein Serine-Threonine Kinases; C495903:Cdk4 protein, rat; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases; D000095043:Cyclin-Dependent Kinase-Activating Kinase