Loss of cell cycle control by deregulation of cyclin-dependent kinase 2 kinase activity in Evi-1 transformed fibroblasts.
The Evi-1 transcriptional repressor protein has two distinct zinc finger DNA binding domains designated ZF1 and ZF2 and is implicated in the progression of human and murine leukemias, in which it is abnormally expressed. In this report, we show that Evi-1-expressing Rat1 fibroblasts are anchorage independent, have an abbreviated G1 phase of the cell cycle, and have a reduced requirement for serum mitogens for S-phase entry. These biological changes are accompanied by a moderately increased production of cell cycle-regulatory proteins cyclin A and cyclin-dependent kinase (Cdk) 2, a dramatic deregulation of Cdk2 kinase activity, and a corresponding increase in the levels of hyperphosphorylated retinoblastoma protein (pRb). We show that the elevated cyclin A-Cdk2 activity is due to the combination of increased accumulation and stabilization of cyclin A bound to a faster-migrating species of Cdk2 believed to be the active threonine 160 phosphorylated form and a substantial reduction in complexed p27. Cyclin E kinase activity is also elevated due to a reduction in p27. A significant reduction in total cellular p27 protein levels and a moderate reduction in p27 mRNA are observed, but no changes in Cdk regulatory kinases and phosphatases occur. The Evi-1 transcriptional repressor domain and the ZF1 DNA binding domain are required for both cell transformation and induction of Cdk2 catalytic activity. We propose that one consequence of Evi-1 expression is to repress the transcription of target genes, which may include p27, that deregulate the normal control of the G1 phase of the cell cycle, providing a cellular proliferative advantage that contributes to transformation in vitro and leukemogenesis in vivo.
Kilbey
A
A
Cancer Research Campaign Beatson Laboratories, Beatson Institute for Cancer Research, Glasgow, Scotland.
Stephens
V
V
Bartholomew
C
C
D000818:Animals; D042846:CDC2-CDC28 Kinases; D018797:Cell Cycle Proteins; D002455:Cell Division / Q000502:physiology; D002461:Cell Line, Transformed; D019925:Cyclin A / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000032:analysis; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D016193:G1 Phase / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D000074008:MDS1 and EVI1 Complex Locus Protein; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011519:Proto-Oncogenes; D051381:Rats; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D020687:cdc25 Phosphatases / Q000378:metabolism
C493962:Cdkn1b protein, rat; D018797:Cell Cycle Proteins; D019925:Cyclin A; D004268:DNA-Binding Proteins; D000074008:MDS1 and EVI1 Complex Locus Protein; C063575:MECOM protein, human; D008869:Microtubule-Associated Proteins; D012097:Repressor Proteins; D016160:Retinoblastoma Protein; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D004247:DNA; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C495892:Cdk2 protein, rat; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; C517004:CDC25A protein, human; C517007:Cdc25a protein, rat; D020687:cdc25 Phosphatases