Induction of p27Kip1 degradation and anchorage independence by Ras through the MAP kinase signaling pathway.
While most untransformed cells require substrate attachment for growth (anchorage dependence), the oncogenic transformed cells lack this requirement (anchorage independence) and are often tumorigenic. However, the mechanism of loss of anchorage dependence is not fully understood. When rat normal fibroblasts were cultured in suspension without substrate attachment, the cell cycle arrested in G1 phase and the cyclin-dependent kinase inhibitor p27Kip1 protein and its mRNA accumulated. Conditional expression of oncogenic Ras induced the G1-S transition of the cell cycle and significantly shortened the half-life of p27Kip1 protein without altering its mRNA level. Inhibition of the activation of mitogen-activated protein (MAP) kinase by cyclic AMP-elevating agents and a MEK inhibitor prevented the oncogenic Ras-induced degradation of p27Kip1. These results suggest that the loss of substrate attachment induces the cell cycle arrest through the up-regulation of p27Kip1 mRNA, but the oncogenic Ras confers anchorage independence by accelerating p27Kip1 degradation through the activation of the MAP kinase signaling pathway. Furthermore, we have found that p27Kip1 is phosphorylated by MAP kinase in vitro and the phosphorylated p27Kip1 cannot bind to and inhibit cdk2.
Kawada
M
M
Department of Bioactive Molecules, National Institute of Health, Tokyo, Japan.
Yamagoe
S
S
Murakami
Y
Y
Suzuki
K
K
Mizuno
S
S
Uehara
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D002448:Cell Adhesion; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D005347:Fibroblasts; D016193:G1 Phase; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011233:Precipitin Tests; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011995:Recombination, Genetic; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins
C493961:Cdkn1b protein, mouse; C493962:Cdkn1b protein, rat; D018797:Cell Cycle Proteins; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D008869:Microtubule-Associated Proteins; D012333:RNA, Messenger; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D000242:Cyclic AMP; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde