Transforming growth factor beta stabilizes p15INK4B protein, increases p15INK4B-cdk4 complexes, and inhibits cyclin D1-cdk4 association in human mammary epithelial cells.
The effects of transforming growth factor beta (TGF-beta) were studied in closely related human mammary epithelial cells (HMEC), both finite-life-span 184 cells and immortal derivatives, 184A1S, and 184A1L5R, which differ in their cell cycle responses to TGF-beta but express type I and type II TGF-beta receptors and retain TGF-beta induction of extracellular matrix. The arrest-resistant phenotype was not due to loss of cyclin-dependent kinase (cdk) inhibitors. TGF-beta was shown to regulate p15INK4B expression at at least two levels: mRNA accumulation and protein stability. In TGF-beta-arrested HMEC, there was not only an increase in p15 mRNA but also a major increase in p5INK4B protein stability. As cdk4- and cdk6-associated p15INK4B increased during TGF-beta arrest of sensitive cells, there was a loss of cyclin D1, p21Cip1, and p27Kip1 from these kinase complexes, and cyclin E-cdk2-associated p27Kip1 increased. In HMEC, p15INK4B complexes did not contain detectable cyclin. p15INK4B from both sensitive and resistant cells could displace in vitro cyclin D1, p21Cip1, and p27Kip1 from cdk4 isolated from sensitive cells. Cyclin D1 could not be displaced from cdk4 in the resistant 184A1L5R cell lysates. Thus, in TGF-beta arrest, p15INK4B may displace already associated cyclin D1 from cdks and prevent new cyclin D1-cdk complexes from forming. Furthermore, p27Kip1 binding shifts from cdk4 to cyclin E-cdk2 during TGF-beta-mediated arrest. The importance of posttranslational regulation of p15INK4B by TGF-beta is underlined by the observation that in TGF-beta-resistant 184A1L5R, although the p15 transcript increased, p15INK4B protein was not stabilized and did not accumulate, and cyclin D1-cdk association and kinase activation were not inhibited.
Sandhu
C
C
Division of Cancer Biology Research, Toronto-Sunnybrook Regional Cancer Centre, Ontario, Canada.
Garbe
J
J
Bhattacharya
N
N
Daksis
J
J
Pan
C H
CH
Yaswen
P
P
Koh
J
J
Slingerland
J M
JM
Stampfer
M R
MR
D001940:Breast / Q000378:metabolism*; D042846:CDC2-CDC28 Kinases; D002352:Carrier Proteins / Q000378:metabolism*; D018797:Cell Cycle Proteins; D019938:Cyclin D1; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D005434:Flow Cytometry; D016147:Genes, Tumor Suppressor; D006801:Humans; D046911:Macromolecular Substances; D015513:Oncogene Proteins / Q000378:metabolism*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D016192:Resting Phase, Cell Cycle; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D025521:Tumor Suppressor Proteins
C493966:CDKN2B protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16; D016213:Cyclins; D004791:Enzyme Inhibitors; D046911:Macromolecular Substances; D015513:Oncogene Proteins; D047428:Protein Kinase Inhibitors; D011518:Proto-Oncogene Proteins; D016212:Transforming Growth Factor beta; D025521:Tumor Suppressor Proteins; D019938:Cyclin D1; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C495900:CDK4 protein, human; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases