Transforming Growth Factor β Stabilizes p15^INK4B Protein, Increases p15^INK4B-cdk4 Complexes, and Inhibits Cyclin D1-cdk4 Association in Human Mammary Epithelial Cells

Abstract

The effects of transforming growth factor β (TGF-β) were studied in closely related human mammary epithelial cells (HMEC), both finite-life-span 184 cells and immortal derivatives, 184A1^S, and 184A1L5^R, which differ in their cell cycle responses to TGF-β but express type I and type II TGF-β receptors and retain TGF-β induction of extracellular matrix. The arrest-resistant phenotype was not due to loss of cyclin-dependent kinase (cdk) inhibitors. TGF-β was shown to regulate p15^INK4B expression at at least two levels: mRNA accumulation and protein stability. In TGF-β-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 p15^INK4B increased during TGF-β arrest of sensitive cells, there was a loss of cyclin D1, p21^Cip1, and p27^Kip1 from these kinase complexes, and cyclin E-cdk2-associated p27^Kip1 increased. In HMEC, p15^INK4B complexes did not contain detectable cyclin. p15^INK4B from both sensitive and resistant cells could displace in vitro cyclin D1, p21^Cip1, and p27^Kip1 from cdk4 isolated from sensitive cells. Cyclin D1 could not be displaced from cdk4 in the resistant 184A1L5^R cell lysates. Thus, in TGF-β arrest, p15^INK4B may displace already associated cyclin D1 from cdks and prevent new cyclin D1-cdk complexes from forming. Furthermore, p27^Kip1 binding shifts from cdk4 to cyclin E-cdk2 during TGF-β-mediated arrest. The importance of posttranslational regulation of p15^INK4B by TGF-β is underlined by the observation that in TGF-β-resistant 184A1L5^R, although the p15 transcript increased, p15^INK4B protein was not stabilized and did not accumulate, and cyclin D1-cdk association and kinase activation were not inhibited.