Transforming growth factor β stabilizes p15(INK4B) protein, increases p15(INK4B)-cdk4 complexes, and inhibits cyclin D1-cdk4 association in human mammary epithelial cells

C. Sandhu, J. Garbe, N. Bhattacharya, J. Daksis, C. H. Pan, P. Yaswen, J. Koh, Joyce M Slingerland, M. R. Stampfer

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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 p15(INK4B) 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.

Original languageEnglish
Pages (from-to)2458-2467
Number of pages10
JournalMolecular and Cellular Biology
Volume17
Issue number5
StatePublished - Jan 1 1997
Externally publishedYes

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ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cell Biology

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