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Evidence for a p23 caspase-cleaved form of p27[KIP1] involved in G1 growth arrest

Oncogene volume 18, pages 3324–3333 (1999)Cite this article

Abstract

p27[KIP1] (p27) is a cyclin dependent kinase inhibitor, involved in the negative regulation of G1 progression in response to a number of anti-proliferative signals. In this study we show, in growing mouse hybridoma (7TD1) and human myeloma (U266) cell lines, that p27 is highly expressed but slightly upregulated when cells are arrested, regardless to the phases of the cell cycle. In contrast, the specific blockade of these cells in early G1 phase reveals the induction of a protein of 23 kDa (p23) specifically recognized by polyclonal anti-p27 antibodies raised against the NH2 terminal part of p27 but not by anti-p21[CIP1] antibodies. Experiments using caspase inhibitors strongly suggest that p23 results from the proteolysis of p27 by a `caspase-3-like' protease. This cleavage leads to the cytosolic sequestration of p23 but does not alter its binding properties to CDK2 and CDK4 kinases. Indeed, p23 associated in vivo with high molecular weight complexes and coprecipitated with CDK2 and CDK4. We demonstrate by transfection experiments in SaOS-2 cells that p23 induces a G1 phase growth arrest by inhibition of cyclin/CDK2 activity. In summary we describe here a caspase-cleaved form of p27, induced in absence of detectable apoptosis and likely involved in cell cycle regulation.

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Abbreviations

Ac-DEVD-CHO:

Acetyl-Asp-Glu-Val-Asp-aldehyde

CDK:

cyclin-dependent kinase

CKI:

cyclin-dependent kinase inhibitor

DMSO:

dimethylsulfoxide

PARP:

Poly(ADP-ribose) polymerase

pRb:

retinoblastoma protein

z-VAD-fmk:

carbobenzoxy-Val-Ala-Asp-fluoremethyl ketone

GFP:

green fluorescent protein

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Acknowledgements

We are extremely grateful to Dr V Dulic for helpful discussion. We thank Dr A Proudfoot (GLAXO, Institute for Molecular Biology, Geneva, Switzerland) for human recombinant interleukin-6 and Dr J Van Snick (Brussels, Belgium) for kindly providing the 7TD1 cell line. We are indebted to A Grima, C Minghelli and R Mescatullo for illustration work, to A Doye and A Spadafora for technical assistance. This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM).

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Authors and Affiliations

  1. U364 INSERM Immunologie Cellulaire et Moléculaire, Faculté de Médecine, Avenue de Valombrose, 06107, Nice, cedex 02, France

    Agnès Loubat, Nathalie Rochet, Laurent Turchi, Dariush Farahi Far, Bernard Rossi & Gilles Ponzio

  2. CJF INSERM 96.05, Activation des Cellules Hématopoïétiques, Faculté de Médecine, Avenue de Valombrose, 06107, Nice, cedex 02, France

    Patrick Auberger

  3. Laboratoire d'Immunologie Clinique, Hôpital Cantonal de Genève, 24 rue Michel-du-Crest, Genève, 14, Switzerland

    Roger Rezzonico

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Loubat, A., Rochet, N., Turchi, L. et al. Evidence for a p23 caspase-cleaved form of p27[KIP1] involved in G1 growth arrest. Oncogene 18, 3324–3333 (1999). https://doi.org/10.1038/sj.onc.1202668

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