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Molecular and Cellular Biology, March 1999, p. 1981-1989, Vol. 19, No. 3
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Induced Expression of p16INK4a Inhibits Both CDK4- and CDK2-Associated Kinase Activity by Reassortment of Cyclin-CDK-Inhibitor Complexes

Beth B. McConnell,dagger Fiona J. Gregory, Francesca J. Stott, Eiji Hara,Dagger and Gordon Peters*

Imperial Cancer Research Fund Laboratories, London WC2A 3PX, United Kingdom

Received 30 July 1998/Returned for modification 17 September 1998/Accepted 19 November 1998

To investigate the mode of action of the p16INK4a tumor suppressor protein, we have established U2-OS cells in which the expression of p16INK4a can be regulated by addition or removal of isopropyl-beta -D-thiogalactopyranoside. As expected, induction of p16INK4a results in a G1 cell cycle arrest by inhibiting phosphorylation of the retinoblastoma protein (pRb) by the cyclin-dependent kinases CDK4 and CDK6. However, induction of p16INK4a also causes marked inhibition of CDK2 activity. In the case of cyclin E-CDK2, this is brought about by reassortment of cyclin, CDK, and CDK-inhibitor complexes, particularly those involving p27KIP1. Size fractionation of the cellular lysates reveals that a substantial proportion of CDK4 participates in active kinase complexes of around 200 kDa. Upon induction of p16INK4a, this complex is partly dissociated, and the majority of CDK4 is found in lower-molecular-weight fractions consistent with the formation of a binary complex with p16INK4a. Sequestration of CDK4 by p16INK4a allows cyclin D1 to associate increasingly with CDK2, without affecting its interactions with the CIP/KIP inhibitors. Thus, upon the induction of p16INK4a, p27KIP1 appears to switch its allegiance from CDK4 to CDK2, and the accompanying reassortment of components leads to the inhibition of cyclin E-CDK2 by p27KIP1 and p21CIP1. Significantly, p16INK4a itself does not appear to form higher-order complexes, and the overwhelming majority remains either free or forms binary associations with CDK4 and CDK6.

* Corresponding author. Mailing address: Imperial Cancer Research Fund Laboratories, P.O. Box 123, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom. Phone: (44) 171 269 3049. Fax: (44) 171 269 3479. E-mail: peters{at}icrf.icnet.uk.

dagger Present address: Department of Radiation Oncology, Emory University School of Medicine, Atlanta, GA 30335.

Dagger Present address: Paterson Institute for Cancer Research, Manchester M20 9BX, United Kingdom.

Molecular and Cellular Biology, March 1999, p. 1981-1989, Vol. 19, No. 3
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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