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Molecular and Cellular Biology, August 2001, p. 5644-5657, Vol. 21, No. 16
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.16.5644-5657.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Akt-Dependent Phosphorylation of p21Cip1 Regulates PCNA Binding and Proliferation of Endothelial Cells

Lothar Rössig, Amir S. Jadidi, Carmen Urbich, Cornel Badorff, Andreas M. Zeiher, and Stefanie Dimmeler*

Molecular Cardiology, Department of Internal Medicine IV, University of Frankfurt, 60590 Frankfurt, Germany

Received 2 March 2001/Returned for modification 13 April 2001/Accepted 24 May 2001

The protein kinase Akt is activated by growth factors and promotes cell survival and cell cycle progression. Here, we demonstrate that Akt phosphorylates the cell cycle inhibitory protein p21Cip1 at Thr 145 in vitro and in intact cells as shown by in vitro kinase assays, site-directed mutagenesis, and phospho-peptide analysis. Akt-dependent phosphorylation of p21Cip1 at Thr 145 prevents the complex formation of p21Cip1 with PCNA, which inhibits DNA replication. In addition, phosphorylation of p21Cip1 at Thr 145 decreases the binding of the cyclin-dependent kinases Cdk2 and Cdk4 to p21Cip1 and attenuates the Cdk2 inhibitory activity of p21Cip1. Immunohistochemistry and biochemical fractionation reveal that the decrease of PCNA binding and regulation of Cdk activity by p21Cip1 phosphorylation is not caused by altered intracellular localization of p21Cip1. As a functional consequence, phospho-mimetic mutagenesis of Thr 145 reverses the cell cycle-inhibitory properties of p21Cip1, whereas the nonphosphorylatable p21Cip1 T145A construct arrests cells in G0 phase. These data suggest that the modulation of p21Cip1 cell cycle functions by Akt-mediated phosphorylation regulates endothelial cell proliferation in response to stimuli that activate Akt.

* Corresponding author. Mailing address: Molecular Cardiology, Department of Internal Medicine IV, University of Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany. Phone: 49-69-6301-7440 or -5789. Fax: 49-69-6301-7113 or -6374. E-mail: Dimmeler{at}em.uni-frankfurt.de.

Molecular and Cellular Biology, August 2001, p. 5644-5657, Vol. 21, No. 16
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.16.5644-5657.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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