Inhibition of Retinoblastoma Protein Degradation by Interaction with the Serpin Plasminogen Activator Inhibitor 2 via a Novel Consensus Motif

Abstract

Plasminogen activator inhibitor-2 (PAI-2) is well documented as an inhibitor of the extracellular serine proteinase urokinase-type plasminogen activator (uPA) and is expressed in activated monocytes and macrophages, differentiating keratinocytes, and many tumors. Here we show that PAI-2 has a novel intracellular function as a retinoblastoma protein (Rb)-binding protein. PAI-2 colocalized with Rb in the nucleus and inhibited the turnover of Rb, which led to increases in Rb protein levels and Rb-mediated activities. Although PAI-2 contains an LXCXE motif, Rb binding was primarily mediated by the C-D interhelical region of PAI-2, which was found to bind to the C pocket of Rb. The C-D interhelical region of PAI-2 contained a novel Rb-binding motif, termed the PENF homology motif, which is shared by many cellular and viral Rb-binding proteins. PAI-2 expression also protected Rb from the accelerated degradation mediated by human papillomavirus (HPV) E7, leading to recovery of Rb and inhibition of E6/E7 mRNA expression. Protection of Rb by PAI-2 begins to explain many of the diverse, uPA-independent phenotypes conferred by PAI-2 expression. These results indicate that PAI-2 may enhance Rb's tumor suppressor activity and suggest a potential therapeutic role for PAI-2 against HPV-transformed lesions.

ACKNOWLEDGMENTS

We thank Biotech Australia for the gift of recombinant PAI-2 and the anti-PAI-2 antibody. Thanks also go to Joy Gardner, Stephanie Kosmala, Kathy Buttigieg, Julie Muddiman, and Victoria Mann for assistance during the project.

This project was funded by the Queensland Institute of Medical Research Trust, the Australian Centre for International and Tropical Health and Nutrition, and the Queensland Cancer Fund. G.A.D. is supported by a University of Queensland Graduate School award. R.W.J. is a Wellcome Trust Senior Research Fellow.

G.A.D. and T.M.A. contributed equally to this work.