Cooperative Signals Governing ARF-Mdm2 Interaction and Nucleolar Localization of the Complex

Abstract

The ARF tumor suppressor protein stabilizes p53 by antagonizing its negative regulator, Mdm2 (Hdm2 in humans). Both mouse p19^ARF and human p14^ARFbind to the central region of Mdm2 (residues 210 to 304), a segment that does not overlap with its N-terminal p53-binding domain, nuclear import or export signals, or C-terminal RING domain required for Mdm2 E3 ubiquitin ligase activity. The N-terminal 37 amino acids of mouse p19^ARF are necessary and sufficient for binding to Mdm2, localization of Mdm2 to nucleoli, and p53-dependent cell cycle arrest. Although a nucleolar localization signal (NrLS) maps within a different segment (residues 82 to 101) of the human p14^ARF protein, binding to Mdm2 and nucleolar import of ARF-Mdm2 complexes are both required for cell cycle arrest induced by either the mouse or human ARF proteins. Because many codons of mouse ARF mRNA are not recognized by the most abundant bacterial tRNAs, we synthesized ARF minigenes containing preferred bacterial codons. Using bacterially produced ARF polypeptides and chemically synthesized peptides conjugated to Sepharose, residues 1 to 14 and 26 to 37 of mouse p19^ARF were found to interact independently and cooperatively with Mdm2, while residues 15 to 25 were dispensable for binding. Paradoxically, residues 26 to 37 of mouse p19^ARF are also essential for ARF nucleolar localization in the absence of Mdm2. However, the mobilization of the p19^ARF-Mdm2 complex into nucleoli also requires a cryptic NrLS within the Mdm2 C-terminal RING domain. The Mdm2 NrLS is unmasked upon ARF binding, and its deletion prevents import of the ARF-Mdm2 complex into nucleoli. Collectively, the results suggest that ARF binding to Mdm2 induces a conformational change that facilitates nucleolar import of the ARF-Mdm2 complex and p53-dependent cell cycle arrest. Hence, the ARF-Mdm2 interaction can be viewed as bidirectional, with each protein being capable of regulating the subnuclear localization of the other.

ACKNOWLEDGMENTS

We thank Karen Vousden for alerting us to the functional significance of the cryptic Mdm2 NrLS, for generously providing the Hdm2 1–440 mutant, and for exchanging manuscripts prior to submission for publication. We thank Esther Van de Kamp, Rose Mathew, and Ming Wang for excellent technical assistance; Jinjun Dang for his help in generating the Hdm2 mutant lacking residues 466 to 473; and John L. Cleveland for insightful criticism of the manuscript.

This work was supported in part by NIH grants P01 CA-71907 (M.F.R.), Cancer Center CORE grant CA-21765, the American Cancer Society (R.W.K.), and the American Lebanese Syrian Associated Charities (ALSAC) of St. Jude Children's Research Hospital. B.B. is supported by a Hal and Alma Reagan Fellowship. C.J.S. is an Investigator and J.D.W. is a Research Associate of the Howard Hughes Medical Institute.