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Depletion of Guanine Nucleotides Leads to the Mdm2-Dependent Proteasomal Degradation of Nucleostemin

¹ Department of Medicine, Divisions of Oncology and Hematology, and the Stanford Cancer Center, Stanford University, Palo Alto, California and ² Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina

Requests for reprints: Beverly S. Mitchell, Stanford Cancer Center, 800 Welch Road, Palo Alto, CA 94305-5796. Phone: 650-725-9621; Fax: 650-736-0607; E-mail: bmitchell{at}stanford.edu.

Key Words: nucleostemin • Mdm2 (used synonymously with Hdm2) • AVN-944 • GTP depletion • proteasomal degradation

Nucleostemin is a positive regulator of cell proliferation and is highly expressed in a variety of stem cells, tumors, and tumor cell lines. The protein shuttles between the nucleolus and the nucleus in a GTP-dependent fashion. Selective depletion of intracellular guanine nucleotides by AVN-944, an inhibitor of the de novo purine synthetic enzyme, IMP dehydrogenase, leads to the rapid disappearance of nucleostemin protein in tumor cell lines, an effect that does not occur with two other nucleolar proteins, nucleophosmin or nucleolin. Endogenous nucleostemin protein is completely stabilized by MG132, an inhibitor of the 26S proteasome, as are the levels of expressed enhanced green fluorescent protein–tagged nucleostemin, both wild-type protein and protein containing mutations at the G₁ GTP binding site. Nutlin-3a, a small molecule that disrupts the binding of the E3 ubiquitin ligase, Mdm2, to p53, stabilizes nucleostemin protein in the face of guanine nucleotide depletion, as does siRNA-mediated knockdown of Mdm2 expression and overexpression of a dominant-negative form of Mdm2. Neither Doxorubicin nor Actinomycin D, which cause the release of nucleostemin from the nucleolus, results in nucleostemin degradation. We conclude that nucleostemin is a target for Mdm2-mediated ubiquitination and degradation when not bound to GTP. Because this effect does not occur with other chemotherapeutic agents, the induction of nucleostemin protein degradation in tumor cells by IMP dehydrogenase inhibition or by other small molecules that disrupt GTP binding may offer a new approach to the treatment of certain neoplastic diseases. [Cancer Res 2009;69(7):3004–12]