Willem den Besten

Myeloid Leukemia-Associated Nucleophosmin Mutants Perturb p53-Dependent and Independent Activities of the Arf Tumor Suppressor Protein

Nucleophosmin (NPM or B23) plays key roles in ribosome biogenesis, centrosome dupli-cation, and maintenance of genomic integrity. Mutations affecting the carboxylterminal domain of NPM occur in a significant percentage of adult patients with acute myeloid leukemia (AML), and these alterations create an additional nuclear export signal that relocalizes much of the protein from its normal nucleolar stores to the cytoplasm. When induced by oncogenic stress, the Arf tumor suppressor protein accumulates within the nucleolus, where it is physically associated with, and stabilized by, NPM. Ectopic overexpression of an NPM cytoplasmic mutant (NPMc) relocalized p19Arf and the endogenous NPM protein to the cytoplasm. NPMc-dependent export of p19Arf from the nucleus inhibited its functional interaction with the p53 negative regulator, Mdm2, and blunted Arf-induced activation of the p53 transcriptional program. Cytoplasmic NPM relocalization also attenuated Arf-induced sumoylation of Mdm2 and NPM and prevented wild type NPM from inhibiting p19Arf protein turnover. However, despite the ability of NPMc to interfere with these p53-dependent and independent activities of Arf, NPMc exhibited anti-proliferative activity in Arf-null NIH-3T3 cells. Overexpression of wild type NPM, but not NPMc, overcame premature senescence of Atm-null cells, a phenotype that can be rescued by inactivation of Arf or p53. Therefore, perturbation of Arf function appears to be insufficient to explain the oncogenic effects of the NPMc mutation. We favor the idea that NPMc also contrib-utes to AML by dominantly perturbing other functions of the wild type NPM protein.

Control of spermatogenesis in mice by the cyclin D-dependent kinase inhibitors p18(Ink4c) and p19(Ink4d).

ABSTRACT Male mice lacking both the Ink4c and Ink4dgenes, which encode two inhibitors of D-type cyclin-dependent kinases (Cdks), are infertile, whereas female fecundity is unaffected. Both p18Ink4c and p19Ink4d are expressed in the seminiferous tubules of postnatal wild-type mice, being largely confined to postmitotic spermatocytes undergoing meiosis. Their combined loss is associated with the delayed exit of spermatogonia from the mitotic cell cycle, leading to the retarded appearance of meiotic cells that do not properly differentiate and instead undergo apoptosis at an increased frequency. As a result, mice lacking bothInk4c and Ink4d produce few mature sperm, and the residual spermatozoa have reduced motility and decreased viability. Whether or not Ink4d is present, animals lackingInk4c develop hyperplasia of interstitial testicular Leydig cells, which produce reduced levels of testosterone. The anterior pituitary of fertile mice lacking Ink4c or infertile mice doubly deficient for Ink4c and Ink4d produces normal levels of luteinizing hormone (LH). Therefore, the failure of Leydig cells to produce testosterone is not secondary to defects in LH production, and reduced testosterone levels do not account for infertility in the doubly deficient strain. By contrast,Ink4d-null or double-null mice produce elevated levels of follicle-stimulating hormone (FSH). Because Ink4d-null mice are fertile, increased FSH production by the anterior pituitary is also unlikely to contribute to the sterility observed inInk4c/Ink4d double-null males. Our data indicate that p18Ink4c and p19Ink4d are essential for male fertility. These two Cdk inhibitors collaborate in regulating spermatogenesis, helping to ensure mitotic exit and the normal meiotic maturation of spermatocytes.

Arf-induced turnover of the nucleolar nucleophosmin-associated SUMO-2/3 protease Senp3

The stabilization and subcellular localization of the p19Arf tumor suppressor protein and the SUMO-2/3 deconjugating protease Senp3 each depend upon their binding to the abundant nucleolar protein nucleophosmin (Npm/B23). Senp3 and p19Arf antagonize each other’s functions in regulating the SUMOylation of target proteins including Npm itself. The p19Arf protein triggers the sequential phosphorylation, polyubiquitination, and rapid proteasomal degradation of Senp3, and this ability of p19Arf to accelerate Senp3 turnover also depends on the presence of Npm. In turn, endogenous p19Arf and Senp3 are both destabilized in viable Npm-null mouse embryo fibroblasts (that also lack p53), and reintroduction of the human NPM protein into these cells reverses this phenotype. NPM mutants that retain their acidic and oligomerization domains can re-stabilize both p19Arf and Senp3 in this setting, but the nucleolar localization of NPM is not strictly required for these effects. Knockdown of Senp3 with shRNAs mimics the anti-proliferative functions of p19Arf in cells that lack p53 alone or in triple knock-out cells that lack the Arf, Mdm2 and p53 genes. These findings reinforce the hypothesis that the p53-independent tumor suppressive functions of p19Arf may be mediated by its ability to antagonize Senp3, thereby inducing cell cycle arrest by abnormally elevating the cellular levels of SUMOylated proteins.

N-Terminal Polyubiquitination of the ARF Tumor Suppressor, A Natural Lysine-Less Protein

Ubiquitin-dependent proteolysis by proteasomes generally depends upon the conjugation of polyubiquitin chains to lysine ?-NH2 groups within the targeted proteins. However, engineered lysine-less mutants of certain viral and cellular proteins can undergo polyubiquitination at their N-termini. Is N-terminal polyubiquitination a physiologic process, and how many cellular proteins can be targeted for proteasomal degradation through this mechanism? Recent work indicates that the turnover of the endogenous lysine-less human ARF tumor suppressor protein and its mouse Arf counterpart (containing a single, non-conserved lysine residue) is regulated in this manner.

Differential post-transcriptional regulation of two Ink4 proteins, p18Ink4c and p19Ink4d

Cyclin(-D-)-dependent kinase (Cdk) inhibitors of the Ink4 family specifically bind to Cdk4 and Cdk6, but not to other Cdks. Ink4c and Ink4d mRNAs are maximally and periodically expressed during the G2/M phase of the cell division cycle, but the abundance of their encoded proteins is regulated through distinct mechanisms. Both proteins undergo polyubiquitination, but the half life of p18Ink4c (~10 hours) is much longer than that of p19Ink4d (~2.5 hours). Lysines 46 and 112 are preferred sites of ubiquitin conjugation in p18Ink4c, although substitution of these and other lysine residues with arginine, particularly in combination, triggers protein misfolding and accelerates p18Ink4c degradation. When tethered to either catalytically active or inactive Cdk4 or Cdk6, polyubiquitination of p18Ink4c is inhibited, and the protein is further stabilized. Conversely, in competing with p18Ink4c for binding to Cdks, cyclin D1 accelerates p18Ink4c turnover. In direct contrast, polyubiquitination of p19Ink4d is induced by its association with Cdks, whereas cyclin D1 overexpression retards p19Ink4d degradation. Although it has been generally assumed that p18Ink4c and p19Ink4d are biochemically similar Cdk inhibitors, the major differences in their stability and turnover are likely key to understanding their distinct biological functions.