Proteins encoded by the human c-myc oncogene: differential expression in neoplastic cells.

To examine myc protein products in the wide variety of human tumor cells having alterations of the c-myc locus, we have prepared an antiserum against a synthetic peptide corresponding to the predicted C-terminal sequence of the human c-myc protein. This antiserum (anti-hu-myc 12C) specifically precipitated two proteins of 64 and 67 kilodaltons in quantities ranging from low levels in normal fibroblasts to 10-fold-higher levels in Epstein-Barr virus-immortalized and Burkitt's lymphoma cell lines, to 20- to 60-fold-higher levels in cell lines having amplified c-myc. The p64 and p67 proteins were found to be highly related by partial V8 proteolytic mapping, and both were demonstrated to be encoded by the c-myc oncogene, using hybrid-selected translation of myc-specific RNA. In addition, the p64 protein was specifically precipitated from cells transfected with a translocated c-myc gene. Both p64 and p67 were found to be nuclear phosphoproteins with extremely short half-lives. In tumor cell lines having alterations at the c-myc locus due to amplification or translocation, we observed a significant change in the expression of p64 relative to p67 when compared with normal or Epstein-Bar virus-immortalized cells.

c-Myc proteolysis by the ubiquitin-proteasome pathway: stabilization of c-Myc in Burkitt's lymphoma cells.

The c-Myc oncoprotein is a transcription factor which is a critical regulator of cellular proliferation. Deregulated expression of c-Myc is associated with many human cancers, including Burkitt's lymphoma. The c-Myc protein is normally degraded very rapidly with a half-life of 20 to 30 min. Here we demonstrate that proteolysis of c-Myc in vivo is mediated by the ubiquitin-proteasome pathway. Inhibition of proteasome activity blocks c-Myc degradation, and c-Myc is a substrate for ubiquitination in vivo. Furthermore, an increase in c-Myc stability occurs in mitotic cells and is associated with inhibited c-Myc ubiquitination. Deletion analysis was used to identify regions of the c-Myc protein which are required for rapid proteolysis. We found that a centrally located PEST sequence, amino acids 226 to 270, is necessary for rapid c-Myc degradation, but not for ubiquitination. Also, N-terminal sequences, located within the first 158 amino acids of c-Myc, are necessary for both efficient c-Myc ubiquitination and subsequent degradation. We found that c-Myc is significantly stabilized (two- to sixfold) in many Burkitt's lymphoma-derived cell lines, suggesting that aberrant c-Myc proteolysis may play a role in the pathogenesis of Burkitt's lymphoma. Finally, mutation of Thr-58, a major phosphorylation site in c-Myc and a mutational hot spot in Burkitt's lymphoma, increases c-Myc stability; however, mutation of c-Myc is not essential for stabilization in Burkitt's lymphoma cells.

Hierarchical phosphorylation at N-terminal transformation-sensitive sites in c-Myc protein is regulated by mitogens and in mitosis.

The N-terminal domain of the c-Myc protein has been reported to be critical for both the transactivation and biological functions of the c-Myc proteins. Through detailed phosphopeptide mapping analyses, we demonstrate that there is a cluster of four regulated and complex phosphorylation events on the N-terminal domain of Myc proteins, including Thr-58, Ser-62, and Ser-71. An apparent enhancement of Ser-62 phosphorylation occurs on v-Myc proteins having a mutation at Thr-58 which has previously been correlated with increased transforming ability. In contrast, phosphorylation of Thr-58 in cells is dependent on a prior phosphorylation of Ser-62. Hierarchical phosphorylation of c-Myc is also observed in vitro with a specific glycogen synthase kinase 3 alpha, unlike the promiscuous phosphorylation observed with other glycogen synthase kinase 3 alpha and 3 beta preparations. Although both p42 mitogen-activated protein kinase and cdc2 kinase specifically phosphorylate Ser-62 in vitro and cellular phosphorylation of Thr-58/Ser-62 is stimulated by mitogens, other in vivo experiments do not support a role for these kinases in the phosphorylation of Myc proteins. Unexpectedly, both the Thr-58 and Ser-62 phosphorylation events, but not other N-terminal phosphorylation events, can occur in the cytoplasm, suggesting that translocation of the c-Myc proteins to the nucleus is not required for phosphorylation at these sites. In addition, there appears to be an unusual block to the phosphorylation of Ser-62 during mitosis. Finally, although the enhanced transforming properties of Myc proteins correlates with the loss of phosphorylation at Thr-58 and an enhancement of Ser-62 phosphorylation, these phosphorylation events do not alter the ability of c-Myc to transactivate through the CACGTG Myc/Max binding site.

Enhanced translation and increased turnover of c-myc proteins occur during differentiation of murine erythroleukemia cells.

To determine whether regulation of c-myc proteins occurs during the differentiation of murine erythroleukemia cells, we examined c-myc protein synthesis and accumulation throughout dimethyl sulfoxide (DMSO)- or hypoxanthine-induced differentiation. c-myc protein expression exhibited an overall biphasic reduction, with an initial concomitant decrease in c-myc RNA, protein synthesis, and protein accumulation early during the commitment phase. However, as the mRNA and protein levels recovered, c-myc protein synthesis levels dissociated from the levels of c-myc mRNA and protein accumulation. This dissociation appears to result from unusual translational and posttranslational regulation during differentiation. Translational enhancement was suggested by the observation that relatively high levels of c-myc proteins were synthesized from relatively moderate levels of c-myc RNA. This translational enhancement was not observed with c-myb. Under certain culture conditions, we also observed a change in the relative synthesis ratio of the two independently initiated c-myc proteins. Posttranslational regulation was evidenced by a dramatic postcommitment decrease in the accumulated c-myc protein levels despite relatively high levels of c-myc protein synthesis. This decrease corresponded with a twofold increase in the turnover of c-myc proteins. The consequence of this regulation was that the most substantial decrease in c-myc protein accumulation occurred during the postcommitment phase of differentiation. This result supports the hypothesis that the reduction in c-myc at relatively late times is most important for completion of murine erythroleukemia cell terminal differentiation.

The c-Myc transactivation domain is a direct modulator of apoptotic versus proliferative signals.

We have assayed the oncogenic, proliferative, and apoptotic activities of the frequent mutations that occur in the c-myc gene in Burkitt's lymphomas. Some alleles have a modest (50 to 60%) increase in transforming activity; however, the most frequent Burkitt's lymphoma allele (T58I) had an unexpected substantial decrease in transforming activity (85%). All alleles restored the proliferation function of c-Myc in cells that grow slowly due to a c-myc knockout. There was discordance for some alleles between apoptotic and oncogenic activities, but only the T58A allele had elevated transforming activity with a concomitant reduced apoptotic potential. We discovered a novel missense mutation, MycS71F, that had a very low apoptotic activity compared to wild-type Myc, yet this mutation has never been found in lymphomas, suggesting that there is no strong selection for antiapoptotic c-Myc alleles. MycS71F also induced very low levels of cytochrome c release from mitochondria, suggesting a mechanism of action for this mutation. Phosphopeptide mapping provided a biochemical basis for the dramatically different biological activities of the transformation-defective T58I and transformation-enhanced T58A c-Myc alleles. Furthermore, the antiapoptotic survival factor insulin-like growth factor 1 was found to suppress phosphorylation of T58, suggesting that the c-Myc transactivation domain is a direct target of survival signals.

Identification of downstream-initiated c-Myc proteins which are dominant-negative inhibitors of transactivation by full-length c-Myc proteins.

The c-myc gene has been implicated in multiple cellular processes including proliferation, differentiation, and apoptosis. In addition to the full-length c-Myc 1 and 2 proteins, we have found that human, murine, and avian cells express smaller c-Myc proteins arising from translational initiation at conserved downstream AUG codons. These c-Myc short (c-Myc S) proteins lack most of the N-terminal transactivation domain but retain the C-terminal protein dimerization and DNA binding domains. As with full-length c-Myc proteins, the c-Myc S proteins appear to be localized to the nucleus, are relatively unstable, and are phosphorylated. Significant levels of c-Myc S, often approaching the levels of full-length c-Myc, are transiently observed during the rapid growth phase of several different types of cells. Optimization of the upstream initiation codons resulted in greatly reduced synthesis of the c-Myc S proteins, suggesting that a "leaky scanning" mechanism leads to the translation of these proteins. In some hematopoietic tumor cell lines having altered c-myc genes, the c-Myc S proteins are constitutively expressed at levels equivalent to that of full-length c-Myc. As predicted, the c-Myc S proteins are unable to activate transcription and inhibited transactivation by full-length c-Myc proteins, suggesting a dominant-negative inhibitory function. While these transcriptional inhibitors would not be expected to function as full-length c-Myc, the occurrence of tumors which express constitutive high levels of c-Myc S and their transient synthesis during rapid cell growth suggest that these proteins do not interfere with the growth-promoting functions of full-length c-Myc.

V-myc- and c-myc-encoded proteins are associated with the nuclear matrix.

A series of extraction procedures were applied to avian nuclei which allowed us to define three types of association of v-myc- and c-myc-encoded proteins with nuclei: (i) a major fraction (60 to 90%) which is retained in DNA- and RNA-depleted nuclei after low- and high-salt extraction, (ii) a small fraction (1%) released during nuclease digestion of DNA in intact nuclei in the presence of low-salt buffer, and (iii) a fraction of myc protein (less than 10%) extractable with salt or detergents and found to have affinity for both single- and double-stranded DNA. Immunofluorescence analysis with anti-myc peptide sera on cells extracted sequentially with nucleases and salts confirmed the idea that myc proteins were associated with a complex residual nuclear structure (matrix-lamin fraction) which also contained avian nuclear lamin protein. Dispersal of myc proteins into the cytoplasm was found to occur during mitosis. Both c-myc and v-myc proteins were associated with the matrix-lamin, suggesting that the function of myc may relate to nuclear structural organization.

Variant Max protein, derived by alternative splicing, associates with c-Myc in vivo and inhibits transactivation.

Max (Myc-associated factor X) is a basic helix-loop-helix/leucine zipper protein that has been shown to play a central role in the functional activity of c-Myc as a transcriptional activator. Max potentiates the binding of Myc-Max heterodimers through its basic region to its specific E-box Myc site (EMS), enabling c-Myc to transactivate effectively. In addition to the alternatively spliced exon a, several naturally occurring forms of alternatively spliced max mRNAs have been reported, but variant protein products from these transcripts have not been detected. Using Western blot (immunoblot) and immunoprecipitation analysis, we have identified a variant form of Max protein (16 to 17 kDa), termed dMax, in detergent nuclear extracts of murine B-lymphoma cells, normal B lymphocytes, and NIH 3T3 fibroblasts. Cloning and sequencing revealed that dMax contains a deletion spanning the basic region and helix 1 and the loop of the helix-loop-helix region, presumably as a result of alternative splicing of max RNA. S1 nuclease analysis confirmed the presence of the mRNA for dMax in cells. The dMax protein, prepared via in vitro transcription and translation, associated with bacterially synthesized Myc-glutathione S-transferase. Coimmunoprecipitation of dMax and c-Myc indicated their intracellular association. In vitro-synthesized dMax failed to bind EMS DNA, presumably because of the absence of the basic region. Coexpression of dMax inhibited EMS-mediated transactivation by c-Myc. Thus dMax, which can interact with c-Myc, appears to function as a dominant negative regulator, providing an additional level of regulation to the transactivation potential of c-Myc.

Disruption of Myc-tubulin interaction by hyperphosphorylation of c-Myc during mitosis or by constitutive hyperphosphorylation of mutant c-Myc in Burkitt's lymphoma.

Somatic mutations at Thr-58 of c-Myc have been detected in Burkitt's lymphoma (BL) tumors and have been shown to affect the transforming potential of the Myc oncoprotein. In addition, the N-terminal domain of c-Myc has been shown to interact with microtubules in vivo, and the binding of c-Myc to alpha-tubulin was localized to amino acids 48 to 135 within the c-Myc protein. We demonstrate that c-Myc proteins harboring a naturally occurring mutation at Thr-58 from BL cell lines have increased stability and are constitutively hyperphosphorylated, which disrupts the in vivo interaction of c-Myc with alpha-tubulin. In addition, we show that wild-type c-Myc-alpha-tubulin interactions are also disrupted during a transient mitosis-specific hyperphosphorylation of c-Myc, which resembles the constitutive hyperphosphorylation pattern of Thr-58 in BL cells.

A link between increased transforming activity of lymphoma-derived MYC mutant alleles, their defective regulation by p107, and altered phosphorylation of the c-Myc transactivation domain.

The c-Myc protein is a transcription factor with an N-terminal transcriptional regulatory domain and C-terminal oligomerization and DNA-binding motifs. Previous studies have demonstrated that p107, a protein related to the retinoblastoma protein, binds to the c-Myc transcriptional activation domain and suppresses its activity. We sought to characterize the transforming activity and transcriptional properties of lymphoma-derived mutant MYC alleles. Alleles encoding c-Myc proteins with missense mutations in the transcriptional regulatory domain were more potent than wild-type c-Myc in transforming rodent fibroblasts. Although the mutant c-Myc proteins retained their binding to p107 in in vitro and in vivo assays, p107 failed to suppress their transcriptional activation activities. Many of the lymphoma-derived MYC alleles contain missense mutations that result in substitution for the threonine at codon 58 or affect sequences flanking this amino acid. We observed that in vivo phosphorylation of Thr-58 was absent in a lymphoma cell line with a mutant MYC allele containing a missense mutation flanking codon 58. Our in vitro studies suggest that phosphorylation of Thr-58 in wild-type c-Myc was dependent on cyclin A and required prior phosphorylation of Ser-62 by a p107-cyclin A-CDK complex. In contrast, Thr-58 remained unphosphorylated in two representative mutant c-Myc transactivation domains in vitro. Our studies suggest that missense mutations in MYC may be selected for during lymphomagenesis, because the mutant MYC proteins have altered functional interactions with p107 protein complexes and fail to be phosphorylated at Thr-58.

Overexpression of c-Myc and cell immortalization alters c-Myc phosphorylation.

Using an extensive series of deletion and site-specific mutation constructs, we have identified five new phosphorylation sites in c-Myc in the N-terminal transactivation domain and near the C-terminal DNA binding/heterodimerization domain. We have also found that Thr-58 phosphorylation is regulated by specific cellular events. When c-Myc is overexpressed in cells Thr-58 phosphorylation was greatly enhanced in the overexpressed, exogenous c-Myc as compared with the endogenous protein. In contrast, an inhibition of Thr-58 phosphorylation and an enhancement of Serine 62 phosphorylation was observed in c-Myc from immortalized cells compared with primary cells. No significant changes in c-Myc phosphorylation were found when transformed and nontransformed cells were compared. Finally, mutations at these phosphorylation sites, either individually or in combination with previously described sites, did not affect the ability of c-Myc to transactivate through the CACGTG Myc/Max DNA binding sites. These results further suggest that either the molecular role for c-Myc phosphorylation does not involve modulating transcriptional activity of c-Myc or that the CACGTG site does not represent a physiological promoter element.

Myc-mediated transformation: the repression connection.

Myc is an important regulator of many cellular processes, including growth promotion, differentiation, and apoptosis. The mechanisms underlying Myc biological activity, however, remain elusive. For many years, research in the field has focused on the idea of Myc as a transactivator of gene expression. More recently, alternative mechanisms of Myc function have been proposed, including gene repression. In this review we present several lines of evidence to support a connection between Myc-mediated transformation and transcriptional repression.

B-Myc is preferentially expressed in hormonally-controlled tissues and inhibits cellular proliferation.

The myc family of genes plays an important role in several cellular processes including proliferation, apoptosis, differentiation, and transformation. B-myc, a relatively new and largely unstudied member of the myc family, encodes a protein that is highly homologous to the N-terminal transcriptional regulatory domain of c-Myc. Here, we show that high level B-myc expression is restricted to specific mouse tissues, primarily hormonally-controlled tissues, with the highest level of expression in the epididymis. We also report the identification of the endogenous B-Myc protein from mouse tissues. Like other Myc family proteins, B-Myc is a short-lived nuclear protein which is phosphorylated on residues Ser-60 and Ser-68. Rapid proteolysis of B-Myc occurs via the ubiquitin-proteasome pathway. Finally, we found that overexpression of B-Myc significantly slows the growth of Rat la fibroblasts and COS cells suggesting B-Myc functions as an inhibitor of cellular proliferation.

Telomerase reverse transcriptase gene is a direct target of c-Myc but is not functionally equivalent in cellular transformation.

The telomerase reverse transcriptase component (TERT) is not expressed in most primary somatic human cells and tissues, but is upregulated in the majority of immortalized cell lines and tumors. Here, we identify the c-Myc transcription factor as a direct mediator of telomerase activation in primary human fibroblasts through its ability to specifically induce TERT gene expression. Through the use of a hormone inducible form of c-Myc (c-Myc-ER), we demonstrate that Myc-induced activation of the hTERT promoter requires an evolutionarily conserved E-box and that c-Myc-ER-induced accumulation of hTERT mRNA takes place in the absence of de novo protein synthesis. These findings demonstrate that the TERT gene is a direct transcriptional target of c-Myc. Since telomerase activation frequently correlates with immortalization and telomerase functions to stabilize telomers in cycling cells, we tested whether Myc-induced activation of TERT gene expression represents an important mechanism through which c-Myc acts to immortalize cells. Employing the rat embryo fibroblast cooperation assay, we show that TERT is unable to substitute for c-Myc in the transformation of primary rodent fibroblasts, suggesting that the transforming activities of Myc extend beyond its ability to activate TERT gene expression and hence telomerase activity.

Differential expression of the mouse mitochondrial genes and the mitochondrial RNA-processing endoribonuclease RNA by androgens.

Using subtractive hybridization to identify genes that are androgen regulated in the mouse epididymis, a number of cDNAs were identified that represented mitochondrial genes including cytochrome oxidase c subunits I, II, and III, cytochrome b, NADH dehydrogenase subunit 5, a region of the displacement loop, and the 16S rRNA. Northern blot analysis of RNA from intact, castrate, or testosterone-replaced epididymides confirmed that these mitochondrial mRNAs as well as the rRNA were androgen regulated with a 2- to 5-fold reduction in expression observed after 4 weeks castration with partial to full recovery to precastrate levels upon 4 weeks of testosterone replacement. In contrast to the mitochondrial genes, the expression of the RNA component of the mitochondrial RNA-processing endoribonuclease (RNAase MRP), a nuclear factor which is thought to be involved in the regulation of mitochondrial DNA synthesis, increased in the epididymis upon castration and then returned to precastrate levels after testosterone replacement. An examination of other androgen-responsive tissues showed that mitochondrial gene expression was also regulated by androgens in the kidney. The RNAase MRP RNA levels, however, showed an increase after castration only in the reproductive tissues (epididymis, vas deferens, and seminal vesicle) and not in the kidney. No correlative increase in mitochondrial DNA levels was observed for any of the tissues. Finally, an analysis of various mouse tissues as well as the different regions of the epididymis revealed large differences in mitochondrial mRNA levels. While for most tissues the mRNA levels correlated with the mitochondrial DNA content, the levels of the RNAase MRP RNA did not. Taken together, these findings not only show the large variations in mitochondrial gene expression between tissues but also demonstrate that the expression of mitochondrial genes and ultimately mitochondrial function are androgen regulated in the epididymis and kidney.

The CRES gene: a unique testis-regulated gene related to the cystatin family is highly restricted in its expression to the proximal region of the mouse epididymis.

As a result of examining regional-specific gene expression in the mouse epididymis, a novel cystatin-related epididymal specific (CRES) gene was identified. Substantial homology between the CRES gene and members of the cystatin family of cysteine proteinase inhibitors was observed at the amino acid level. This homology included the presence of four highly conserved cysteine residues in exact alignment with the cystatins as well as other regions of sequence characteristic of the cystatins. However, unlike the cystatins, the CRES gene does not contain specific highly conserved sequence motifs thought to be necessary for cysteine proteinase inhibitory activity. Also, in contrast to the ubiquitous expression of the cystatin C gene, Northern blot analysis and in situ hybridization demonstrated that the CRES gene is very restricted in its expression. The 0.75-kilobase CRES transcript is dramatically restricted to the very proximal caput region of the epididymis with 15- to 20-fold less expression in the testis and no expression detected in any of the other 24 tissues examined. In addition, the CRES transcript disappears 2-3 weeks after castration, suggesting a dependence on androgens. However, its expression remained undetectable even after the administration of testosterone or dihydrotestosterone. Unilateral castration also resulted in the disappearance of the CRES mRNA from the castrate epididymis, but not from the intact epididymis, suggesting that testicular factors or hormones other than androgens may be involved in the regulation of CRES gene expression. Therefore, the unique sequence of the CRES gene as well as its highly restricted expression and unusual regulation by the testis suggests that it has a very specialized role in the epididymis.

Regulation and function of non-AUG-initiated proto-oncogenes.

A small, yet growing, number of cellular eukaryotic mRNAs encoding important regulatory proteins, such as c-myc and other proto-oncogenes, initiate translation from a non-AUG codon, usually in addition to initiating at a downstream AUG. The efficiency of non-AUG initiation on these natural cellular mRNAs varies considerably and appears to be governed by several features, including the codon sequence, the context surrounding the codon and the secondary structure of the transcript. In addition to factors which control the overall efficiency of c-myc non-AUG initiation, the relative efficiency of the upstream non-AUG initiation compared with the AUG initiation changes during the growth of cells. As lymphoid and fibroblast cells approach high densities in culture there is a sustained 5-10-fold induction in the synthesis of the non-AUG-initiated c-Myc 1 protein to levels comparable to or greater than the AUG-initiated c-Myc 2 protein. This increased efficiency of c-myc non-AUG initiation, due to methionine depletion of the growth medium, suggests that the scanning preinitiation complex can be regulated to enhance the recognition of a suboptimal non-AUG codon. The significance of non-AUG initiation for the growth-regulatory genes is illustrated by the different localizations of the int-2, bFGF and hck non-AUG-initiated proteins, the disruption of the c-myc and lyl-1 non-AUG initiation in tumor-derived cell lines, and the distinct biological function of the non-AUG-initiated forms of bFGF.

Methionine deprivation regulates the translation of functionally-distinct c-Myc proteins.

Numerous studies have demonstrated a critical role for the c-myc gene in the control of cellular growth. Alterations of the c-myc gene have been found associated with many different types of tumors in several species, including humans. The increased synthesis of one of the major forms of c-Myc protein, c-Myc 1, upon methionine deprivation provides a link between the regulation of oncogenes and the nutritional status of the cell. While deregulation or overexpression of the other major form, c-Myc 2, has been shown to cause tumorigenesis, the synthesis of c-Myc 1 protein is lost in many tumors. This suggests that the c-Myc 1 protein is necessary to keep the c-Myc 2 protein "in check" and prevent certain cells from becoming tumorigenic. Indeed, we have shown that overproduction of c-Myc 1 can inhibit cell growth. We have also shown that c-Myc 1 and 2 proteins have a differential molecular function in the regulation of transcription through a new binding site of Myc/Max heterodimers. We have also recently identified new translational forms of the c-Myc protein which we term delta-c-Myc. These proteins arise from translational initiation at downstream start sites which yield N-terminally-truncated c-Myc proteins. Since these proteins lack a significant portion of the transactivation domain of c-Myc, they behave as dominant-negative inhibitors of the full-length c-Myc 1 and 2 proteins. The synthesis of delta-c-Myc proteins is also regulated during cell growth and is repressed by methionine deprivation. Therefore, the synthesis of c-Myc 1 and delta-c-Myc proteins are reciprocally regulated by methionine availability. We have also found some tumor cell lines which synthesize high levels of the delta-c-Myc proteins. Taken together, our data suggest that c-Myc function is dependent on the levels of these different translational forms of c-Myc protein which are regulated by the nutritional status of the cell during growth. Numerous reports have demonstrated a fundamental and diverse role for the myc gene in cellular events, including proliferation, differentiation and apoptosis (Cole 1986; Spencer and Groudine 1991; Askew et al. 1991; Evan et al. 1992). This is dramatically illustrated by the frequent occurrence of a variety of tumors in many species having alterations of myc genes and the transduction of c-myc sequences by retroviruses (Spencer and Groudine 1991).4+ Eisenman 1990).(ABSTRACT TRUNCATED AT 400 WORDS)

Nucleophosmin is essential for c-Myc nucleolar localization and c-Myc-mediated rDNA transcription.

The transcription factor c-Myc has a critical role in cell proliferation and growth. The control of ribosome biogenesis by c-Myc through the regulation of transcription mediated by all three RNA polymerases is essential for c-Myc-driven proliferation. Specifically, in the nucleolus, c-Myc has been shown to be recruited to ribosomal DNA and activate RNA polymerase (pol) I-mediated transcription of ribosomal RNA (rRNA) genes. In addition, c-Myc accumulates in nucleoli upon inhibition of the proteasome, suggesting nucleolar localization also has a role in c-Myc proteolysis. Nucleophosmin (NPM), a predominantly nucleolar protein, is also critical in ribosome biogenesis and, like c-Myc, is found overexpressed in many types of tumors. Previously, we demonstrated that NPM directly interacts with c-Myc and controls c-Myc-induced hyperproliferation and transformation. Here, we show that NPM is necessary for the localization of c-Myc protein to nucleoli, whereas c-Myc nucleolar localization is independent of p53, Mdm2 and ARF. Conversely, high transient NPM expression enhances c-Myc nucleolar localization, leading to increased c-Myc proteolysis. In addition, NPM is necessary for the ability of c-Myc to induce rRNA synthesis in the nucleolus, and constitutive NPM overexpression stimulates c-Myc-mediated rRNA synthesis. Taken together, these results demonstrate an essential role for NPM in c-Myc nucleolar localization and c-Myc-mediated rDNA transcription.