Csx/Nkx2.5 is a vertebrate homeobox gene with a sequence homology to the Drosophila tinman, which is required for the dorsal mesoderm specification. Recently, heterozygous mutations of this gene were found to cause human congenital heart disease (Schott, J.-J., Benson, D. W., Basson, C. T., Pease, W., Silberbach, G. M., Moak, J. P., Maron, B. J., Seidman, C. E. and Seidman, J. G. (1998) Science 281, 108-111). To investigate the functions of Csx/Nkx2.5 in cardiac and extracardiac development in the vertebrate, we have generated and analyzed mutant mice completely null for Csx/Nkx2.5. Homozygous null embryos showed arrest of cardiac development after looping and poor development of blood vessels. Moreover, there were severe defects in vascular formation and hematopoiesis in the mutant yolk sac. Interestingly, TUNEL staining and PCNA staining showed neither enhanced apoptosis nor reduced cell proliferation in the mutant myocardium. In situ hybridization studies demonstrated that, among 20 candidate genes examined, expression of ANF, BNP, MLC2V, N-myc, MEF2C, HAND1 and Msx2 was disturbed in the mutant heart. Moreover, in the heart of adult chimeric mice generated from Csx/Nkx2.5 null ES cells, there were almost no ES cell-derived cardiac myocytes, while there were substantial contributions of Csx /Nkx2.5-deficient cells in other organs. Whole-mount &bgr;-gal staining of chimeric embryos showed that more than 20% contribution of Csx/Nkx2. 5-deficient cells in the heart arrested cardiac development. These results indicate that (1) the complete null mutation of Csx/Nkx2.5 did not abolish initial heart looping, (2) there was no enhanced apoptosis or defective cell cycle entry in Csx/Nkx2.5 null cardiac myocytes, (3) Csx/Nkx2.5 regulates expression of several essential transcription factors in the developing heart, (4) Csx/Nkx2.5 is required for later differentiation of cardiac myocytes, (5) Csx/Nkx2. 5 null cells exert dominant interfering effects on cardiac development, and (6) there were severe defects in yolk sac angiogenesis and hematopoiesis in the Csx/Nkx2.5 null embryos.

It has been shown recently in China that arsenic trioxide (As2O3) is a very effective treatment for acute promyelocytic leukemia (APL). APL patients resistant to all-trans retinoic acid (ATRA) and conventional chemotherapy can still respond to AS2O3. In this study, we addressed the possible cellular and molecular mechanisms of this treatment by using NB4 cells as a model. The results show that: (1) As2O3 triggers relatively specific NB4 cell apoptosis at micromolar concentration, as proved by morphology, histogramic related nuclear DNA contents, and DNA gel eletrophoresis. (2) As2O3 does not influence bax, bcl-x, c-myc, and p53 gene expression, but downregulates bcl-2 gene expression at both mRNA and protein levels. (3) As2O3 induces a significant modulation of the PML staining pattern in NB4 cells and HL-60 cells. The micropunctates characteristic of PML-RAR alpha in NB4 cells dissappear after treatment with As2O3, whereas a diffuse PML staining occurs in the perinuclear cytoplasmic region. In addition, a low percentage of untreated NB4 cells exhibits an accumulation of PML positive particles in a compartment of cytoplasm. The percentage of these cells can be significantly increased after As2O3 treatment. A similar PML staining pattern is observed in apoptotic cells. (4) ATRA pretreatment does not influence As2O3-induced apoptosis. These results suggest that induction of cell apoptosis can be one of the mechanisms of the therapeutic effect of As2O3. Moreover, this apoptosis induction occurs independently of the retinoid pathway and may be mediated, at least partly, through the modulation of bcl-2, as well as PML-RAR alpha and/ or PML proteins.

Human colonic epithelial cell renewal, proliferation, and differentiation are stringently controlled by numerous regulatory pathways. To identify genetic programs of human colonic epithelial cell differentiation in vivo as well as candidate marker genes that define colonic epithelial stem/progenitor cells and the stem cell niche, we applied gene expression analysis of normal human colon tops and basal crypts by using expression microarrays with 30,000 genes. Nine hundred and sixty-nine cDNA clones were found to be differentially expressed between human colon crypts and tops. Pathway analysis revealed the differential expression of genes involved in cell cycle maintenance and apoptosis, as well as genes in bone morphogenetic protein (BMP), Notch, Wnt, EPH, and MYC signaling pathways. BMP antagonists gremlin 1, gremlin 2, and chordin-like 1 were found to be expressed by colon crypts. In situ hybridization and RT-PCR confirmed that these BMP antagonists are expressed by intestinal cryptal myofibroblasts and smooth muscle cells at the colon crypt. In vitro analysis demonstrated that gremlin 1 partially inhibits Caco-2 cell differentiation upon confluence and activates Wnt signaling in normal rat intestinal epithelial cells. Collectively, the expression data set provides a comprehensive picture of human colonic epithelial cell differentiation. Our study also suggests that BMP antagonists are candidate signaling components that make up the intestinal epithelial stem cell niche.

We have cloned a 2.0-kb EcoRI fragment of human genomic DNA (NB-19-21) which has homology to the v-myc oncogene but is distinct from the classical c-myc gene. This sequence is amplified from 25- to 700-fold in eight of nine tested human neuroblastoma cell lines which contain either homogeneously staining regions or double minutes (HSRs or DMs), the caryological manifestations of amplified genes. In the remaining line, the c-myc proto-oncogene is amplified approximately 30-fold. NB-19-21 hybridizes to a 3.2-kb cytoplasmic, poly(A)+ RNA species that is abundant only in lines in which the sequence is amplified. We propose that the gene encoding the NB-19-21-related RNA species may represent a new oncogene, which we call N-myc. NB-19-21 derives from chromosome 2; but in the five HSR-containing lines that have amplified this sequence, none has HSRs on chromosome 2. NB-19-21 is associated with DMs in a DM-containing line. A second, randomly cloned, amplified DNA segment from the HSR of one of the neuroblastoma lines is amplified in a subset of the lines in which NB-19-21 is amplified. In addition, this probe identifies a novel joint in the amplification unit of one line relative to that of the others. We suggest that, in the eight lines which have amplified NB-19-21, the amplification units are overlapping, but not identical, and that transposition of the common sequences may occur prior to amplification.

The sequencing of cancer genomes may enable tailoring of therapeutics to the underlying biological abnormalities driving a particular patient's tumor. However, sequencing-based strategies rely heavily on representative sampling of tumors. To understand the subclonal structure of primary breast cancer, we applied whole-genome and targeted sequencing to multiple samples from each of 50 patients' tumors (303 samples in total). The extent of subclonal diversification varied among cases and followed spatial patterns. No strict temporal order was evident, with point mutations and rearrangements affecting the most common breast cancer genes, including PIK3CA, TP53, PTEN, BRCA2 and MYC, occurring early in some tumors and late in others. In 13 out of 50 cancers, potentially targetable mutations were subclonal. Landmarks of disease progression, such as resistance to chemotherapy and the acquisition of invasive or metastatic potential, arose within detectable subclones of antecedent lesions. These findings highlight the importance of including analyses of subclonal structure and tumor evolution in clinical trials of primary breast cancer.

Comparative nucleotide sequence analysis of a rearranged c-myc gene in a murine plasmacytoma and c-myc cDNA from normal spleen reveals that chromosomal translocation in the plasmacytoma breaks the c-myc gene within the first exon or intron. In the plasmacytoma truncated c-myc RNAs initiate from newly exposed promoter sites. Nevertheless, the myc polypeptide produced in the plasmacytoma is probably the same as that from the intact c-myc gene because the exon lost by breakage and translocation is non-coding. The second and third exons of the mouse c-myc gene are substantially conserved in the v-myc gene of the avian retrovirus, MC29.

The muE3 motif within the immunoglobulin heavy-chain enhancer is required for full enhancer activity and is known to bind one, or perhaps a family, of related ubiquitous nuclear proteins. Here, we present the isolation of a cDNA that encodes an apparently novel microE3-binding protein designated TFE3. The major open reading frame of the cDNA predicts a protein of 59 kD, with a leucine zipper situated adjacent to an myc-related motif that has been proposed to assume a helix-loop-helix structure. Both of these motifs have been shown (for other proteins) to facilitate protein-protein interactions and DNA binding. Expression of the cDNA in 3T3 cells stimulates transcription from an artificial promoter consisting of four muE3 sites linked to a TATA box and also augments transcription of a reporter gene when it is linked to multiple copies of a particular heavy-chain enhancer subfragment but not when it is linked to the intact enhancer. Using GAL4 fusion proteins, we mapped a strong transcription activation domain within TFE3 that is distinct from the leucine zipper and helix-loop-helix motifs and includes a potential negative amphipathic helix. Like the other muE3-binding proteins detected in nuclear extracts, in vitro-synthesized TFE3 also binds to the USF/MLTF site found in the adenovirus major late promoter.

Myn, a novel murine approximately 18 kd basic/helix-loop-helix/"leucine zipper" (B/HLH/LZ) protein, forms a specific DNA-binding complex with the c-Myc oncoprotein through the HLH/LZ motif in both proteins. c-Myc/Myn recognizes a c-Myc-binding site (GACCACGTGGTC) with higher affinity than either protein by itself. CpG methylation of the recognition site greatly inhibits DNA binding, suggesting that DNA methylation may regulate the c-Myc/Myn complex in vivo. In 3T3 fibroblasts, Myn mRNA levels are induced several-fold by serum with delayed early kinetics, suggesting regulation by immediate-early gene products. Coexpression of Myn in a myc/ras rat embryo fibroblast focus formation assay specifically augmented c-myc transforming activity. We suggest that interaction of Myn with c-Myc stabilizes sequence-specific DNA binding in vivo.

Plants must effectively defend against biotic and abiotic stresses to survive in nature. However, this defense is costly and is often accompanied by significant growth inhibition. How plants coordinate the fluctuating growth-defense dynamics is not well understood and remains a fundamental question. Jasmonate (JA) and gibberellic acid (GA) are important plant hormones that mediate defense and growth, respectively. Binding of bioactive JA or GA ligands to cognate receptors leads to proteasome-dependent degradation of specific transcriptional repressors (the JAZ or DELLA family of proteins), which, at the resting state, represses cognate transcription factors involved in defense (e.g., MYCs) or growth [e.g. phytochrome interacting factors (PIFs)]. In this study, we found that the coi1 JA receptor mutants of rice (a domesticated monocot crop) and Arabidopsis (a model dicot plant) both exhibit hallmark phenotypes of GA-hypersensitive mutants. JA delays GA-mediated DELLA protein degradation, and the della mutant is less sensitive to JA for growth inhibition. Overexpression of a selected group of JAZ repressors in Arabidopsis plants partially phenocopies GA-associated phenotypes of the coi1 mutant, and JAZ9 inhibits RGA (a DELLA protein) interaction with transcription factor PIF3. Importantly, the pif quadruple (pifq) mutant no longer responds to JA-induced growth inhibition, and overexpression of PIF3 could partially overcome JA-induced growth inhibition. Thus, a molecular cascade involving the COI1-JAZ-DELLA-PIF signaling module, by which angiosperm plants prioritize JA-mediated defense over growth, has been elucidated.

The c-Jun and c-Myc oncogenic transcription factors are highly unstable proteins due to polyubiquitination. Similar to c-Myc, we report here that phosphorylation of c-Jun by GSK3 creates a high-affinity binding site for the E3 ligase Fbw7, which targets c-Jun for polyubiquitination and proteasomal degradation. In keeping with this, we found that c-Jun levels were inversely related to GSK3 activity in mammalian cells that had entered the cell cycle. Importantly, phosphorylation of c-Jun by GSK3 requires a priming phosphorylation event at Ser-243. Ser-243 is mutated to phenylalanine in v-Jun and allows it to escape recognition by Fbw7. These findings explain the enhanced stability and oncogenicity of v-Jun relative to its cellular counterpart and reveal that GSK3 and Fbw7 coordinately regulate c-Jun and c-Myc.

The product of the MYC oncogene is widely deregulated in cancer and functions as a regulator of gene transcription. Despite an extensive profile of regulated genes, the transcriptional targets of c-Myc essential for transformation remain unclear. In this study, we show that c-Myc significantly induces the expression of the H19 noncoding RNA in diverse cell types, including breast epithelial, glioblastoma, and fibroblast cells. c-Myc binds to evolutionarily conserved E-boxes near the imprinting control region to facilitate histone acetylation and transcriptional initiation of the H19 promoter. In addition, c-Myc down-regulates the expression of insulin-like growth factor 2 (IGF2), the reciprocally imprinted gene at the H19/IGF2 locus. We show that c-Myc regulates these two genes independently and does not affect H19 imprinting. Indeed, allele-specific chromatin immunoprecipitation and expression analyses indicate that c-Myc binds and drives the expression of only the maternal H19 allele. The role of H19 in transformation is addressed using a knockdown approach and shows that down-regulation of H19 significantly decreases breast and lung cancer cell clonogenicity and anchorage-independent growth. In addition, c-Myc and H19 expression shows strong association in primary breast and lung carcinomas. This work indicates that c-Myc induction of the H19 gene product holds an important role in transformation.

To directly assess c-myc function in cellular proliferation, differentiation, and embryogenesis, we have used homologous recombination in embryonic stem cells to generate both heterozygous and homozygous c-myc mutant ES cell lines. The mutation is a null allele at the protein level. Mouse chimeras from seven heterozygous cell lines transmitted the mutant allele to their offspring. The analysis of embryos from two clones has shown that the mutation is lethal in homozygotes between 9.5 and 10.5 days of gestation. The embryos are generally smaller and retarded in development compared with their littermates. Pathologic abnormalities include the heart, pericardium, neural tube, and delay or failure in turning of the embryo. Heterozygous females have reduced fertility owing to embryonic resorption before 9.5 days of gestation in 14% of implanted embryos. c-Myc protein is necessary for embryonic survival beyond 10.5 days of gestation; however, it appears to be dispensable for cell division both in ES cell lines and in the embryo before that time.

Although the p53 tumor suppressor acts in a plethora of processes that influence cellular proliferation and survival, it remains unclear which p53 functions are essential for tumor suppression and, as a consequence, are selected against during tumor development. Using a mouse model harboring primary, genetically modified myc-driven lymphomas, we show that disruption of apoptosis downstream of p53 by Bcl2 or a dominant-negative caspase 9 confers-like p53 loss-a selective advantage, and completely alleviates pressure to inactivate p53 during lymphomagenesis. Despite their p53-null-like aggressive phenotype, apoptosis-defective lymphomas that retain intact p53 genes do not display the checkpoint defects and gross aneuploidy that are characteristic of p53 mutant tumors. Therefore, apoptosis is the only p53 function selected against during lymphoma development, whereas defective cell-cycle checkpoints and aneuploidy are mere byproducts of p53 loss.

Multipotent neural cell lines were generated via retrovirus-mediated v-myc transfer into murine cerebellar progenitor cells. When transplanted back into the cerebellum of newborn mice, these cells integrated into the cerebellum in a nontumorigenic, cytoarchitecturally appropriate manner. Cells from the same clonal line differentiated into neurons or glia in a manner appropriate to their site of engraftment. Engrafted cells, identified by lacZ expression and PCR-mediated detection of a unique sequence arrangement, could be identified in animals up to 22 months postengraftment. Electron microscopic and immunohistochemical analysis demonstrated that some engrafted cells were similar to host neurons and glia. Some transplant-derived neurons received appropriate synapses and formed normal intercellular contacts. These data indicate that generating immortalized cell lines for repair of, or transport of genes into, the CNS may be feasible. Such lines may also provide a model for commitment and differentiation of cerebellar progenitor cells.

Somatic reprogramming induced by defined transcription factors is a low-efficiency process that is enhanced by p53 deficiency. So far, p21 is the only p53 target shown to contribute to p53 repression of iPSC (induced pluripotent stem cell) generation, indicating that additional p53 targets may regulate this process. Here, we demonstrate that miR-34 microRNAs (miRNAs), particularly miR-34a, exhibit p53-dependent induction during reprogramming. Mir34a deficiency in mice significantly increased reprogramming efficiency and kinetics, with miR-34a and p21 cooperatively regulating somatic reprogramming downstream of p53. Unlike p53 deficiency, which enhances reprogramming at the expense of iPSC pluripotency, genetic ablation of Mir34a promoted iPSC generation without compromising self-renewal or differentiation. Suppression of reprogramming by miR-34a was due, at least in part, to repression of pluripotency genes, including Nanog, Sox2 and Mycn (also known as N-Myc). This post-transcriptional gene repression by miR-34a also regulated iPSC differentiation kinetics. miR-34b and c similarly repressed reprogramming; and all three miR-34 miRNAs acted cooperatively in this process. Taken together, our findings identified miR-34 miRNAs as p53 targets that play an essential role in restraining somatic reprogramming.

In the mouse neocortex, neural progenitor cells generate both differentiating neurons and daughter cells that maintain progenitor fate. Here, we show that the TRIM-NHL protein TRIM32 regulates protein degradation and microRNA activity to control the balance between those two daughter cell types. In both horizontally and vertically dividing progenitors, TRIM32 becomes polarized in mitosis and is concentrated in one of the two daughter cells. TRIM32 overexpression induces neuronal differentiation while inhibition of TRIM32 causes both daughter cells to retain progenitor cell fate. TRIM32 ubiquitinates and degrades the transcription factor c-Myc but also binds Argonaute-1 and thereby increases the activity of specific microRNAs. We show that Let-7 is one of the TRIM32 targets and is required and sufficient for neuronal differentiation. TRIM32 is the mouse ortholog of Drosophila Brat and Mei-P26 and might be part of a protein family that regulates the balance between differentiation and proliferation in stem cell lineages.

The APC tumor suppressor controls the stability and nuclear export of beta-catenin (beta-cat), a transcriptional coactivator of LEF-1/TCF HMG proteins in the Wnt/Wg signaling pathway. We show here that beta-cat and APC have opposing actions at Wnt target genes in vivo. The beta-cat C-terminal activation domain associates with TRRAP/TIP60 and mixed-lineage-leukemia (MLL1/MLL2) SET1-type chromatin-modifying complexes in vitro, and we show that beta-cat promotes H3K4 trimethylation at the c-Myc gene in vivo. H3K4 trimethylation in vivo requires prior ubiquitination of H2B, and we find that ubiquitin is necessary for transcription initiation on chromatin but not nonchromatin templates in vitro. Chromatin immunoprecipitation experiments reveal that beta-cat recruits Pygopus, Bcl-9/Legless, and MLL/SET1-type complexes to the c-Myc enhancer together with the negative Wnt regulators, APC, and betaTrCP. Interestingly, APC-mediated repression of c-Myc transcription in HT29-APC colorectal cancer cells is initiated by the transient binding of APC, betaTrCP, and the CtBP corepressor to the c-Myc enhancer, followed by stable binding of the TLE-1 and HDAC1 corepressors. Moreover, nuclear CtBP physically associates with full-length APC, but not with mutant SW480 or HT29 APC proteins. We conclude that, in addition to regulating the stability of beta-cat, APC facilitates CtBP-mediated repression of Wnt target genes in normal, but not in colorectal cancer cells.

Jasmonates (JA) are important regulators of plant defense responses that activate expression of many wound-induced genes including the tomato proteinase inhibitor II (pin2) and leucine aminopeptidase (LAP) genes. Elements required for JA induction of the LAP gene are all present in the -317 to -78 proximal promoter region. Using yeast one-hybrid screening, we have identified the bHLH-leu zipper JAMYCMYCMYCMYCMYC overexpression enhanced JA-induced expression of these defense genes in potato, but did not result in constitutive transcript accumulation. Using footprinting assays, an additional protected element was identified, located directly adjacent to the T/G-box motif. Mutation of this element abolishes JA response, showing that recognition of this duplicated element is also required for gene expression. Knockout mutants in the AtMYCMYC/AtMYCMYC-based regulatory system conserved in dicotyledonous plants with a key role in JA-induced defense gene activation.

Despite recent emerging evidence suggesting that cancer stem cells subsist in a variety of tumors, it is not yet fully elucidated whether postnatal stem cells are directly involved in tumorigenesis. We used murine bone marrow-derived mesenchymal stem cells (BMMSCs) as a model to test a hypothesis that tumorigenesis may originate from spontaneous mutation of stem cells. In this study, we demonstrated that murine BMMSCs, after numerous passages, obtained unlimited population doublings and proceeded to a malignant transformation state, resulting in fibrosarcoma formation in vivo. Transformed BMMSCs colonized to multiple organs when delivered systemically through the tail vein. Fibrosarcoma cells formed by transformed BMMSCs contained cancer progenitors, which were capable of generating colony clusters in vitro and fibrosarcoma in vivo by the second administration. The mechanism by which BMMSCs transformed to malignant cells was associated with accumulated chromosomal abnormalities, gradual elevation in telomerase activity, and increased c-myc expression. Moreover, BMMSCs and their transformed counterpart, fibrosarcoma-forming cells, demonstrated different sensitivity to anti-cancer drugs. BMMSCs/fibrosarcoma transformation system may provide an ideal system to elucidate the mechanism of how stem cells become cancer cells and to screen anti-sarcoma drugs.

Overexpression of myc protooncogenes has been implicated in the genesis of many human tumors. Myc proteins seem to regulate diverse biological processes, but their role in tumorigenesis remains enigmatic. Here we use Drosophila imaginal discs to mimic situations in which cells with unequal levels of Myc protein are apposed and show that this invariably elicits a win/lose situation reminiscent of cell competition; cells with lower levels of dMyc are eliminated by apoptosis whereas cells with higher levels of dMyc over-proliferate. We find that this competitive behavior correlates with, and can be corrected by, the activation of the BMP/Dpp survival signaling pathway. Hence the heritable increase in dMyc levels causes cells to behave as "super-competitors" and reveals a novel mode of clonal expansion that causes, but also relies on, the killing of surrounding cells.

Before quiescent cells can respond to mitogens and progress through the G1 phase of cell growth, new messenger RNA synthesis is required. The G1 phase seems to be a critical point of control in the cell cycle, where normal cells deprived of growth factors halt cycling while transformed cells do not, suggesting that regulatory genes, uncontrolled in the neoplastic phenotype, are expressed during the G0 to G1 transition. Some of these may code for nuclear proteins that participate in the transactivation of genes required for the progression through G1. The observed changes in expression of the proto-oncogenes c-fos and c-myc, following stimulation of fibroblasts with growth factors, support this notion as recent evidence suggests that c-FOS and c-MYC proteins can function as transactivating factors. Moreover, the rapid induction of several genes in fibroblasts coding for putative transacting factors during the G0 to G1 transition has been recently reported. Here we present the nucleotide sequence of a mouse cDNA clone coding for a 334 residue protein which shows 80% similarity with v-JUN and more than 98% similarity with the human c-JUN sequence. We have demonstrated that in quiescent fibroblasts c-jun transcription is rapidly induced during the G0 to G1 transition.

Transforming growth factor-beta (TGF-beta) potently inhibits cell cycle progression at the G1 phase. Smad3 has a key function in mediating the TGF-beta growth-inhibitory response. Here we show that Smad3 is a major physiological substrate of the G1 cyclin-dependent kinases CDK4 and CDK2. Except for the retinoblastoma protein family, Smad3 is the only CDK4 substrate demonstrated so far. We have mapped CDK4 and CDK2 phosphorylation sites to Thr 8, Thr 178 and Ser 212 in Smad3. Mutation of the CDK phosphorylation sites increases Smad3 transcriptional activity, leading to higher expression of the CDK inhibitor p15. Mutation of the CDK phosphorylation sites of Smad3 also increases its ability to downregulate the expression of c-myc. Using Smad3(-/-) mouse embryonic fibroblasts and other epithelial cell lines, we further show that Smad3 inhibits cell cycle progression from G1 to S phase and that mutation of the CDK phosphorylation sites in Smad3 increases this ability. Taken together, these findings indicate that CDK phosphorylation of Smad3 inhibits its transcriptional activity and antiproliferative function. Because cancer cells often contain high levels of CDK activity, diminishing Smad3 activity by CDK phosphorylation may contribute to tumorigenesis and TGF-beta resistance in cancers.

The proliferation of mammalian cells is under strict control, and the cyclin-dependent-kinase inhibitory protein p27Kip1 is an essential participant in this regulation both in vitro and in vivo. Although mutations in p27Kip1 are rarely found in human tumours, reduced expression of the protein correlates well with poor survival among patients with breast or colorectal carcinomas, suggesting that disruption of the p27Kip1 regulatory mechanisms contributes to neoplasia. The abundance of p27Kip1 in the cell is determined either at or after translation, for example as a result of phosphorylation by cyclinE/Cdk2 complexes, degradation by the ubiquitin/proteasome pathway, sequestration by unknown Myc-inducible proteins, binding to cyclinD/Cdk4 complexes, or inactivation by the viral E1A oncoprotein. We have found that a mouse 38K protein (p38) encoded by the Jab1 gene interacts specifically with p27Kip1 and show here that overexpression of p38 in mammalian cells causes the translocation of p27Kip1 from the nucleus to the cytoplasm, decreasing the amount of p27Kip1 in the cell by accelerating its degradation. Ectopic expression of p38 in mouse fibroblasts partially overcomes p27Kip1-mediated arrest in the G1 phase of the cell cycle and markedly reduces their dependence on serum. Our findings indicate that p38 functions as a negative regulator of p27Kip1 by promoting its degradation.

Smad3 is a direct mediator of transcriptional activation by the TGFbeta receptor. Its target genes in epithelial cells include cyclin-dependent kinase inhibitors that generate a cytostatic reponse. We defined how, in the same context, Smad3 can also mediate transcriptional repression of the growth-promoting gene c-myc. A complex containing Smad3, the transcription factors E2F4/5 and DP1, and the corepressor p107 preexists in the cytoplasm. In response to TGFbeta, this complex moves into the nucleus and associates with Smad4, recognizing a composite Smad-E2F site on c-myc for repression. Previously known as the ultimate recipients of cdk regulatory signals, E2F4/5 and p107 act here as transducers of TGFbeta receptor signals upstream of cdk. Smad proteins therefore mediate transcriptional activation or repression depending on their associated partners.