Ras family GTPases (RFGs) are known to share many regulatory and effector proteins. How signaling and biological specificity is achieved is poorly understood. Using a proteomics approach, we have identified a complex comprised of Shoc2/Sur-8 and the catalytic subunit of protein phosphatase 1 (PP1c) as a highly specific M-Ras effector. M-Ras targets Shoc2-PP1c to stimulate Raf activity by dephosphorylating the S259 inhibitory site of Raf proteins bound to other molecules of M-Ras or Ras. Therefore, distinct RFGs, through independent effectors, can regulate different steps in the activation of Raf kinases. Shoc2 function is essential for activation of the MAPK pathway by growth factors. Furthermore, in tumor cells with Ras gene mutations, inhibition of Shoc2 expression inhibits MAPK, but not PI3K activity. We propose that the Shoc2-PP1c holoenzyme provides an attractive therapeutic target for inhibition of the MAPK pathway in cancer.

Binding of Src family kinases to membrane-associated polyoma virus middle T-antigen (PyMT) can result in the phosphorylation of PyMT tyrosine 250, which serves as a docking site for the binding of Shc and subsequent activation of the Raf-MEK-ERK (MAP) kinase cascade. In a screen for PyMT variants that could not activate the ARF tumor suppressor, we isolated a cytoplasmic nontransforming mutant (MTA) that encoded a C-terminal truncated form of the PyMT protein. Surprisingly, MTA was able to strongly activate the MAP kinase pathway in the absence of Src family kinase and Shc binding. Interestingly, the polyoma small T-antigen (PyST), which shares with MTA both partial amino acid sequence homology and cellular location, also activates the MAP kinase cascade. Activation of the MAP kinase cascade by both MTA and PyST has been demonstrated to be PP2A-dependent. Neither MTA nor PyST activate the phosphorylation of AKT. The SV40 small T-antigen, which is similar to PyST in containing a J domain and in binding to the PP2A AC dimer, does not activate the MAP kinase cascade, but does stimulate phosphorylation of AKT in a PP2A-dependent manner. These findings highlight a novel role of PP2A in stimulating the MAP kinase cascade and indicate that the similar polyoma and SV40 small T-antigens influence PP2A to activate discrete cellular signaling pathways involved in growth control.

We have analyzed the transcriptional start sites of the rat ARF gene and the amino acid sequence of the rat ARF tumor suppressor protein. The 5' end of the ratARF gene is similar to that of a number of cellular housekeeping genes in that it is CG-rich and does not contain an upstream TATA box motif to define a precise transcriptional start site. The transcription of the rat ARF gene is initiated at multiple start sites with one major start site accounting for 41% of transcription. The rat ARF protein contains two methionine ATG codons at its amino terminus separated by 10 amino acids. The translation of the major endogenous ARF protein species is initiated from the upstream methionine ATG codon. The upstream methionine ATG codon is predominantly used, despite the fact that it is both very close to the major transcriptional start site (6 bases downstream) and is in a less favorable nucleic acid sequence context than the downstream ATG, relative to the ideal sequence postulated for efficient initiation of translation. The downstream, inefficient rat ARF ATG is equivalent to the major mouse ARF ATG start codon. Both of these closely spaced ATGs can be utilized as a translational start codon to produce a nucleolar-localized ARF protein which can induce a p53-dependent inhibition of cell division and transcriptional activation of p53 in the absence of p53 stabilization.

We have isolated spontaneous mutants of polyoma virus middle T-antigen (PyMT) that do not activate the ARF-p53 pathway based on their inability to block REF52 cell division. The REF52 cells containing these mutants have a flat untransformed morphological phenotype and do not express the ARF protein. The PyMT mutations in the different cell isolates so far analysed occur at a mutational hotspot in the PyMT sequence between nucleotides 1241 and 1249, which contains nine consecutive cytosines. In one set of mutants a single cytosine was deleted, while in another mutant set an additional cytosine was inserted. Both these mutations result in frameshifts, generating altered PyMT proteins containing amino-acid sequences derived from each of the two other alternative reading frames of the polyoma virus early region. Both types of mutations result in the loss of the C-terminal PyMT region containing the membrane-binding hydrophobic region and result is mislocalization of the PyMT mutant proteins. Revertant wild-type PyMT (containing nine cytosines) was easily detected in transformants generated after infection of REF52 cells expressing high amounts of dominant negative p53 with retroviruses containing either mutation. We demonstrate that wild-type PyMT revertants are derived from mutations in the hotspot sequence of the integrated mutant PyMT sequences.

The small DNA tumor viruses encode proteins that subvert many of the pivotal growth regulatory pathways within the cell to facilitate their own replication. The cell responds to viral infection/proteins by activating the p53 tumor suppressor pathway. Activation of p53 could impair a productive viral infection at many levels, including the inhibition of viral DNA replication and/or the premature death of infected cells. Therefore, DNA viruses encode proteins that inactivate the p53 tumor suppressor pathway. Understanding how DNA viral proteins activate/ inactivate the p53 pathway has provided invaluable insights into tumorigenesis. Recent studies with polyoma virus have identified a viral protein (PyST) that inhibits ARF-mediated activation of p53, and revealed a novel role for PP2A in the regulation of the ARF-p53 tumor suppressor pathway.

Activation of the ARF–p53 tumor suppressor pathway is one of the cell’s major defense mechanisms against cancer induced by oncogenes. The ARF–p53 pathway is dysfunctional in a high proportion of human cancers. The regulation of the ARF–p53 signaling pathway has not yet been well characterized. In this study polyoma virus (Py) is used as a tool to better define the ARF–p53 signaling pathway. Py middle T-antigen (PyMT) induces ARF, which consequently up-regulates p53. We show that Py small T-antigen (PyST) blocks ARF-mediated activation of p53. This inhibition requires the small T-antigen PP2A-interacting domain. Our results reveal a previously unrecognized role of PP2A in the modulation of the ARF–p53 tumor suppressor pathway.

Activation of the ARF-p53 tumor suppressor pathway is one of the cell's major defense mechanisms against cancer induced by oncogenes. The ARF-p53 pathway is dysfunctional in a high proportion of human cancers. The regulation of the ARF-p53 signaling pathway has not yet been well characterized. In this study polyoma virus (Py) is used as a tool to better define the ARF-p53 signaling pathway. Py middle T-antigen (PyMT) induces ARF, which consequently up-regulates p53. We show that Py small T-antigen (PyST) blocks ARF-mediated activation of p53. This inhibition requires the small T-antigen PP2A-interacting domain. Our results reveal a previously unrecognized role of PP2A in the modulation of the ARF-p53 tumor suppressor pathway.

The Ink4a/Arf locus encodes two distinct proteins, both of which may contribute to senescence and tumor suppression. We find that human diploid fibroblasts (HDFs) that are specifically deficient for p16INK4a achieve anchorage independence when transduced with retroviruses encoding telomerase (hTERT) and either Ras or Myc. Significantly, Ras and Myc together enable the cells to form tumors in nude mice but at a frequency that suggests additional genetic changes. All five tumors analyzed expressed high levels of Ras and retained functional p53, although two showed downregulation of Arf. Cytogenetic analyses identified clonal chromosomal alterations that may have contributed to tumorigenesis, but the tumor cells were essentially diploid.

The INK4a/ARF tumor suppressor locus is implicated in the senescence-like growth arrest provoked by oncogenic Ras in primary cells. INK4a and ARF are distinct proteins encoded by transcripts in which a shared exon is decoded in alternative reading frames. Here we analyze dermal fibroblasts (designated Q34) from an individual carrying independent missense mutations in each copy of the common exon. Both mutations alter the amino acid sequence of INK4a and functionally impair the protein, although they do so to different degrees. Only one of the mutations affects the sequence of ARF, causing an apparently innocuous change near its carboxy terminus. Unlike normal human fibroblasts, Q34 cells are not permanently arrested by Ras or its downstream effectors Ets1 and Ets2. Moreover, ectopic Ras enables the cells to grow as anchorage-independent colonies, and in relatively young Q34 cells anchorage independence can be achieved without addition of telomerase or perturbation of the p53 pathway. Whereas ARF plays the principal role in Ras-induced arrest of mouse fibroblasts, our data imply that INK4a assumes this role in human fibroblasts.

The INK4A/ARF/INK4B locus, conserved in mammals, encodes three polypeptides that regulate cell proliferation via the pRb and p53 tumour suppressor pathways. The locus is mutated in many cancers. The related, tandemly-linked INK4A and INK4B genes encode the p16INK4A and p15INK4B members of the INK4 family of cyclin-dependent kinase inhibitors which block phosphorylation of pRb, whereas the third product, ARF, derived from an alternative reading frame of INK4A, regulates p53 activity. We assessed the status of this unusual locus in the puffer fish, Fugu rubripes, and identified two INK4 genes using degenerate PCR and hybridization analyses. Sequence conservation and conservation of synteny between human and Fugu predict one gene to be an INK4A or INK4B homologue and the other an INK4D homologue. Analysis of the Fugu INK4A/B gene and the surrounding 40-kb of genomic DNA did not reveal the presence of any ARF-encoding potential or another related INK4 gene. We conclude that the gene duplication event that generated adjacent INK4A and INK4B genes and the association of ARF with the ancestral INK4A gene occurred after the divergence of the lineage leading to mammals from fish. Thus, unlike mammals, the fish p53 and pRb tumour suppressor pathways are not regulated by a single locus.

Polyoma virus (Py) differs from other small DNA tumor viruses in not encoding a protein that inactivates p53. The complete Py early region encoding the large T-antigen (PyLT), middle T-antigen (PyMT) and small T-antigen (PyST) will transform primary rodent cells and REF52 cells, but PyMT, the main Py oncogene, by itself will only transform these cells when p53 or ARF is inactivated. We have related Py oncogene cooperation with the effects of the Py T-antigens on the ARF-p53 signaling pathway. PyMT activates an ARF-induced p53-mediated block to cell division explaining the inability of PyMT alone to generate dividing transformed cells. In contrast, in REF52 cells transformed by the whole Py early region (PyREF52), ARF is upregulated but p53 is not activated. Thus PyLT and/or PyST negates the PyMT-induced ARF-mediated block to cell division by disrupting the signaling pathway from ARF to p53. Although there is no detectable interaction or co-localization of endogenous ARF (nucleoli) and MDM2 (nucleoplasm) in PyREF52 cells, expression of transfected ectopic ARF results in an MDM2/ARF interaction and sequestration of MDM2 into the nucleoli. Sequestration of MDM2 by ARF in the nucleoli is not essential for a p53 response in REF52 cells as activation of Raf in REF52Raf-ER cells results in an ARF-induced p53-mediated cell cycle block in the absence of a detectable ARF-MDM2 interaction. Py may provide new insights into the cellular ARF-p53 signaling pathway.

Rat-1 cells are used in many studies on transformation, cell cycle, and apoptosis. Whereas UV treatment of Rat-1 cells results in apoptosis, X-ray treatment does not induce either apoptosis or a cell cycle block. X-ray treatment of Rat-1 cells results in both an increase of p53 protein and expression of the p53-inducible gene MDM2 but not the protein or mRNA of the p53-inducible p21(WAF1/CIP1) gene, which in other cells plays an important role in p53-mediated cell cycle block. The lack of p21(WAF1/CIP1) expression appears to be the result of hypermethylation of the p21(WAF1/CIP1) promoter region, as p21(WAF1/CIP1) protein expression could be induced by growth of Rat-1 cells in the presence of 5-aza-2-deoxycytidine. Furthermore, sequence analysis of bisulfite-treated DNA demonstrated extensive methylation of cytosine residues in CpG dinucleotides in a CpG-rich island in the promoter region of the p21(WAF1/CIP1) gene. Stable X-ray-induced p53-dependent p21(WAF1/CIP1) expression and cell cycle block were restored to a Rat-1 clone after transfection with a P1 artificial chromosome (PAC) DNA clone containing a rat genomic copy of the p21(WAF1/CIP1) gene. The absence of expression of the p21(WAF1/CIP1) gene may contribute to the suitability of Rat-1 cells for transformation, cell cycle, and apoptosis studies.

The human Surfeit locus contains at least six tightly clustered genes (Surf-1 to Surf-6) of which five (Surf-1 to Surf-5) have been characterised and found not to share any sequence homology. The organisation and juxtaposition of the Surfeit genes are conserved between human and mouse. The Surf-6 gene that encodes a novel nucleolar-matrix protein with nucleic-acid binding properties has been characterised in mouse. In this work, we have isolated and analysed the human Surf-6 homologue and determined its genomic organisation in the Surfeit locus. The human Surf-6 gene has five exons spread over a distance of 4.3kb and has features of a housekeeping gene being ubiquitously expressed, having its 5′ end located within a CpG rich island and lacking a canonical TATA box. The intragenic region between the 3′ end of the Surf-5 gene and the 5′ end of the Surf-6 gene is 3.2kb and contains a pseudogene of the ribosomal protein gene rpL21. The putative human Surf-6 protein is 361 amino acids long and includes motifs found in both the mouse and fish Surf-6 homologues, which may underlie the functions of Surf-6. Three amino acid polymorphisms have been detected at codons 163, 175 and 311 by SSCP analysis.

Following exposure to DNA damaging agents the p53 tumour-suppressor gene induces either a growth arrest (primarily in G1) or apoptosis. The factors governing which response a cell undertakes, however, are unclear. We find that the osteosarcoma cell line, U2OS, (wild-type for p53) is capable of undergoing either p53 dependent apoptosis or cell cycle arrest in response to distinct forms of radiation. Following exposure to UVC, the majority of U2OS cells were apoptotic within 2 days and cells continued to cycle even as viability was being lost. In contrast, after X-ray treatment, U2OS cells exhibited a cell cycle arrest. Western analysis showed that p53 protein was stabilized to a greater extent by UVC than X-ray. Treatment with X-rays induced p21WAF1/CIP1 whereas p21WAF1/CIP1 expression was specifically repressed at the post-transcriptional level after exposure to UVC. Ectopic expression of high levels of p21WAF1/CIP1, which arrested U2OS cells in G1 and G2, initially conferred considerable protection against UVC-induced apoptosis. Ultimately, however, cells underwent apoptosis indicating that a high level of p21WAF1/CIP1 delays but does not block apoptosis. Taken together, these results show that cell cycle arrest and apoptosis can occur in the same cell type in response to different forms of radiation and that the repression of p21WAF1/CIP1 after UVC may contribute to the efficient induction of apoptosis in response to this particular insult.

The recently identified novel protein SURF-6 is shown to be a component of the nucleolar matrix. Immunofluorescence analysis demonstrated that SURF-6 was localized in residual nucleoli of in situ nuclear matrix preparations of mouse fibroblast cells (NIH 3T3), which were depleted of soluble and chromatin related proteins. Immunoblot analysis of biochemical nucleolar subtractions confirmed that SURF-6 was present in the nucleolar matrix fraction, and was absent from the fractions of soluble proteins released by DNase or RNase. The capacity of SURF-6 to bind nucleic acids was investigated in vitro. Both endogenous SURF-6 from nuclear extracts and recombinant SURF-6 exhibited a strong binding capacity for nucleic acids. It was shown that SURF-6 bound to both DNA and RNA, however, it showed stronger binding to RNA. The presence and nuclear distribution of SURF-6 during the cell cycle was explored by immunofluorescence analysis. It was shown that SURF-6 was always found in the nucleolus regardless of the phase of the cell cycle suggesting that it is a structural protein constitutively present in nucleolar substructures. The colocalization of SURF-6 with the major nucleolar proteins B23 and fibrillarin, which are known to be involved in the processing of ribosomal RNA (rRNA), was examined both in interphase and mitosis by double immunolabeling of cells. SURF-6 was found to be largely coincident with both proteins in interphase and it was distributed in the same cellular locations, namely the perichromosomal layer, the cytoplasm and prenucleolar bodies, in mitosis. However, colocalization of SURF-6 with fibrillarin and B23 was only partial in interphase, and the dynamics of its localization was not completely the same as those of either fibrillarin or B23 during mitosis. Taken together, these results indicate that SURF-6 is a novel nucleolar matrix component and imply that SURF-6 might support nucleolar matrix structure and function(s) via its association with nucleic acids. We propose that SURF-6 may be involved in processing of rRNA, based on its cytological characteristics, but at stages in ribosomal biogenesis which are different from those for fibrillarin and B23.

The organization of the human Surfeit locus containing the six sequence-unrelated housekeeping genes Surf-1 to Surf-6 (HGMW-approved symbols SURF1-SURF6) has been determined. The human surfeit locus occupies about 60 kb of DNA, and the tightly clustered gene organization and the juxtaposition of the human genes are similar to the mouse and chicken surfeit loci with the 5' end of each gene associated with a CpG-rich island. Whereas in the mouse the Surf-2 and Surf-4 genes overlap at their 3' ends, the human Surf-2 and Surf-4 genes have been found to be separated by 302 bp due to a much shorter 3' untranslated region in the human Surf-2 gene. The distance between the 3' ends of the human Surf-1 and Surf-3 genes is 374 bp, and the distance between the 5' ends of the human Surf-3 and Surf-5 genes is only 112 bp. Unusually the human Surf-5 gene contains an intron in its 5' untranslated region not found in the mouse or rat Surf-5 genes. This additional intron is also found in the Surf-5 gene of both Old and New World monkeys, being generated before the divergence of human and prosimians but after the divergence of primates and rodents. A contig of 200 kb containing the human Surfeit locus has been constructed from overlapping cosmid, P1, and PAC clones. Approximately 40 kb proximal to the 3' end of the Surf-6 gene, the 5' region of the ABO glycosyltransferase gene has been detected. This allows us to determine the orientation of the Surfeit and ABO loci with respect to each other and to the telomere and centromere of human chromosome 9.

U24 and U36 are members of the box C/D-containing group of antisense snoRNAs which possess long (9-21 nucleotide) conserved stretches of sequence complementarity to 18S and 28S rRNA and act as guides for the site-specific ribose methylation of rRNA. Both U24 and two variants of U36 are encoded within introns of the human and chicken rpL7a genes. We now report that an additional U36 variant is encoded within intron 4 of the human rpL7a gene and that murine homologs of the three human U36 variants are encoded within the same adjacent introns (4, 5, and 6) of the mouse rpL7a gene. We also show that, like that of the chicken, the Fugu rubripes rpL7a gene possesses only two U36-like sequences within introns 4 and 5. Whereas the two U36 variants in chicken and Fugu possess stretches of complementarity to both 18S and 28S rRNAs, it is noted that only one mammalian variant (U36b) possesses both. Unusually, the stretch of complementarity to 18S rRNA in the mammalian U36a variants and the stretch of complementarity to 28S rRNA in the mammalian U36c variants are not present, appearing to have diverged extensively from their consensus sequence. Additionally, the mammalian U36 variants show a unique heterogeneity in their potential to form a terminal stembox structure predicted for many other box C/D-containing antisense snoRNAs. Finally, the Saccharomyces cerevisiae small nuclear RNA, snR47, is shown to be homologous to the vertebrate U36 snoRNA.