Identification and characterization of the herpesvirus saimiri oncoprotein STP-C488.

The protein encoded by herpesvirus saimiri transforming gene STP-C488 was identified and characterized. Antibodies were produced in rabbits by immunization with keyhole limpet hemocyanin-conjugated synthetic peptides specific for the predicted sequence of STP-C488. STP-C488-encoded protein was detected in recombinant Escherichia coli, transformed Rat-1 cells, transfected COS-1 cells, and in common marmoset T lymphocytes immortalized by herpesvirus saimiri strain 488. STP-C488 protein was sensitive to treatment by bacterial collagenase, consistent with the 18 uninterrupted collagenlike repeats predicted by the DNA sequence. The apparent molecular size of STP-C488 in sodium dodecyl sulfate (SDS)-polyacrylamide gels (20 to 22 kDa) was considerably larger than that predicted from the DNA sequence (9.9 kDa). Using indirect immunofluorescence tests and subcellular fractionation, STP-C488 was found to be membrane bound, primarily in perinuclear compartments. The 18 uninterrupted collagenlike repeats, sensitivity to collagenase, location in the cell, and anomalous migration through SDS-polyacrylamide gels suggest an unusual, membrane-associated, fibrous structure for this transforming herpesvirus oncoprotein.     

Herpesvirus saimiri oncogene STP-C488 encodes a phosphoprotein.

The STP-C488 open reading frame of herpesvirus saimiri encodes an oncoprotein that has transforming and tumor-inducing activities independent of the rest of the herpesvirus genome. STP-C488 protein has an unusual, membrane-associated, fibrous structure and is located primarily in perinuclear compartments. We now report that STP-C488 is phosphorylated in vivo. The phosphorylated form, which accounted for about 15% of STP-C488 in transformed cells, migrated slightly more slowly through sodium dodecyl sulfate-polyacrylamide gels than unphosphorylated STP-C488. A serine residue near the amino terminus was shown to be the site of phosphorylation. However, phosphorylation was not required for transformation of Rat-1 cells by STP-C488.     

Role of cellular tumor necrosis factor receptor-associated factors in NF-kappaB activation and lymphocyte transformation by herpesvirus Saimiri STP

The STP oncoproteins of the herpesvirus saimiri (HVS) subgroup A strain 11 and subgroup C strain 488 are now found to be stably associated with tumor necrosis factor receptor-associated factor (TRAF) 1, 2, or 3. Mutational analyses identified residues of PXQXT/S in STP-A11 as critical for TRAF association. In addition, a somewhat divergent region of STP-C488 is critical for TRAF association. Mutational analysis also revealed that STP-C488 induced NF-kappaB activation that was correlated with its ability to associate with TRAFs. The HVS STP-C488 P10-->R mutant was deficient in human T-lymphocyte transformation to interleukin-2-independent growth but showed wild-type phenotype for marmoset T-lymphocyte transformation in vitro and in vivo. The STP-C488 P10-->R mutant was also defective in Rat-1 fibroblast transformation, and fibroblast cell transformation was blocked by a TRAF2 dominant-negative mutant. These data implicate TRAFs in STP-C488-mediated transformation of human lymphocytes and rodent fibroblasts. Other factors are implicated in immortalization of common marmoset T lymphocytes and may also be critical in the transformation of human lymphocytes and rodent fibroblasts.     

STP-C, an oncoprotein of herpesvirus saimiri augments the activation of NF-kappaB through ubiquitination of TRAF6

Herpesvirus saimiri (HVS), a member of the gamma-herpesvirus family, encodes an oncoprotein called Saimiri Transforming Protein (STP) which is required for lymphoma induction in non-human primates. Previous study has shown that STP-C, an oncoprotein of HVS, activates NF-kappaB signaling pathway. However, the detailed mechanism of STP-C-mediated NF-kappaB activation has not been reported yet. We first report that STP-C interacts with TRAF6 protein in vivo and in vitro and further investigation shows that Glu(12) residue of STP-C is critical for binding to TRAF6. Introduction of ubiquitin together with STP-C augments NF-kappaB activity compared to that of STP-C expression alone. STP-C expression further induces ubiquitination of endogenous TRAF6. In addition, either a deubiquitination enzyme, CYLD or a dominant negative E2-conjugation enzyme reduced NF-kappaB activity in spite of the presence of STP-C, supporting that the interaction between STP-C and TRAF6 induces ubiquitination of TRAF6. NF-kappaB activation by STP-C through the ubiquitinated TRAF6 causes the increased production of IL-8, an inflammatory chemokine and the enhanced expression of costimulatory molecule ICAM, which might ultimately contribute cellular transformation by the exposure of HVS-infected cells with inflammatory microenvironment and chronic activation.     

Activation of cellular p21ras by myristoylation

p21ras is palmitoylated on a cysteine residue near the C-terminus. Changing Cys-186 to Ser in oncogenic forms produces a non-palmitoylated protein that fails to associate with membranes and does not transform NIH 3T3 cells. To examine whether palmitate acts in a general way to increase ras protein hydrophobicity, or is involved in more specific interactions between p21ras and membranes, we constructed genes that encode non-palmitoylated ras proteins containing myristic acid at their N-termini. Myristoylated, activated ras, without palmitate (61Leu/186Ser) exhibited both efficient membrane association and full transforming activity. Unexpectedly, we found that myristoylated forms of normal cellular ras were also potently transforming. Myristoylated c-ras retained the high GTP binding and GTPase characteristic of the cellular protein and, moreover, bound predominantly GDP in vivo. This implied that it continued to interact with GAP (GTPase-activating protein). While the membrane binding induced by myristate permitted transformation, only palmitate produced a normal (non-transforming) association of ras with membranes and must therefore regulate ras function by some unique property that myristate does not mimic. Myristoylation thus represents a novel mechanism by which the ras proto-oncogene protein can become transforming.     

STP and Tip Are Essential for Herpesvirus Saimiri Oncogenicity

Mutant forms of herpesvirus saimiri (HVS) subgroup C strain 488 with deletions in either STP-C488 or Tip were constructed. The transforming potentials of the HVS mutants were tested in cell culture and in common marmosets. Parental HVS subgroup C strain 488 immortalized common marmoset T lymphocytes in vitro to interleukin-2-independent growth, but neither of the deletion mutants produced such growth transformation. Wild-type HVS produced fatal lymphoma within 19 to 20 days of experimental infection of common marmosets, while HVS ΔSTP-C488 and HVS ΔTip were nononcogenic. Virus was repeatedly isolated from the peripheral blood of marmosets infected with mutant virus for more than 5 months. These results demonstrate that STP-C488 and Tip are not required for replication or persistence, but each is essential for transformation in cell culture and for lymphoma induction in common marmosets.     

PEA-15 is inhibited by adenovirus E1A and plays a role in ERK nuclear export and Ras-induced senescence

Oncogenic ras activates multiple signaling pathways to enforce cell proliferation in tumor cells. The ERK1/2 mitogen-activated protein kinase pathway is required for the transforming effects of ras, and its activation is often sufficient to convey mitogenic stimulation. However, in some settings oncogenic ras triggers a permanent cell cycle arrest with features of cellular senescence. How the Ras/ERK1/2 pathway activates different cellular programs is not well understood. Here we show that ERK1/2 localize predominantly in the cytoplasm during ras-induced senescence. This cytoplasmic localization seems to be dependent on an active nuclear export mechanism and can be rescued by the viral oncoprotein E1A. Consistent with this hypothesis, we showed that E1A dramatically down-regulated the expression of the ERK1/2 nuclear export factor PEA-15. Also, RNA interference against PEA-15 restored the nuclear localization of phospho-ERK1/2 in Ras-expressing primary murine embryo fibroblasts and stimulated their escape from senescence. Because senescence prevents the transforming effect of oncogenic ras, our results suggest a tumor suppressor function for PEA-15 that operates by means of controlling the localization of phospho-ERK1/2.     

Bradykinin-induced oscillations of cell membrane potential in cells expressing the Ha-ras oncogene

Products of ras genes are putative elements of growth factor signal transduction. However, the mechanism of action of these proteins in normal and malignant growth is as yet obscure. To test for functional consequences of ras oncogene expression, electrophysiological experiments were performed on NIH-3T3 fibroblasts transfected with a transforming Ha-ras MMTV-LTR construct expressing the oncogene on treatment with dexamethasone (+ras). Transfected cells in the absence of dexamethasone (-ras) and nontransfected cells in the presence of dexamethasone (oras) served as controls. In -ras and oras, bradykinin induces a single, transient hyperpolarization. In +ras, bradykinin elicits oscillations of cell membrane potential throughout the presence of the hormone by activation of calcium-sensitive K+ channels. The oscillations of cell membrane potential are abolished in the absence of extracellular calcium. As evident from fura 2 fluorescence, bradykinin leads to a transient increase of intracellular calcium both in the presence and absence of extracellular calcium. Oscillations of intracellular calcium could be observed in +ras cells, if bradykinin was applied at reduced extracellular sodium concentration possibly to impair calcium extrusion via the sodium/calcium exchange. Bradykinin induces oscillations of cell membrane potential similarly in -ras cells loaded with GTP[S], a nonhydrolyzable analogue of GTP. Thus, the altered response of ras oncogene expressing cells to bradykinin relates to the GTP binding property of the ras protein. It is concluded that in cells expressing ras oncogene but not in other fibroblasts bradykinin mimicks the effect of growth factors on the cell membrane.     

Transformation by pp60src or stimulation of cells with epidermal growth factor induces the stable association of tyrosine-phosphorylated cellular proteins with GTPase-activating protein.

GTPase-activating protein (GAP) is a cytosolic protein that stimulates the rate of hydrolysis of GTP (GTP to GDP) bound to normal p21ras, but does not catalyze the hydrolysis of GTP bound to oncogenic, activated forms of the ras protein. Transformation of cells with v-src or activated transforming variants of c-src or stimulation of cells with epidermal growth factor resulted in the stable association of GAP with two tyrosine-phosphorylated cellular proteins of 64 kDa (p64) and 190 kDa (p190). Analysis of GAP immune complexes isolated from extracts of metabolically labeled src-transformed cells and epidermal growth factor-stimulated cells indicated that tyrosine phosphorylation of p64 and p190 appeared to be coincident with the stable association of these proteins with GAP. Quantitation of the amount of p64 associated with GAP in v-src-transformed cells, however, indicated that only 15 to 25% of tyrosine-phosphorylated p64 was found in complex with GAP. Mutations within the SH2 region of pp60src that render activated pp60src defective for transformation inhibited the efficient formation of complexes between GAP and the tyrosine-phosphorylated forms of p64 and p190. From these data, we suggest that tyrosine phosphorylation and stable association of p64 with GAP is an important step in mediating cellular signaling through the p21ras-GAP pathway.     

Mutation is required to activate the p53 gene for cooperation with the ras oncogene and transformation.

Previous experiments have brought into question which amino acid sequence of the p53 oncogene product should be considered wild type and whether the normal protein is capable of cooperating with the ras oncogene to transform cells in culture. To address these questions, a series of p53 cDNA-genomic hybrid clones have been compared for the ability to cooperate with the ras oncogene in transformation assays. From these experiments, it has become clear that the amino acid alanine at position 135, in either the genomic clone or the cDNA clone, failed to produce a p53 protein that cooperated with the ras oncogene and transformed cells. Replacing alanine with valine at this position in either the genomic or the cDNA clone activated for transformation in this assay. Using restriction enzyme polymorphisms in the p53 gene, it was shown that normal mouse DNA encodes alanine at position 135 in the p53 protein. Thus, mutation is required to activate the p53 protein for cooperation with the ras oncogene. After cotransfection with the activated ras gene, the genomic p53 DNA clone always produced more transformed cell foci (1.7-fold) than similar cDNA clones and these foci were more readily cloned (3.6-fold) into permanent cell lines. A series of deletion mutants of the genomic p53 clone were employed to show that the presence of intron 4 in the p53 gene was sufficient to provide much enhanced clonability of transformed foci from culture dishes. The presence of introns in the p53 gene constructions also resulted in elevated levels of p53 protein in the p53-plus-ras-transformed cell lines. Thus, qualitative changes in the p53 protein are required to activate p53 for transformation with the oncogene ras. Quantitative improvements of transformation frequencies are associated with the higher expression levels of altered p53 protein that are provided by having one of the p53 introns in the transforming plasmid.     

Susceptibility of Xeroderma pigmentosum cells to transformation with oncogenes

Oncogenes capable of transforming 3T3-Vill cells were not detected in 'normal' Xeroderma pigmentosum (XP) fibroblasts but were detected in two out of six XP epitheliomas. Preliminary results concerning the transfection of 'normal' XP fibroblasts with activated ras genes seem to indicate that these cells are as resistant as the healthy controls to the transforming action of the group II oncogenes. However, after transfection with v-myb oncogene in XP fibroblasts several cellular clones have been isolated showing some new phenotypic characteristics.     

Activation of a human c-K-ras oncogene

The human lung carcinomas PR310 and PR371 contain activated c-K-ras oncogenes. The oncogene of PR371 was found to present a mutation at codon 12 of the first coding exon which substitutes cysteine for glycine in the encoded p21 protein. We report here that the transforming gene of PR310 tumor contains a mutation in the second coding exon. An A----T transversion at codon 61 results in the incorporation of histidine instead of glutamine in the c-K-ras gene product. By constructing c-K-ras/c-H-ras chimeric genes we show that this point mutation is sufficient to confer transforming potential to ras genes, and that a hybrid ras gene coding for a protein mutant at both codons 12 and 61 is also capable of transforming NIH3T3 cells. The relative transforming potency of p21 proteins encoded by ras genes mutant at codons 12, 61 or both has been analyzed. Our studies also show that the coding exons of ras genes, including the fourth, can be interchanged and the chimeric p21 ras proteins retain their oncogenic ability in normal rodent established cell lines.     

Biphasic activation of p21ras by endothelin-1 sequentially activates the ERK cascade and phosphatidylinositol 3-kinase.

Endothelin-1 (ET-1) induces cell proliferation and differentiation through multiple G-protein-linked signaling systems, including p21ras activation. Whereas p21ras activation and desensitization by receptor tyrosine kinases have been extensively investigated, the kinetics of p21ras activation induced by engagement of G-protein-coupled receptors remains to be fully elucidated. In the present study we show that ET-1 induces a biphasic activation of p21ras in rat glomerular mesangial cells. The first peak of activation of p21ras, at 2-5 min, is mediated by immediate association of phosphorylated Shc with the guanosine exchange factor Sos1 via the adaptor protein Grb2. This initial activation of p21ras results in activation of the extracellular signal-regulated kinase (ERK) cascade. We demonstrate that ET-1 signaling elicits a negative feedback mechanism, modulating p21ras activity through ERK-dependent Sos1 phosphorylation, findings which were confirmed using an adenovirus MEK construct. Subsequent to p21ras and ERK deactivation, Sos1 reverts to the non-phosphorylated condition, enabling it to bind again to the Grb2/Shc complex, which is stabilized by persistent Shc phosphorylation. However, the resulting secondary activation of p21ras at 30 min does not lead to ERK activation, correlating with intensive, ET-1-induced expression of MAP kinase phosphatase-1, but does result in increased p21ras-associated phosphatidylinositol 3-kinase activity. Our data provide evidence that ET-1-induced biphasic p21ras activation causes sequential stimulation of divergent downstream signaling pathways.