Double-stranded short interfering RNAs (siRNA) induce post-transcriptional silencing in a variety of biological systems. In the present study we have investigated the structural requirements of chemically synthesised siRNAs to mediate efficient gene silencing in mammalian cells. In contrast to studies with Drosophila extracts, we found that synthetic, double-stranded siRNAs without specific nucleotide overhangs are highly efficient in gene silencing. Blocking of the 5'-hydroxyl terminus of the antisense strand leads to a dramatic loss of RNA interference activity, whereas blocking of the 3' terminus or blocking of the termini of the sense strand had no negative effect. We further demonstrate that synthetic siRNA molecules with internal 2'-O-methyl modification, but not molecules with terminal modifications, are protected against serum-derived nucleases. Finally, we analysed different sets of siRNA molecules with various 2'-O-methyl modifications for stability and activity. We demonstrate that 2'-O-methyl modifications at specific positions in the molecule improve stability of siRNAs in serum and are tolerated without significant loss of RNA interference activity. These second generation siRNAs will be better suited for potential therapeutic application of synthetic siRNAs in vivo.

The pharmaceutical industry is currently facing several challenges to identify and develop novel drug targets. Traditional drug discovery focussed on a small number of well-characterized gene products. Recently, this picture has changed with the completion of the draft sequence of the human genome, which has led to the identification of thousands of novel genes with unknown or poorly understood function. To cope with this overwhelming number of potential drug target candidates, new strategies for the elucidation of gene function, as well as their involvement in intracellular pathways, are required.

Genetic studies indicated that the Drosophila melanogaster protein REAPER (RPR) controls apoptosis during embryo development. Induction of RPR expression in Drosophila Schneider cells rapidly stimulated apoptosis. RPR-mediated apoptosis was blocked by N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk), which suggests that an interleukin-1β converting enzyme (ICE)-like protease is required for RPR function. RPR-induced apoptosis was associated with increased ceramide production that was also blocked by Z-VAD-fmk, which suggests that ceramide generation requires an ICE-like protease as well. Thus, the intracellular RPR protein uses cell death signaling pathways similar to those used by the vertebrate transmembrane receptors Fas (CD95) and tumor necrosis factor receptor type 1.

We have used two approaches to identify possible substrates of the insulin receptor (IR) tyrosine kinase. First, we used a potent tyrosine phosphatase inhibitor, phenylarsine oxide (PAO), which is reported to be specific for the insulin-induced signal transduction route, to augment tyrosine phosphorylation. Second, we used src homology 2 (SH2) domains fused to glutathione S-transferase as high affinity binding agents for tyrosine-phosphorylated proteins. Using the SH2 domain-containing region of p120 GTPase-activating protein and growth factor-bound protein 2, we observed a tyrosine-phosphorylated M(r) 70,000 protein in insulin- plus PAO-treated NIH3T3 cells overexpressing the IR, This M(r) 70,000 protein, which migrated as a doublet on SDS-polyacrylamide gels, efficiently bound to polyuridylic acid-Sepharose but is distinct from similar-size RNA-binding proteins such as p62 (sam68) and heterogeneous nuclear ribonucleoproteins I, K, L, and M, In addition, it differs from other M(r) 70,000 tyrosine-phosphorylated proteins, such as SH2-containing tyrosine phosphatase, raf1, and paxillin, Tyrosine phosphorylation of this protein was hardly observed after epidermal growth factor treatment, This suggests that the M(r) 70,000 protein is a novel and specific substrate for the IR kinase or an insulin-induced tyrosine kinase, The requirement for PAO to identify this tyrosine phosphorylation indicates a high turnover rate of the tyrosine phosphate.

Association of the p21ras guanine nucleotide exchange factor mSOS with tyrosine-phosphorylated Shc has been implicated in the activation of p21ras. In addition, after growth factor stimulation mSOS becomes phosphorylated as indicated by the appearance of a form of mSOS with reduced electrophoretic mobility. This phosphorylation is delayed with respect to Shc-Grb2-mSOS complex formation and activation of p21ras. To investigate the role of mSOS phosphorylation in further detail we have investigated the effect of phosphorylation on mSOS complex formation and p21ras activation. We found that Shc is associated with the unphosphorylated, faster migrating form of mSOS. Furthermore, although there is a correlation between the amount of complexes formed and the activation of p21ras, there is no such a correlation between mSOS phosphorylation and p21ras activation. In addition, inhibition of mSOS phosphorylation did not affect complex formation of mSOS with tyrosine phosphorylated Shc. Also, induction of mSOS phosphorylation prior to complex formation did not affect EGF-induced association of mSOS with Shc significantly, and Shc still associated predominantly with the faster migrating form of mSOS. From these results we conclude that the unphosphorylated form of mSOS is associated with Shc and that perhaps a phosphorylation-dephosphorylation step is part of the mSOS activation-inactivation cycle.

The notion that ras proteins are required for the stimulation of mitogenesis by different receptor tyrosine kinases (RTKs) has spurred researchers to investigate the precise role of p21ras in signal transduction. A large number of stimuli can drive p21ras in the active conformation, and several proteins that play an important role in regulating the GTP/GDP balance on p21ras have been identified. Indeed, activation of p21ras has been demonstrated to occur by stimulation of guanine nucleotide-releasing proteins (GNRPs) or inhibition of GTPase-activating proteins (GAPs). Moreover, a number of SH2-containing proteins have been implicated in this signaling pathway, such as shc and sem-5/grb2. On the other hand, downstream signaling from p21ras involves an important protein kinase cascade. This pathway seems to be conserved in evolution, and analogous routes have been described in organisms such as yeast, nematodes, and fruit flies. Nevertheless, the direct effector molecule of p21ras that could couple to this kinase cascade is still unknown. Some indications have been obtained that suggest that this function might be partially performed by p120GAP. This review gives an overview of the role of p21ras in signaling from diverse RTKs. Elucidation of this pathway will improve our understanding of mitogenic signaling pathways and the basis of cancer.

Shc proteins are phosphorylated on tyrosine residues and associate with growth factor receptor-bound protein 2 (Grb2) upon treatment of cells with epidermal growth factor (EGF) or insulin. We have studied the role of Shc in insulin- and EGF-induced activation of p21ras in NIH 3T3 cells overexpressing human insulin receptors (A14 cells). A14 cells are equally responsive to insulin and EGF with respect to activation of p21ras. Analysis of Shc immunoprecipitates revealed that (i) both insulin and EGF treatment resulted in Shc tyrosine phosphorylation and (ii) Shc antibodies coimmunoprecipitated both Grb2 and mSOS after insulin and EGF treatment. The induction of tyrosine phosphorylation of Shc and the presence of Grb2 and mSOS in Shc immunoprecipitates followed similar time courses, with somewhat higher levels after EGF treatment. In mSOS immunoprecipitates, Shc could be detected as well. Furthermore, Shc immune complexes contained guanine nucleotide exchange activity toward p21ras in vitro. From these results, we conclude that after insulin and EGF treatment, Shc associates with both Grb2 and mSOS and therefore may mediate, at least in part, insulin- and EGF-induced activation of p21ras. In addition, we investigated whether the Grb2-mSOS complex associates with the insulin receptor or with insulin receptor substrate 1 (IRS1). Although we observed association of Grb2 with IRS1, we did not detect complex formation between mSOS and IRS1 in experiments in which the association of mSOS with Shc was readily detectable. Furthermore, whereas EGF treatment resulted in the association of mSOS with the EGF receptor, insulin treatment did not result in the association of mSOS with the insulin receptor. These results indicate that the association of Grb2-nSOS with Shc may be an important event in insulin-induced, mSOS-mediated activation of p21ras.

The activation of p21ras by receptor tyrosine kinases involves the translocation of the growth factor receptor bound protein 2-mammalian son of sevenless protein (Grb2-SOS) complex to the plasma membrane where p21ras is localized. Insulin receptors induce p21ras-GTP formation by two possible mechanisms: tyrosine phosphorylation of insulin receptor substrate 1 (IRS1) and its subsequent association with Grb2, or Shc phosphorylation and its subsequent association with Grb2. We investigated the contribution of the major tyrosine autophosphorylation sites Tyr1158, Tyr1162, and Tyr1163 of the insulin receptor to IRS1.Grb2 and Shc.Grb2 association and the formation of p21ras-GTP. Chinese hamster ovary-derived cell lines were used overexpressing mutant insulin receptors in which the major tyrosine autophosphorylation sites were stepwise replaced by phenylalanines. In cell lines expressing wild type or mutant Y1158F,Y1162,Y1163 (FYY) receptors, insulin stimulated tyrosine phosphorylation of IRS1 and Shc and the formation of IRS1.Grb2 and Shc.Grb2 protein complexes, together with an increase in p21ras-GTP. Cell lines expressing mutant Y1158,Y1162F,Y1163F (YFF) receptors showed insulin-induced tyrosine phosphorylation of Shc, Shc.Grb2 complex formation, and p21ras-GTP formation, whereas tyrosine phosphorylation of IRS1 was strongly decreased and formation of IRS1.Grb2 complexes was undetectable. The activity of FYY and YFF receptors to mediate p21ras-GTP formation correlated with their activity to induce Shc phosphorylation and Shc.Grb2 association. The mutant insulin receptors Y1158F,Y1162F,Y1163 and Y1158F,Y1162F,Y1163F were inactive in inducing any of these responses. We conclude that phosphorylation of Tyr1158 and Tyr1162 of the insulin receptor is linked to distinct post-receptor processes and that YFF receptors generate p21ras-GTP via the Shc.Grb2 pathway rather than one involving IRS1.Grb2 interaction.

Conference Article| November 01 1993 Role of p21ras in growth factor signal transduction Boudewijn M. Th. Burgering; Boudewijn M. Th. Burgering 1Laboratory of Physiological Chemistry, Utrecht University, 3521 GG Utrecht, The Netherlands Search for other works by this author on: This Site PubMed Google Scholar Gijsbertus J. Pronk; Gijsbertus J. Pronk 1Laboratory of Physiological Chemistry, Utrecht University, 3521 GG Utrecht, The Netherlands Search for other works by this author on: This Site PubMed Google Scholar Jan Paul Medema; Jan Paul Medema 1Laboratory of Physiological Chemistry, Utrecht University, 3521 GG Utrecht, The Netherlands Search for other works by this author on: This Site PubMed Google Scholar Loesje van der Voorn; Loesje van der Voorn 1Laboratory of Physiological Chemistry, Utrecht University, 3521 GG Utrecht, The Netherlands Search for other works by this author on: This Site PubMed Google Scholar Alida M. M. de Vries Smits; Alida M. M. de Vries Smits 1Laboratory of Physiological Chemistry, Utrecht University, 3521 GG Utrecht, The Netherlands Search for other works by this author on: This Site PubMed Google Scholar Pascale C. van Weeren; Pascale C. van Weeren 1Laboratory of Physiological Chemistry, Utrecht University, 3521 GG Utrecht, The Netherlands Search for other works by this author on: This Site PubMed Google Scholar Johannes L. Bos Johannes L. Bos 1Laboratory of Physiological Chemistry, Utrecht University, 3521 GG Utrecht, The Netherlands Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1993) 21 (4): 888–894. https://doi.org/10.1042/bst0210888 Article history Received: August 05 1993 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation Boudewijn M. Th. Burgering, Gijsbertus J. Pronk, Jan Paul Medema, Loesje van der Voorn, Alida M. M. de Vries Smits, Pascale C. van Weeren, Johannes L. Bos; Role of p21ras in growth factor signal transduction. Biochem Soc Trans 1 November 1993; 21 (4): 888–894. doi: https://doi.org/10.1042/bst0210888 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: EGF, epidermal growth factor, ERK2, extracellular-signal-regulated kinase 2, GAP, GTPase-activating protein, GRF, guanine-nucleotide releasing factor, MAP kinase, mitogen-activated protein kinase, PDGF, platelet-derived growth factor, PKC, protein kinase C, PMA, phorbol 12-myristate 13-acetate This content is only available as a PDF. © 1993 Biochemical Society1993 Article PDF first page preview Close Modal You do not currently have access to this content.

In fibroblasts, stimulation of receptor tyrosine kinases results in the activation of the extracellular signal-regulated kinase 2 (ERK2). The major signalling pathway employed by these receptors involves the activation of p21ras and raf-1 kinase. Here we show that in NIH3T3 and rat-1 fibroblasts, elevation of the intracellular cAMP level results in the inhibition of ERK2 activation induced by PDGF, EGF and insulin treatment. Analysis of various signalling intermediates shows that cAMP interferes at a site downstream of p21ras, but upstream of raf-1 kinase. Inhibition by cAMP depends on both the cAMP concentration and the absolute amount of p21ras molecules bound to GTP, suggesting a mechanism of competitive inhibition. Also TPA-induced, p21ras-independent, activation of raf-1 kinase and ERK2 is inhibited by cAMP. We have used the inhibitory effect of cAMP to investigate whether phosphorylation of mSos, a p21ras nucleotide exchange factor, is dependent on the activity of the raf-1 kinase/ERK2 pathway. We found that phosphorylation of mSos, as monitored by a mobility shift, is delayed with respect to p21ras and ERK2 activation and is inhibited by cAMP in a similar cell type- and concentration-dependent manner as the inactivation of ERK2. These results provide evidence for a model of p21ras-directed signalling towards ERK2 that feeds back on mSos by regulating its phosphorylation status and that can be negatively modulated by protein kinase A and positively modulated by protein kinase C action.

The products of the shc gene appear to be substrates for activated oncogenic tyrosine kinases, such as v-Src and v-Fps and activated tyrosine kinase receptors like the epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) receptors. We investigated whether the Shc proteins are targets for the activated insulin receptor tyrosine kinase. Here we show that the 46- and 52-kDa Shc proteins are rapidly phosphorylated upon insulin receptor activation in fibroblasts expressing elevated levels of human insulin receptors. Furthermore, we observed insulin-induced association of a 23-kDa protein with the Shc proteins. These effects on Shc proteins are similar to those observed after EGF and PDGF treatment. In contrast to the observed Shc-EGF receptor association, we did not detect association between the Shc proteins and the insulin receptor. We conclude that the Shc proteins are common elements in a signal transduction pathway that is shared by EGF, PDGF, and insulin.

We have investigated whether complex formation between the p21ras GTPase-activating protein (GAP) and the phosphotyrosine-containing proteins p62 and p190 is dependent on functional p21ras, to test the hypothesis that binding of p21rasGTP to GAP enables GAP to associate with these phosphoproteins. The formation of p21rasGTP was inhibited by a dominant interfering mutant of p21ras, p21ras(Asn-17), which was introduced with a vaccinia virus expression system. We used NIH3T3 cells in which complex formation between GAP and tyrosine-phosphorylated p62 and p190 can be induced either by v-src transformation, by incubating the cells with the phosphotyrosine phosphatase inhibitor pervanadate or by activation of a growth factor receptor tyrosine kinase. In all cases, expression of p21ras(Asn-17) did not affect the presence or the formation of the GAP-phosphoprotein complexes. To monitor the effectiveness of p21ras inhibition, we measured p21ras-mediated phosphorylation of extracellular signal-regulated kinase 2 (ERK2). In all cases, expression of p21ras(Asn-17) completely blocked signalling to ERK2. From these data we conclude that p21rasGTP formation is not essential for complex formation between GAP and tyrosine-phosphorylated p62 and p190, and thus complex formation does not depend on interaction of GAP with p21rasGTP.

We investigated the involvement of the p21ras-GTPase activating protein (GAP) in insulin-induced signal transduction. In cells overexpressing the insulin receptor, we did not observe association between GAP and the insulin receptor after insulin treatment nor the phosphorylation of GAP on tyrosine residues. However, after insulin treatment in the presence of the phosphotyrosine phosphatase inhibitor phenylarsine oxide (PAO), 5-10% of GAP was found to be associated with the insulin receptor, and, in addition, a fraction of total GAP was phosphorylated on tyrosine. Using in vitro binding we showed that the N-terminal part of GAP containing the src-homology domains 2 and 3 (SH2-SH3-SH2 region) is involved in binding to the autophosphorylated insulin receptor beta-chain. In vitro binding between GAP and the autophosphorylated insulin receptor occurred independently of PAO pretreatment. These results suggest that GAP can transiently interact with the insulin receptor after insulin treatment, and this interaction is arrested after PAO pretreatment.

p21ras GAP is phosphorylated on tyrosine residues and associates with 62 kDa and 190 kDa tyrosine phosphorylated proteins in v-src-transformed fibroblasts. We were interested in identifying the tyrosine kinase responsible for phosphorylation of GAP and the two associated proteins. Here, we report that GAP-immunoprecipitates from v-src transformed cells contain a tyrosine kinase activity that phosphorylates GAP, p62 and p190. Tryptic peptide analysis indicated that the sites phosphorylated in vitro and in vivo are indistinguishable, suggesting that the precipitated kinase could be responsible for tyrosine phosphorylation of GAP in vivo. The GAP-associated kinase activity might be due to v-src itself, because pp60v-src is able to associate with GAP in vitro and GAP can be phosphorylated by pp60v-src immunecomplexes.

We have cloned the immediate-early serum-reponsive JE gene from the rat in order to study the regulation of this gene. We show that sequences of the JE promoter region confer serum-inducibility to a reporter gene. Analysis of the promoter in transient assays reveals that: i) the -141/-88 region is required for the response to the phorbol ester TPA, ii) the -70/-38 region is essential for basal activity. This latter region harbors the sequence TGACTCC, which resembles the consensus site for AP-1 binding, TGACTCA. DNA-protein binding assays indicate that the JE AP-1 site and the consensus AP-1 site have an overlapping but not identical binding spectrum for AP-1 proteins. Our data suggest that the inability of some AP-1 sites to respond to TPA is caused by subtle differences in affinity for AP-1 proteins.

Primary baby rat kidney cells, primary human embryonic retinoblast cells, established NIH3T3 and established normal rat kidney (NRK) cells, expressing Ell A and/or E1 B gene regions of adenovirus 5 (Ad5) or Ad 12, were investigated for susceptibility to the cytotoxic/cytostatic action of Tumor Necrosis Factor (TNF). In the primary cells and in the NRK cells, there was no correlation between TNF sensitivity and E1 gene expression; neither did sensitivity to TNF correlate with the oncogenicity of the Ad serotype. In contrast, the expression of Ad El gene regions in NIH3T3 cells was found to enhance TNF sensitivity of this cell line. Differences in El A expression levels between cell types cannot explain this discrepancy regarding modulation of TNF sensitivity by E1A.