Global expression analysis identified a preferentially nerve growth factor-induced transcriptional program regulated by sustained mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) and AP-1 protein activation during PC12 cell differentiation.

Neuronal differentiation of PC12 cells in response to NGF is a prototypical model in which signal duration determines a biological response. Sustained ERK activity induced by NGF, as compared with transient activity induced by EGF, is critical to the differentiation of these cells. To characterize the transcriptional program activated preferentially by NGF, we compared global gene expression profiles between cells treated with NGF and EGF for 2-4 h, when sustained ERK signaling in response to NGF is most distinct from the transient signal elicited by EGF. This analysis identified 69 genes that were preferentially up-regulated in response to NGF. As expected, up-regulation of these genes was mediated by sustained ERK signaling. In addition, they were up-regulated in response to other neuritogenic treatments (pituitary adenylate cyclase-activating polypeptide and 12-O-tetradecanoylphorbol-13-acetate plus dbcAMP) and were enriched for genes related to neuronal differentiation/function. Computational analysis and chromatin immunoprecipitation identified binding of CREB and AP-1 family members (Fos, FosB, Fra1, JunB, JunD) upstream of >30 and 50%, respectively, of the preferentially NGF-induced genes. Expression of several AP-1 family members was induced by both EGF and NGF, but their induction was more robust and sustained in response to NGF. The binding of Fos family members to their target genes was similarly sustained in response to NGF and was reduced upon MEK inhibition, suggesting that AP-1 contributes significantly to the NGF transcriptional program. Interestingly, Fra1 as well as two other NGF-induced AP-1 targets (HB-EGF and miR-21) function in positive feedback loops that may contribute to sustained AP-1 activity.

The E-box binding factors Max/Mnt, MITF, and USF1 act coordinately with FoxO to regulate expression of proapoptotic and cell cycle control genes by phosphatidylinositol 3-kinase/Akt/glycogen synthase kinase 3 signaling.

Phosphatidylinositol (PI) 3-kinase/Akt signaling plays a critical role in cell proliferation and survival, partly by regulation of FoxO transcription factors. Previous work using global expression profiling indicated that inhibition of PI 3-kinase in proliferating cells led to induction of genes that promote cell cycle arrest and apoptosis. The upstream regulatory regions of these genes had binding sites not only for FoxO, but also for Myc/Max transcription factors. In the present study, we have addressed the role of Myc family members and related E-box-binding proteins in the regulation of these genes. Chromatin immunoprecipitations and RNA interference indicated that transcription was repressed by Max-Mnt-Sin3a-histone deacetylase complexes in proliferating cells. Inhibition of PI 3-kinase led to a loss of Max/Mnt binding and transcriptional induction by MITF and USF1, as well as FoxO. Both MITF and USF1 were activated by glycogen synthase kinase (GSK) 3, with GSK3 phosphorylation sites on USF1 identified as the previously described activating site threonine 153 as well as serine 186. siRNA against MITF as well as against FoxO3a protected cells from apoptosis following PI 3-kinase inhibition. These results define a novel E-box-regulated network that functions coordinately with FoxO to regulate transcription of apoptotic and cell cycle regulatory genes downstream of PI 3-kinase/Akt/GSK3 signaling.

GSK-3 represses growth factor-inducible genes by inhibiting NF-kappaB in quiescent cells.

GSK-3 is active in the absence of growth factor stimulation and generally acts to induce apoptosis or inhibit cell proliferation. We previously identified a subset of growth factor-inducible genes that can also be induced in quiescent T98G cells solely by inhibition of GSK-3 in the absence of growth factor stimulation. Computational predictions verified by chromatin immunoprecipitation assays identified NF-kappaB binding sites in the upstream regions of 75% of the genes regulated by GSK-3. p50 bound to most of these sites in quiescent cells, and for one-third of the genes, binding of p65 to the predicted sites increased upon inhibition of GSK-3. The functional role of p65 in gene induction following inhibition of GSK-3 was demonstrated by RNA interference experiments. Furthermore, inhibition of GSK-3 in quiescent cells resulted in activation of IkappaB kinase, leading to phosphorylation and degradation of IkappaB alpha and nuclear translocation of p65 and p50. Taken together, these results indicate that the high levels of GSK-3 activity in quiescent cells repress gene expression by negatively regulating NF-kappaB through inhibition of IkappaB kinase. This inhibition of NF-kappaB is consistent with the role of GSK-3 in the induction of apoptosis or cell cycle arrest in cells deprived of growth factors.

Prolyl isomerase Pin1 regulates transcription factor LSF (TFCP2) by facilitating dephosphorylation at two serine-proline motifs.

Transcription factor LSF is essential for cell cycle progression, being required for activating expression of the thymidylate synthase (Tyms) gene at the G1/S transition. We previously established that phosphorylation of LSF in early G1 at Ser-291 and Ser-309 inhibits its transcriptional activity and that dephosphorylation later in G1 is required for its reactivation. Here we reveal the role of prolyl cis-trans isomerase Pin1 in activating LSF, by facilitating dephosphorylation at both Ser-291 and Ser-309. We demonstrate that Pin1 binds LSF both in vitro and in vivo. Using coimmunoprecipitation assays, we identify three SP/TP motifs in LSF (at residues Ser-291, Ser-309, and Thr-329) that are required and sufficient for association with Pin1. Co-expression of Pin1 enhances LSF transactivation potential in reporter assays. The Pin1-dependent enhancement of LSF activity requires residue Thr-329 in LSF, requires both the WW and PPiase domains of Pin1, and correlates with hypophosphorylation of LSF at Ser-291 and Ser-309. These findings support a model in which the binding of Pin1 at the Thr-329-Pro-330 motif in LSF permits isomerization by Pin1 of the peptide bonds at the nearby phosphorylated SP motifs (Ser-291 and Ser-309) to the trans configuration, thereby facilitating their dephosphorylation.

Phosphatidylinositol 3-kinase signaling in proliferating cells maintains an anti-apoptotic transcriptional program mediated by inhibition of FOXO and non-canonical activation of NFkappaB transcription factors.

BACKGROUND: Phosphatidylinositol (PI) 3-kinase is activated by a variety of growth factor receptors and the PI 3-kinase/Akt signaling pathway is a key regulator of cell proliferation and survival. The downstream targets of PI 3-kinase/Akt signaling include direct regulators of cell cycle progression and apoptosis as well as a number of transcription factors. Growth factor stimulation of quiescent cells leads to robust activation of PI 3-kinase, induction of immediate-early genes, and re-entry into the cell cycle. A lower level of PI 3-kinase signaling is also required for the proliferation and survival of cells maintained in the presence of growth factors, but the gene expression program controlled by PI 3-kinase signaling in proliferating cells has not been elucidated. RESULTS: We used microarray analyses to characterize the changes in gene expression resulting from inhibition of PI 3-kinase in proliferating cells. The genes regulated by inhibition of PI 3-kinase in proliferating cells were distinct from genes induced by growth factor stimulation of quiescent cells and highly enriched in genes that regulate programmed cell death. Computational analyses followed by chromatin immunoprecipitations demonstrated FOXO binding to both previously known and novel sites in promoter regions of approximately one-third of the up-regulated genes, consistent with activation of FOXO1 and FOXO3a in response to inhibition of PI 3-kinase. NFkappaB binding sites were similarly identified in promoter regions of over one-third of the down-regulated genes. RelB was constitutively bound to promoter regions in cells maintained in serum, however binding decreased following PI 3-kinase inhibition, indicating that PI 3-kinase signaling activates NFkappaB via the non-canonical pathway in proliferating cells. Approximately 70% of the genes targeted by FOXO and NFkappaB regulate cell proliferation and apoptosis, including several regulators of apoptosis that were not previously known to be targeted by these transcription factors. CONCLUSION: PI 3-kinase signaling in proliferating cells regulates a novel transcriptional program that is highly enriched in genes that regulate apoptosis. At least one-third of these genes are regulated either by FOXO transcription factors, which are activated following PI 3-kinase inhibition, or by RelB, which is activated by PI 3-kinase via the non-canonical pathway in proliferating cells.

Role of translation initiation factor 2B in control of cell survival by the phosphatidylinositol 3-kinase/Akt/glycogen synthase kinase 3beta signaling pathway.

The phosphatidylinositol 3-kinase (PI 3-kinase)/Akt signaling pathway is an important mediator of growth factor-dependent survival of mammalian cells. A variety of targets of the Akt protein kinase have been implicated in cell survival, including the protein kinase glycogen synthase kinase 3beta (GSK-3beta). One of the targets of GSK-3beta is translation initiation factor 2B (eIF2B), linking global regulation of protein synthesis to PI 3-kinase/Akt signaling. Because of the central role of protein synthesis, we have investigated the involvement of eIF2B, which is inhibited as a result of GSK-3beta phosphorylation, in programmed cell death. We demonstrate that expression of eIF2B mutants lacking the GSK-3beta phosphorylation or priming sites is sufficient to protect both Rat-1 and PC12 cells from apoptosis induced by overexpression of GSK-3beta, inhibition of PI 3-kinase, or growth factor deprivation. Consistent with these effects on cell survival, expression of nonphosphorylatable eIF2B prevented inhibition of protein synthesis following treatment of cells with the PI 3-kinase inhibitor LY294002. Conversely, cycloheximide induced apoptosis of PC12 and Rat-1 cells, further indicating that protein synthesis was required for cell survival. Inhibition of translation resulting from treatment with cycloheximide led to the release of cytochrome c from mitochondria, similar to the effects of inhibition of PI 3-kinase. Expression of nonphosphorylatable eIF2B prevented cytochrome c release resulting from PI 3-kinase inhibition but did not affect cytochrome c release or apoptosis induced by cycloheximide. Regulation of translation resulting from phosphorylation of eIF2B by GSK-3beta thus appears to contribute to the control of cell survival by the PI 3-kinase/Akt signaling pathway, acting upstream of mitochondrial cytochrome c release.

Role for Egr1 in the Transcriptional Program Associated with Neuronal Differentiation of PC12 Cells.

PC12 cells are a well-established model to study how differences in signal transduction duration can elicit distinct cell behaviors. Epidermal growth factor (EGF) activates transient ERK signaling in PC12 cells that lasts 30-60 min, which in turn promotes proliferation; nerve growth factor (NGF) activates more sustained ERK signaling that lasts 4-6 h, which in turns induces neuronal differentiation. Data presented here extend a previous study by Mullenbrock et al. (2011) that demonstrated that sustained ERK signaling in response to NGF induces preferential expression of a 69-member gene set compared to transient ERK signaling in response to EGF and that the transcription factors AP-1 and CREB play a major role in the preferential expression of several genes within the set. Here, we examined whether the Egr family of transcription factors also contributes to the preferential expression of the gene set in response to NGF. Our data demonstrate that NGF causes transient induction of all Egr family member transcripts, but a corresponding induction of protein was detected for only Egr1 and 2. Chromatin immunoprecipitation experiments provided clearest evidence that, after induction, Egr1 binds 12 of the 69 genes that are preferentially expressed during sustained ERK signaling. In addition, Egr1 expression and binding upstream of its target genes were both sustained in response to NGF versus EGF within the same timeframe that its targets are preferentially expressed. These data thus provide evidence that Egr1 contributes to the transcriptional program activated by sustained ERK signaling in response to NGF, specifically by contributing to the preferential expression of its target genes identified here.

A GSK-3-mediated transcriptional network maintains repression of immediate early genes in quiescent cells.

Glycogen synthase kinase-3 (GSK-3) plays a central role in cell survival and proliferation, in part by the regulation of transcription. Unlike most protein kinases, GSK-3 is active in quiescent cells in the absence of growth factor signaling. In a recent series of studies, we employed a systems-level approach to understanding the transcription network regulated by GSK-3 in a quiescent cell model. We identified a group of immediate early genes that were upregulated in quiescent cells solely by the inhibition of GSK-3 in the absence of growth factor stimulation. Computational analysis of the upstream sequences of these genes identified statistically over-represented binding sites for the transcription factors CREB, NFκB and AP-1, and the roles of these factors in regulating expression of GSK-3 target genes were verified by chromatin immunoprecipitation and RNA interference. In quiescent cells, GSK-3 inhibits CREB, NFκB and AP-1, thereby maintaining repression of their target genes and contributing to maintenance of cell cycle arrest.

AP-1 is a component of the transcriptional network regulated by GSK-3 in quiescent cells.

BACKGROUND: The protein kinase GSK-3 is constitutively active in quiescent cells in the absence of growth factor signaling. Previously, we identified a set of genes that required GSK-3 to maintain their repression during quiescence. Computational analysis of the upstream sequences of these genes predicted transcription factor binding sites for CREB, NFκB and AP-1. In our previous work, contributions of CREB and NFκB were examined. In the current study, the AP-1 component of the signaling network in quiescent cells was explored. METHODOLOGY/PRINCIPAL FINDINGS: Using chromatin immunoprecipitation analysis, two AP-1 family members, c-Jun and JunD, bound to predicted upstream regulatory sequences in 8 of the 12 GSK-3-regulated genes. c-Jun was phosphorylated on threonine 239 by GSK-3 in quiescent cells, consistent with previous studies demonstrating inhibition of c-Jun by GSK-3. Inhibition of GSK-3 attenuated this phosphorylation, resulting in the stabilization of c-Jun. The association of c-Jun with its target sequences was increased by growth factor stimulation as well as by direct GSK-3 inhibition. The physiological role for c-Jun was also confirmed by siRNA inhibition of gene induction. CONCLUSIONS/SIGNIFICANCE: These results indicate that inhibition of c-Jun by GSK-3 contributes to the repression of growth factor-inducible genes in quiescent cells. Together, AP-1, CREB and NFκB form an integrated transcriptional network that is largely responsible for maintaining repression of target genes downstream of GSK-3 signaling.

mRNA degradation plays a significant role in the program of gene expression regulated by phosphatidylinositol 3-kinase signaling.

Control of gene expression by the phosphatidylinositol (PI) 3-kinase/Akt pathway plays an important role in mammalian cell proliferation and survival, and numerous transcription factors and genes regulated by PI 3-kinase signaling have been identified. Because steady-state levels of mRNA are regulated by degradation as well as transcription, we have investigated the importance of mRNA degradation in controlling gene expression downstream of PI 3-kinase. We previously performed global expression analyses that identified a set of approximately 50 genes that were downregulated following inhibition of PI 3-kinase in proliferating T98G cells. By blocking transcription with actinomycin D, we found that almost 40% of these genes were regulated via effects of PI 3-kinase on mRNA stability. Analyses of ß-globin-3' untranslated region (UTR) fusion transcripts indicated that sequences within 3' UTRs were the primary determinants of rapid mRNA decay. Small interfering RNA (siRNA) experiments further showed that knockdown of BRF1 or KSRP, both ARE binding proteins (ARE-BPs) regulated by Akt, stabilized the mRNAs of a majority of the downregulated genes but that knockdown of ARE-BPs that are not regulated by PI 3-kinase did not affect degradation of these mRNAs. These results show that PI 3-kinase regulation of mRNA stability, predominantly mediated by BRF1, plays a major role in regulating gene expression.

Phosphorylation by cyclin C/cyclin-dependent kinase 2 following mitogenic stimulation of murine fibroblasts inhibits transcriptional activity of LSF during G1 progression.

Transcription factor LSF is required for progression from quiescence through the cell cycle, regulating thymidylate synthase (Tyms) expression at the G(1)/S boundary. Given the constant level of LSF protein from G(0) through S, we investigated whether LSF is regulated by phosphorylation in G(1). In vitro, LSF is phosphorylated by cyclin E/cyclin-dependent kinase 2 (CDK2), cyclin C/CDK2, and cyclin C/CDK3, predominantly on S309. Phosphorylation of LSF on S309 is maximal 1 to 2 h after mitogenic stimulation of quiescent mouse fibroblasts. This phosphorylation is mediated by cyclin C-dependent kinases, as shown by coimmunoprecipitation of LSF and cyclin C in early G(1) and by abrogation of LSF S309 phosphorylation upon suppression of cyclin C with short interfering RNA. Although mouse fibroblasts lack functional CDK3 (the partner of cyclin C in early G(1) in human cells), CDK2 compensates for this absence. By transient transfection assays, phosphorylation at S309, mediated by cyclin C overexpression, inhibits LSF transactivation. Moreover, overexpression of cyclin C and CDK3 inhibits induction of endogenous Tyms expression at the G(1)/S transition. These results identify LSF as only the second known target (in addition to pRb) of cyclin C/CDK activity during progression from quiescence to early G(1). Unexpectedly, this phosphorylation prevents induction of LSF target genes until late G(1).

Rapid turnover of mcl-1 couples translation to cell survival and apoptosis.

Inhibition of translation plays a role in apoptosis induced by a variety of stimuli, but the mechanism by which it promotes apoptosis has not been established. We have investigated the hypothesis that selective degradation of anti-apoptotic regulatory protein(s) is responsible for apoptosis resulting from translation inhibition. Induction of apoptosis by cycloheximide was detected within 2-4 h and blocked by proteasome inhibitors, indicating that degradation of short-lived protein(s) was required. Caspase inhibition and overexpression of Bcl-x(L) blocked cycloheximide-induced apoptosis. In addition, cycloheximide induced rapid activation of Bak and Bax, which required proteasome activity. Mcl-1 was degraded by the proteasome with a half-life of approximately 30 min following inhibition of protein synthesis, preceding Bak/Bax activation and the onset of apoptosis. Overexpression of Mcl-1 blocked apoptosis induced by cycloheximide, whereas RNA interference knockdown of Mcl-1 induced apoptosis. Knockdown of Bim and Bak, downstream targets of Mcl-1, inhibited cycloheximide-induced apoptosis, as did knockdown of Bax. Apoptosis resulting from inhibition of translation thus involves the rapid degradation of Mcl-1, leading to activation of Bim, Bak, and Bax. Because of its rapid turnover, Mcl-1 may serve as a convergence point for signals that affect global translation, coupling translation to cell survival and the apoptotic machinery.

Immediate-early and delayed primary response genes are distinct in function and genomic architecture.

The transcriptional program induced by growth factor stimulation is classically described in two stages as follows: the rapid protein synthesis-independent induction of immediate-early genes, followed by the subsequent protein synthesis-dependent induction of secondary response genes. In this study, we obtained a comprehensive view of this transcriptional program. As expected, we identified both rapid and delayed gene inductions. Surprisingly, however, a large fraction of genes induced with delayed kinetics did not require protein synthesis and therefore represented delayed primary rather than secondary response genes. Of 133 genes induced within 4 h of growth factor stimulation, 49 (37%) were immediate-early genes, 58 (44%) were delayed primary response genes, and 26 (19%) were secondary response genes. Comparison of immediate-early and delayed primary response genes revealed functional and regulatory differences. Whereas many immediate-early genes encoded transcription factors, transcriptional regulators were not prevalent among the delayed primary response genes. The lag in induction of delayed primary response compared with immediate-early mRNAs was because of delays in both transcription initiation and subsequent stages of elongation and processing. Consistent with increased abundance of RNA polymerase II at their promoters, immediate-early genes were characterized by over-representation of transcription factor binding sites and high affinity TATA boxes. Immediate-early genes also had short primary transcripts with few exons, whereas delayed primary response genes more closely resembled other genes in the genome. These findings suggest that genomic features of immediate-early genes, in contrast to the delayed primary response genes, are selected for rapid induction, consistent with their regulatory functions.

Glycogen synthase kinase-3 represses cyclic AMP response element-binding protein (CREB)-targeted immediate early genes in quiescent cells.

Despite its central role in cell survival and proliferation, the transcriptional program controlled by GSK-3 is poorly understood. We have employed a systems level approach to characterize gene regulation downstream of PI 3-kinase/Akt/GSK-3 signaling in response to growth factor stimulation of quiescent cells. Of 31 immediate-early genes whose induction was dependent on PI 3-kinase signaling, 12 were induced directly by inhibition of GSK-3. Most of the GSK-3-regulated genes encoded transcription factors, growth factors, and signaling molecules. Binding sites for CREB were highly over-represented in the upstream regions of these genes, with 9 genes containing CREB sites that were conserved in mouse orthologs. Binding sites predicted in 6 genes were confirmed by CREB chromatin immunoprecipitation and forskolin induction of CBP binding. Moreover, CREB siRNA substantially blocked induction of 5 genes by forskolin and of 3 genes following inhibition of GSK-3. These results indicate that GSK-3 actively represses gene expression in quiescent cells, with inhibition of CREB playing a key role in this transcriptional response.

Mouse embryonic stem cells and preimplantation embryos require signaling through the phosphatidylinositol 3-kinase pathway to suppress apoptosis.

Whereas most mammalian cells require extracellular signals to suppress apoptosis, preimplantation embryos can survive and develop to the blastocyst stage in defined medium without added serum or growth factors. Since cells of these embryos are capable of undergoing apoptosis, it has been suggested that their lack of dependence upon exogenous growth factors results from the production of endogenous growth factors that suppress apoptosis by an autocrine signaling mechanism. In the present study, we have examined the growth factor requirements and intracellular signaling pathways that suppress apoptosis in both mouse preimplantation embryos and embryonic stem (ES) cells, which are derived from the blastocyst inner cell mass. Cultured ES cells, in contrast to intact embryos, required serum growth factors to prevent apoptosis. Suppression of ES cell apoptosis by serum growth factors required the phosphatidylinositol 3-kinase (PI 3-kinase) signaling pathway, since apoptosis was rapidly induced by inhibition of PI 3-kinase with LY294002. In contrast, inhibition of MEK/ERK signaling with U0126 or of mTOR with rapamycin had no detectable effect on ES cell survival. Thus, like most mammalian cells, the survival of ES cells is mediated by growth factor stimulation of PI 3-kinase signaling. Treatment with LY294002 (but not with U0126 or rapamycin) similarly induced apoptosis of mouse blastocysts in serum-free medium, indicating that intact preimplantation embryos are also dependent upon PI 3-kinase signaling for survival. These results demonstrate that PI 3-kinase signaling is required to suppress apoptosis of both ES cells and intact preimplantation embryos, consistent with the hypothesis that survival of preimplantation embryos is maintained by endogenous growth factors that stimulate the PI 3-kinase pathway.

A binding site for germ cell nuclear factor within c-mos regulatory sequences.

The proto-oncogene c-mos is specifically expressed in male and female germ cells. Previous studies have shown that the orphan nuclear receptor chicken ovalbumin upstream promoter transcription factor (COUP-TF) contributes to the repression of c-mos in somatic cells by binding to an inverted hexamer repeat within the c-mos regulatory region. In the present study, we demonstrate that another nuclear receptor superfamily member, germ cell nuclear factor (GCNF), binds to a sequence overlapping the c-mos COUP-TF binding site. Electrophoretic mobility shift assays with recombinant GCNF and both wild-type and mutant c-mos oligonucleotides demonstrated the binding of GCNF to an extended half site, CCAAGTTCA, which overlaps the first hexamer of the COUP-TF binding site. Transient transfection assays in NIH 3T3 cells further demonstrated that GCNF fused to a VP16 activation domain stimulated transcription from reporter constructs containing the c-mos GCNF binding site. Since GCNF is expressed in male and female germ cells at the same stages of development at which c-mos is transcribed, these results suggest that GCNF may serve as a regulator of c-mos transcription. Mol. Reprod. Dev. 67: 55-64, 2004.

Functional analysis of sperm from c-mos(-/-) mice.

The c-mos protooncogene, which is expressed predominantly in male and female germ cells, is crucial for normal oocyte meiosis and female fertility in mice. Inactivation of c-mos results in abnormal oocyte development and leads to ovarian cysts and tumors in vivo. In contrast to the severe effects of c-mos ablation in females, targeted inactivation of c-mos has not been reported to affect spermatogenesis in male mice. However, previously reported studies of male c-mos(-/-) mice have been limited to histological analyses of testes and in vivo matings, both of which are relatively insensitive indicators of sperm production and function. Therefore, we assayed sperm function of c-mos(-/-) males under in vitro conditions to determine whether the absence of Mos during development affected sperm production or fertilizing ability. We found no significant differences between the number of sperm collected from c-mos(-/-) and wild type mice. Additionally, sperm from c-mos(-/-) and c-mos(+/+) males performed equally well in assays of in vitro fertilization (IVF) and fertilization-associated events including zona pellucida (ZP) penetration, sperm/egg plasma membrane fusion, and sperm chromatin remodeling. Therefore, we suggest that the function of Mos in spermatogenesis is either not related to the ultimate fertilizing potential of the sperm, or else the absence of Mos is masked by a redundant kinase.

Role of mTOR in the degradation of IRS-1: regulation of PP2A activity.

We have investigated the role of PI 3-kinase and mTOR in the degradation of IRS-1 induced by insulin. Inhibition of mTOR with rapamycin resulted in approximately 50% inhibition of the insulin-induced degradation of IRS-1. In contrast, inhibition of PI-3 kinase, an upstream activator of mTOR, leads to a complete block of the insulin-induced degradation. Inhibition of either PI-3 kinase or mTOR prevented the mobility shift in IRS-1 in response to insulin, a shift that is caused by Ser/Thr phosphorylation. These results indicate that insulin stimulates PI 3-kinase-mediated degradation of IRS-1 via both mTOR-dependent and -independent pathways. Platelet-derived growth factor (PDGF) stimulation leads to a lower level of degradation, but significant phosphorylation of IRS-1. Both the degradation and phosphorylation of IRS-1 in response to PDGF are completely inhibited by rapamycin, suggesting that PDGF stimulates IRS-1 degradation principally via the mTOR-dependent pathway. Inhibition of the serine/threonine phosphatase PP2A with okadaic acid also induced the phosphorylation and degradation of IRS-1. IRS-1 phosphorylation and degradation in response to okadaic acid were not inhibited by rapamycin, suggesting that the action of mTOR in the degradation of IRS-1 results from inhibition of PP2A. Consistent with this, treatment of cells with rapamycin stimulated PP2A activity. While the role of mTOR in the phosphorylation of IRS-1 appears to proceed primarily through the regulation of PP2A, we also provide evidence that the regulation of p70S6 kinase phosphorylation requires the direct activity of mTOR.

Mammalian transcription factor LSF is a target of ERK signaling.

LSF is a mammalian transcription factor that is rapidly and quantitatively phosphorylated upon growth induction of resting, peripheral human T cells, as assayed by a reduction in its electrophoretic mobility. The DNA-binding activity of LSF in primary T cells is greatly increased after this phosphorylation event (Volker et al. [1997]: Genes Dev 11:1435-1446). We demonstrate here that LSF is also rapidly and quantitatively phosphorylated upon growth induction in NIH 3T3 cells, although its DNA-binding activity is not significantly altered. Three lines of experimentation established that ERK is responsible for phosphorylating LSF upon growth induction in both cell types. First, phosphorylation of LSF by ERK is sufficient to cause the reduced electrophoretic mobility of LSF. Second, the amount of ERK activity correlates with the extent of LSF phosphorylation in both primary human T cells and NIH 3T3 cells. Finally, specific inhibitors of the Ras/Raf/MEK/ERK pathway inhibit LSF modification in vivo. This phosphorylation by ERK is not sufficient for activation of LSF DNA-binding activity, as evidenced both in vitro and in mouse fibroblasts. Nonetheless, activation of ERK is a prerequisite for the substantial increase in LSF DNA-binding activity upon activation of resting T cells, indicating that ERK phosphorylation is necessary but not sufficient for activation of LSF in this cell type.

Identification of transcription factor binding sites upstream of human genes regulated by the phosphatidylinositol 3-kinase and MEK/ERK signaling pathways.

We have taken an integrated approach in which expression profiling has been combined with the use of small molecule inhibitors and computational analysis of transcription factor binding sites to characterize regulatory sequences of genes that are targets of specific signaling pathways in growth factor-stimulated human cells. T98G cells were stimulated with platelet-derived growth factor (PDGF) and analyzed by DNA microarrays, which identified 74 immediate-early gene transcripts. Cells were then treated with inhibitors to identify subsets of genes that are targets of the phosphatidylinositol 3-kinase (PI3K) and MEK/ERK signaling pathways. Four groups of PDGF-induced genes were defined: independent of PI3K and MEK/ERK signaling, dependent on PI3K signaling, dependent on MEK/ERK signaling, and dependent on both pathways. The upstream regions of all genes in the four groups were scanned using TRANSFAC for putative cis-elements as compared with a background set of non-induced genes. Binding sites for 18 computationally predicted transcription factors were over-represented in the four groups of co-expressed genes compared with the background sequences (p < 0.01). Many of the cis-elements identified were conserved in orthologous mouse genes, and many of the predicted elements and their cognate transcription factors were consistent with previous experimental data. In addition, chromatin immunoprecipitation assays experimentally verified nine predicted SRF binding sites in T98G cells, including a previously unknown SRF site upstream of DUSP5. These results indicate that groups of human genes regulated by discrete intracellular signaling pathways share common cis-regulatory elements.