Biomedical subjects

M R Rosner

Publications and source records attributed to M R Rosner.

At least 37 records · Page 2Linked to original sources

PubMed1997

Identification of the cleavage sites of transforming growth factor alpha by insulin-degrading enzymes.

Insulin-degrading enzyme (IDE) is a sulfhydryl-dependent metalloproteinase with a zinc binding site unique to a new class of proteinases. The enzyme is relatively specific for a number of hormones/growth factors, such as insulin, atrial natriuretic peptide, IGF-II, and proinsulin. In this study we have identified the amino-acid bonds cleaved by IDE in transforming growth factor-alpha. High-performance liquid chromatography was used to separate the peptides generated by the degradation of 125I-TGF-alpha. The peptides were then submitted to sequential Edman degradation to determine the peptide bond broken. Cleavage sites were found at amino acids, 10-11 (Asp-Ser), 25-26 (Val-Gln), 28-29 (Asp-Lys), and 30-31 (Pro-Ala). In agreement with studies of cleavage sites of other hormones by this enzyme, no clear amino-acid specificity was seen. However, examination of the sites on a three-dimensional model of TGF-alpha suggest the primary mechanism used by IDE for determining cleavage sites is the tertiary structure of the substrate.

Animals↗

PubMed1997

Insulin gene expression in immortalized rat hippocampal and pheochromocytoma-12 cell lines.

Employing reverse transcription-polymerase chain reaction and clonal cell lines derived by retroviral transduction of the temperature sensitive simian virus 40 large T-antigen into dispersed rat embryonic hippocampal cells, we detected the ancestral gene-insulin II mRNA in three progenitor subcloned cell lines. These cell lines upon differentiation are known to express markers indicative of commitment to either neuronal (H19-7; NF + , GFAP -), glial (H19-5; GFAP +, NF -), or bipotential (H583-5, NF +, GFAP + ) lineages. No duplicated, i.e., insulin I gene expression, was observed in any of the three cell lines. Induction of differentiation was associated with the persistence of insulin II mRNA and in the cells expressing a neuronal phenotype (H19-7; NF +, GFAP -) a relative doubling in insulin II mRNA level was present (P < 0.05). Minimal cellular insulin immunoreactivity was detected only in a subpopulation of cells with a differentiated neuronal phenotype. Radioimmunoassayable insulin peptide in the H19-7 cellular conditioned medium revealed a 5-fold increase in the differentiated state. In contrast, peripheral sympathetic PC-12 neuronal cells both in the undifferentiated and nerve growth factor-driven differentiated states, failed to express both insulin I and insulin II genes. We conclude that insulin II is expressed by cultured rat hippocampal clonal cell lines, and not by the peripheral sympathetic PC-12 neuronal cell line.

Animals↗

PubMed1997

Role of cyclins in neuronal differentiation of immortalized hippocampal cells.

The proto-oncogene cyclin D1 and the neuron-specific cyclins p35 and p39 are expressed during brain maturation. To investigate the role of these cyclins in neuronal differentiation, we used a conditionally immortalized rat hippocampal cell line, H19-7, that expresses cyclin-dependent kinases 4 and 5 (cdk4 and -5). Cyclin D1, which activates cdk4 and binds but does not activate cdk5, was increased upon differentiation of the H19-7 cells. However, microinjection of either sense or antisense cyclin D1 cDNA or anti-cyclin D1 antibodies had no effect on morphological differentiation of the cells. On the other hand, neurite outgrowth was stimulated by expression of p35 or p39, both of which activate cdk5. A dominant-negative mutant of cdk5 blocked both p35- and p39-induced neurite extension as well as basic fibroblast growth factor (bFGF)-induced neuronal differentiation. However, of these cyclins, only antisense p39 prevented bFGF-induced neurite outgrowth. These studies indicate that cyclin D1 is neither necessary nor sufficient for morphological differentiation, that p35 is sufficient but not required, and that p39 is both necessary and sufficient for neurite outgrowth in the hippocampal cells. Taken together, these results represent the first demonstration of a specific role for p39 in neuronal differentiation, implicate the cyclin-activated kinase cdk5 in this process, and indicate that p39 is able to mediate neurite outgrowth in the presence or absence of cyclin D1.

Animals↗

PubMed1997

Differentiation of central nervous system neuronal cells by fibroblast-derived growth factor requires at least two signaling pathways: roles for Ras and Src.

To evaluate the role of mitogen-activated protein (MAP) kinase and other signaling pathways in neuronal cell differentiation by basic fibroblast-derived growth factor (bFGF), we used a conditionally immortalized cell line from rat hippocampal neurons (H19-7). Previous studies have shown that activation of MAP kinase kinase (MEK) is insufficient to induce neuronal differentiation of H19-7 cells. To test the requirement for MEK and MAP kinase (ERK1 and ERK2), H19-7 cells were treated with the MEK inhibitor PD098059. Although the MEK inhibitor blocked the induction of differentiation by constitutively activated Raf, the H19-7 cells still underwent differentiation by bFGF. These results suggest that an alternative pathway is utilized by bFGF for differentiation of the hippocampal neuronal cells. Expression in the H19-7 cells of a dominant-negative Ras (N17-Ras) or Raf (C4-Raf) blocked differentiation by bFGF, suggesting that Ras and probably Raf are required. Expression of dominant-negative Src (pcSrc295Arg) or microinjection of an anti-Src antibody blocked differentiation by bFGF in H19-7 cells, indicating that bFGF also signals through a Src kinase-mediated pathway. Although neither constitutively activated MEK (MEK-2E) nor v-Src was sufficient individually to differentiate the H19-7 cells, coexpression of constitutively activated MEK and v-Src induced neurite outgrowth. These results suggest that (i) activation of MAP kinase (ERK1 and ERK2) is neither necessary nor sufficient for differentiation by bFGF; (ii) activation of Src kinases is necessary but not sufficient for differentiation by bFGF; and (iii) differentiation of H19-7 neuronal cells by bFGF requires at least two signaling pathways activated by Ras and Src.

Animals↗

PubMed1997

Cyclin D1 is required for S phase traversal in bovine tracheal myocytes.

We examined the role of cyclin D1 in cultured bovine tracheal myocyte mitogenesis. Immunoblots using a polyclonal antibody against cyclin D1 showed the appearance of this protein 4 h after treatment with platelet-derived growth factor (PDGF), a potent mitogen for these cells. Immunoblots utilizing an antibody against the 110-kDa retinoblastoma protein (Rb), a downstream phosphorylation target of the cyclin D1/cyclin-dependent kinase 4 (cdk4) complex, showed reduced electrophoretic mobility of this protein as early as 8 h after PDGF treatment, suggesting phosphorylation of Rb by the cyclin D1/cdk4 dimer in vivo. Epidermal growth factor (EGF), a nonmitogen, failed to induce either cyclin D1 synthesis or Rb phosphorylation. PDGF treatment of cells transiently transfected with the full-length cyclin D1 promoter subcloned into a luciferase reporter increased luciferase activity almost threefold, demonstrating transcriptional activation of the cyclin D1 promoter with mitogenic stimulation. Finally, microinjection of individual myocytes with an affinity-purified antibody against cyclin D1 reduced the percentage of cells traversing S phase after serum stimulation, as assessed by fractional bromodeoxyuridine labeling (isotype control antibody, 0.74 +/- 0.10; anti-cyclin D1, 0.22 +/- 0.04; P = 0.0001). We conclude that 1) mitogenic stimulation of cultured bovine tracheal myocytes by PDGF induces cyclin D1 transcriptional activation and protein expression, 2) cyclin D1 expression is accompanied by Rb phosphorylation, which is evidence of increased cyclin D1-associated kinase activity in vivo, and 3) microinjection of anti-cyclin D1 antibodies inhibits cellular DNA synthesis, which is evidence that cyclin D1 is required for airway smooth muscle S phase traversal.

Animals↗

PubMed1997

Insulin-degrading enzyme does not require peroxisomal localization for insulin degradation.

Although considerable evidence implicates insulin-degrading enzyme (IDE) in the cellular metabolism of insulin in many cell types, its mechanism and site of action are not clear. In this study, we have examined the relationship between insulin-degrading enzyme's peroxisomal location and its ability to degrade insulin by mutation of its peroxisomal targeting signal (PTS), the carboxy terminal A/S-K-L tripeptide. Site-directed mutagenesis was used to destroy the peroxisomal targeting signal of human insulin-degrading enzyme by changing alanine to leucine (AL.pts), leucine to valine (LV.pts), or by deleting the entire tripeptide (DEL.pts). The alanine or leucine mutants, when expressed in COS cells, were indistinguishable from wild-type insulin-degrading enzyme with respect to size (110 kDa), amount of immunoreactive material, ability to bind insulin, in vitro activity, and cellular degradation of insulin. In contrast, the deletion mutant was shorter in size (approximately 0 kDa) and unable to bind the hormone. Thus, although the tripeptide at insulin-degrading enzyme's carboxy terminus appeared to confer enzyme stability, the conserved sequence was not required for insulin degradation. Finally, an immunocytofluorescence study showed that, whereas a significant amount of the wild-type protein was localized in peroxisomes, none of the peroxisomal targeting mutants could be detected in these organelles. These findings indicate that insulin-degrading enzyme does not require peroxisomal localization for insulin degradation and suggest that this enzyme has multiple cellular functions.

Alanine↗

PubMed1996

Mutation of Di-leucine residues in the juxtamembrane region alters EGF receptor expression.

Di-leucine motifs have been implicated in the internalization or degradation of many membrane proteins. The epidermal growth factor receptor (EGFR) contains two di-leucine residues at 658 (TLRRLLQER) and 679 (NQALLRIL). To determine the role of these di-leucine motifs in regulating EGF receptor expression, activity, or ligand-induced degradation, the di-leucine residues at positions 658 or 679 were mutated to di-alanine residues, and the mutant receptors were stably expressed in CHO cells. The results indicate that mutation of either di-leucine motif generates and promotes cell surface expression of carboxy-truncated EGF receptors (M(r) 120, 140 kDa) that do not undergo EGF-induced autophosphorylation or degradation. In contrast, full-length EGF receptors (170 kDa) containing di-alanine substitutions resemble wild type receptors in that they respond to EGF by autophosphorylation, their tyrosine kinase activity is inhibited by protein kinase C, and they are degraded. The level of autophosphorylation of the 170 kDa mutant receptors and EGF-induced tyrosine phosphorylation of other cellular proteins is lower than that of the wild type receptor, consistent with formation of kinase-inactive heterodimers between the truncated and full-length mutant receptors. These results demonstrate that removal of either of the di-leucines leads to generation of inactivating carboxy-truncated receptors, suggesting that the two di-leucine motifs within the juxtamembrane region of the EGFR are important for ensuring normal receptor expression.

Alanine↗

PubMed1996

Role of mitogen-activated protein kinase kinase in regulation of the epidermal growth factor receptor by protein kinase C.

The epidermal growth factor receptor (EGFR) is regulated by at least two mechanisms involving protein kinase C (PKC), inhibition of EGF binding and inhibition of EGF-stimulated tyrosine kinase activity. In this study we investigated whether mitogen-activated protein kinase (MAPK) mediates the inhibitory effects of PKC on EGFR binding or kinase activity by pretreating NIH3T3 and Chinese hamster ovary cells expressing the EGFR with PD98059, an inhibitor of MAPK/extracellular signal-regulated kinase kinase (MEK). We also determined whether substitution of cysteine for threonine at residue 669, the site of MAPK phosphorylation of the EGFR, alters the inhibition of kinase activity by PKC. The results indicate that 1) PKC down-regulates EGFR tyrosine kinase activity by an MEK-dependent mechanism presumably involving MAPK; 2) the inhibition by PKC is not a direct result of phosphorylation of the EGFR by PKC or MAPK; 3) activation of MAPK is not sufficient to regulate EGFR kinase activity; and 4) PKC-mediated down-regulation of EGF binding and EGFR kinase activity occur by different mechanisms. These data are consistent with a model for regulation of the EGFR by other receptors whereby their activation of PKC, in conjunction with MAPK, results in the phosphorylation of a protein(s) that modulates EGFR kinase activity.

3T3 Cells↗

PubMed1996

Apoptosis induced by differentiation or serum deprivation in an immortalized central nervous system neuronal cell line.

To characterize the nature of programmed cell death (PCD) induced in neuronal cells during development, three regulators of apoptosis were investigated: one, the bcl-2-related genes, modulate cell survival, and the other two, the interleukin-1 beta converting enzyme (ICE)-related enzymes and the tumor suppressor protein p53, have been implicated as mediators of apoptosis. These regulators were studied in H19-7 cells, an SV40 Tts-immortalized rat hippocampal neuronal cell line that can be differentiated with basic fibroblast growth factor at the nonpermissive temperature, resulting in a rapid attrition of cells by apoptosis. PCD occurred by two mechanisms in H19-7 cells: The first was initiated by removal of serum from undifferentiated cells, and the second was a consequence of neuronal differentiation. In differentiated H19-7 cells, the survival time was increased by both human bcl-2 and bcl-xL, and this could be reversed by bcl-xs. Addition of a peptide inhibitor of the ICE enzyme family to H19-7 cells resulted in a transient protection against differentiation-associated apoptosis, whereas no further protection was observed in the BCL-2- or BCL-XL-expressing cells. Shifting the differentiated cells to 33 degrees C to inactivate p53 did not significantly affect the apoptotic process, indicating that apoptosis induced by neuronal differentiation is not dependent on the continued presence of p53. By contrast, in undifferentiated cells, cell loss induced by transfer to serum-free media occurred more rapidly on inactivation of large T, consistent with p53 involvement. This medium-induced decrease in cell survival could not be rescued by the ICE inhibitor but was partially rescued by BCL-2 or BCL-XL. Furthermore, studies involving expression of BCL-2 and BCL-XL alone or together revealed differences in the survival dependent on the cellular environment. These results suggest that apoptosis of neuronal cells occurs by at least two processes: one in undifferentiated cells initiated by removal of serum and one linked to differentiation. The data implicate the ICE enzyme family but not p53 in apoptosis induced by differentiation and demonstrate that either BCL-2 or BCL-XL can prolong the survival of differentiated neuronal cells.

Amino Acid Chloromethyl Ketones↗

PubMed1996

Raf, but not MEK or ERK, is sufficient for differentiation of hippocampal neuronal cells.

To elucidate signal transduction pathways leading to neuronal differentiation, we have investigated a conditionally immortalized cell line from rat hippocampal neurons (H19-7) that express a temperature sensitive simian virus 40 large T antigen. Treatment of H19-7 cells with the differentiating agent basic fibroblast growth factor at 39 degrees C, the nonpermissive temperature for T function, resulted in the activation of c-Raf-1, MEK, and mitogen-activated protein (MAP) kinases (ERK1 and -2). To evaluate the role of Raf-1 in neuronal cell differentiation, we stably transfected H19-7 cells with v-raf or an oncogenic human Raf-1-estrogen receptor fusion gene (deltaRaf-1:ER). deltaRaf-1:ER transfectants in the presence of estradiol for 1 to 2 days expressed a differentiation phenotype only at the nonpermissive temperature. However, extended exposure of the deltaRaf-1:ER transfectants to estradiol or stable expression of the v-raf construct yielded cells that extended processes at the permissive as well as the nonpermissive temperature, suggesting that cells expressing the large T antigen are capable of responding to the Raf differentiation signal. deltaRaf-1:ER, MEK, and MAP kinase activities in the deltaRaf-1:ER cells were elevated constitutively for up to 36 h of estradiol treatment at the permissive temperature. At the nonpermissive temperature, MEK and ERKs were activated to a significantly lesser extent, suggesting that prolonged MAP kinase activation may not be sufficient for differentiation. To test this possibility, H19-7 cells were transfected or microinjected with constitutively activated MEK. The results indicate that prolonged activation of MEK or MAP kinases (ERK1 and -2) is not sufficient for differentiation of H19-7 neuronal cells and raise the possibility that an alternative signaling pathway is required for differentiation of H19-7 cells by Raf.

Animals↗

PubMed1995

WT1 suppresses synthesis of the epidermal growth factor receptor and induces apoptosis.

The Wilms tumor suppressor gene WT1 encodes a developmentally regulated transcription factor that is mutated in a subset of embryonal tumors. To test its functional properties, we developed osteosarcoma cell lines expressing WT1 under an inducible tetracycline-regulated promoter. Induction of WT1 resulted in programmed cell death. This effect, which was differentially mediated by the alternative splicing variants of WT1, was independent of p53. WT1-mediated apoptosis was associated with reduced synthesis of the epidermal growth factor receptor (EGFR), but not of other postulated WT1-target genes, and it was abrogated by constitutive expression of EGFR. WT1 repressed transcription from the EGFR promoter, binding to two TC-rich repeat sequences. In the developing kidney, EGFR expression in renal precursor cells declined with the onset of WT1 expression. Repression of EGFR and induction of apoptosis by mechanism that may contribute to its critical role in normal kidney development and to the immortalization of tumor cells with inactivated WT1 alleles.

Alternative Splicing↗

PubMed1995

Histamine antagonizes serotonin and growth factor-induced mitogen-activated protein kinase activation in bovine tracheal smooth muscle cells.

We examined the effects of the bronchoconstrictor agonists serotonin (5-hydroxytryptamine; 5-HT) and histamine on mitogen-activated protein (MAP) kinase activation in cultured bovine tracheal myocytes. Kinase renaturation assays demonstrated activation of the 42- and 44-kDa MAP kinases within 2 min of 5-HT exposure. MAP kinase activation was mimicked by alpha-methyl-5-HT and reduced by pretreatment with either phorbol 12,13-dibutyrate or forskolin, suggesting activation of the 5-HT2 receptor, protein kinase C, and Raf-1, respectively. Raf-1 activation was confirmed by measurement of Raf-1 activity, and the requirement of Raf-1 for 5-HT-induced MAP kinase activation was demonstrated by transient transfection of cells with a dominant-negative allele of Raf-1. Histamine pretreatment significantly inhibited 5-HT and insulin-derived growth factor-1-induced MAP kinase activation. Attenuation of MAP kinase activation was reversed by cimetidine, mimicked by forskolin, and accompanied by cAMP accumulation and inhibition of Raf-1, suggesting activation of the H2 receptor and cAMP-dependent protein kinase A. However, histamine treatment inhibited Raf-1 but not MAP kinase activation following treatment with either platelet-derived growth factor or epidermal growth factor, implying a Raf-1-independent MAP kinase activation pathway. In summary, our data suggest a model whereby 5-HT activates MAP kinase via a protein kinase C/Raf-1 pathway, and histamine attenuates MAP kinase activation by serotonin via activation of cAMP-dependent protein kinase A and inhibition of Raf-1.

Animals↗

PubMed1995

Role of MAP kinase activation in bovine tracheal smooth muscle mitogenesis.

Abnormal growth of airway smooth muscle may play an important role in the pathogenesis of human airway diseases. Little is known about the proliferative responses of cultured airway smooth muscle cells, nor of the precise pathways responsible for mitogenesis in these cells. We assessed DNA synthesis, cell proliferation, and mitogen-activated protein (MAP) kinase activation in bovine tracheal myocytes after exposure to four potential mitogens: platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), and 5-hydroxytryptamine (5-HT). Stimulation with either PDGF or IGF-1 induced substantial increases in DNA synthesis and cell number, as reflected by [3H]thymidine incorporation, flow cytometry, and methylene blue staining. Treatment with EGF or 5-HT, on the other hand, induced only modest DNA synthesis and no increase in cell number. Immunoblots and kinase renaturation assays of cell extracts demonstrated activation of both the 42- and 44-kDa MAP kinases within minutes of either PDGF, IGF-1, EGF, or 5-HT exposure. However, relative to EGF and 5-HT stimulation, late-phase MAP kinase activation was significantly greater after treatment with the mitogens PDGF and IGF-1. We conclude that in cultured bovine tracheal myocytes 1) PDGF and IGF-1 are potent mitogens; 2) MAP kinase may be activated subsequent to stimulation of either receptor tyrosine kinases (PDGF, EGF, IGF-1) or G protein-linked receptors lacking in known tyrosine kinase activity (5-HT); and 3) unsustained MAP kinase activation is insufficient for mitogenesis. Finally, the finding that mitogenicity correlates with the late phase of MAP kinase activation is consistent with the notion that sustained MAP kinase activation is important for bovine tracheal myocyte proliferation.

Animals↗

PubMed1994

Identification of zinc ligands of the insulin-degrading enzyme.

The insulin degrading enzyme (IDE), a nonlysosomal enzyme involved in the metabolism of internalized insulin, is a member of a new family of metalloproteases which has an HXXEH active site motif. We have previously shown that both His108 and Glu111 within the HXCEH domain of human IDE are necessary for catalytic activity. Comparison to the prototypic zinc metalloprotease thermolysin, which contains an inversion of this motif, would predict that His112, as well as a downstream glutamate, serves as the second and third zinc ligands of IDE. To examine the role of His112, we mutated this residue to glutamine, leucine, or arginine. To identify a downstream zinc ligand, we substituted a glutamine for glutamate at either Glu182 or Glu189, both of which are conserved in human, rat, and Drosophila IDE. Vectors containing wild type or mutant IDE genes were transfected into COS cells, and the enzymes were analyzed for insulin degradation, insulin cross-linking, and zinc binding. Our results suggest that His108, His112, and Glu189 are the zinc ligands of human IDE, and Glu182 can influence zinc binding. In addition to a catalytic role, zinc binding to these residues appears to play a role in stabilizing the structure of the enzyme.

Amino Acid Sequence↗

PubMed1994

Conditional immortalization of neuronal cells from postmitotic cultures and adult CNS.

To determine whether postmitotic neurons can be immortalized by oncogenic transduction, we used two approaches involving conditional expression of a temperature-sensitive SV40 large T antigen (Tts). Initially, Tts was introduced into E17 rat embryonal hippocampal cells that were then cultured at the non-permissive temperature to enrich for postmitotic pyramidal neurons, and subsequently cloned at the permissive temperature. One clonal line (HMR10-3) expressed neuron-specific proteins upon differentiation, was capable of generating action potentials, and formed synapses with primary rat neurons in co-culture. Replating of these postmitotic cells at the permissive temperature resulted in reversible loss of neurofilament expression. Conditionally immortalized cell lines were also generated from the brain of an adult mouse carrying an inducible Tts transgene. These lines proliferated in a T antigen-dependent manner and expressed neuron-specific proteins upon differentiation at the non-permissive temperature. These results suggest that postmitotic neurons can be induced to enter the cell cycle without losing their commitment to a neuronal lineage.

Animals↗

PubMed1994

Inducible expression and cellular localization of insulin-degrading enzyme in a stably transfected cell line.

Insulin degrading enzyme (IDE) is an evolutionarily conserved, nonlysosomal metalloprotease that has been implicated in the cellular degradation and processing of insulin. However, the site and the mode of the action of this enzyme are unclear. We have addressed these questions by establishing several Ltk- cell lines that can overexpress human insulin-degrading enzyme (hIDE) upon glucocorticoid induction. The level of overexpression of hIDE protein and transcripts in these lines correlates well with an increase in insulin degradation in both cell lysates and intact cells. Comparison of the deduced amino acid sequences of mammalian and Drosophila IDEs reveals a conserved carboxyl-terminal peroxisomal targeting sequence (A/S-K-L), suggesting that IDE may be localized in peroxisomes. To test this possibility, we determined the cellular location of the stably transfected hIDE by both immunofluorescence and immunocryoelectron microscopy. The overexpressed hIDE predominantly colocalized with catalase in peroxisomes, although IDE was also found in the cytosol at a much lower concentration. These results demonstrate that stably transfected IDE catalyzes a rate-limiting step in cellular insulin degradation and is localized predominantly in peroxisomes.

Amino Acid Sequence↗

PubMed1994

Differential Raf requirement for activation of mitogen-activated protein kinase by growth factors, phorbol esters, and calcium.

Although a pathway that requires sequential activation of Ras, Raf, and MAP kinase kinase has been proposed as the major mechanism for stimulation of mitogen-activated protein kinase (MAP kinase), alternative pathways also exist. A wide variety of extracellular stimuli have been shown to activate MAP kinase; however, the precise mechanisms by which these stimuli mediate the signaling events have not been elucidated. Using a Balb/c-derived cell line expressing a dominant-negative mutant of Raf, we determined whether Raf is required for the activation of MAP kinase by growth factors, phorbol esters, and calcium. Insulin-like growth factor I (IGF-I), epidermal growth factor (EGF), and phorbol 12,13-dibutyrate activated Ras in both mutant and control cells. However, stimulation of MAP kinase by IGF-I was nearly abolished in the dominant-negative Raf mutant. Stimulation of MAP kinase by the Ca2+ mobilizer thapsigargin was also inhibited in the presence of the Raf mutant. In contrast, EGF and phorbol 12,13-dibutyrate remained potent stimulators of MAP kinase in the dominant-negative Raf cells. The activation of MAP kinase by these stimuli can be further distinguished by differential requirements for Ca2+ and protein kinase C. These results suggest that Raf is required for the activation of MAP kinase by IGF-I and calcium, whereas EGF and possibly phorbol esters may employ alternative Raf-independent pathways for MAP kinase activation.

3T3 Cells↗

PubMed1994

Identification of an epidermal growth factor receptor transcriptional repressor.

Identification of the factors controlling transcription of the epidermal growth factor (EGF) receptor gene is essential for understanding regulation of the EGF receptor and its overexpression in human carcinomas. In this study, we have identified a 60-base pair (bp) region (-919 to -860) relative to the AUG translation initiation codon in the EGF receptor 5' promoter that functions as a cis-acting EGF receptor transcriptional repressor (ETR). This fragment also acted as a repressor when linked to the thymidine kinase promoter. Gel mobility shift assays demonstrated that trans-acting factors bind to 60- and 19-bp fragments. Competition and chloramphenicol acetyltransferase assays with oligonucleotides containing mutations and deletions in this region indicate that the TTCGAGGG sequence (-877 to -870) is required for binding as well as repressor activity. While the ETR-protected region contains consensus sequences for the E2F binding site, no competition was observed with an E2F binding fragment. However, DNA-protein blot analysis indicates that both the 60- and 19-bp fragments specifically bind a 128-kDa polypeptide in extracts from HeLa or A431 human epidermoid carcinoma cells. These results suggest that a novel transcription factor(s) negatively regulates EGF receptor gene expression through binding to the ETR element.

Base Sequence↗