scholarly journals Gab1 and SHP-2 promote Ras/MAPK regulation of epidermal growth and differentiation

2002 ◽  
Vol 159 (1) ◽  
pp. 103-112 ◽  
Author(s):  
Ti Cai ◽  
Keigo Nishida ◽  
Toshio Hirano ◽  
Paul A. Khavari
Keyword(s):  
Epidermal Cell ◽  
Mapk Signaling ◽  
Dominant Negative ◽  
Mapk Activity ◽  
Ras Activation ◽  
Proliferation And Differentiation ◽  
Epidermal Proliferation ◽  
Epidermal Growth ◽  
Docking Protein

În epidermis, Ras can influence proliferation and differentiation; however, regulators of epidermal Ras function are not fully characterized, and Ras effects on growth and differentiation are controversial. EGF induced Ras activation in epidermal cells along with phosphorylation of the multisubstrate docking protein Gab1 and its binding to SHP-2. Expression of mutant Gab1Y627F deficient in SHP-2 binding or dominant-negative SHP-2C459S reduced basal levels of active Ras and downstream MAPK proteins and initiated differentiation. Differentiation triggered by both Gab1Y627F and SHP-2C459S could be blocked by coexpression of active Ras, consistent with Gab1 and SHP-2 action upstream of Ras in this process. To study the role of Gab1 and SHP-2 in tissue, we generated human epidermis overexpressing active Gab1 and SHP-2. Both proteins stimulated proliferation. In contrast, Gab1Y627F and SHP-2C459S inhibited epidermal proliferation and enhanced differentiation. Consistent with a role for Gab1 and SHP-2 in sustaining epidermal Ras/MAPK activity, Gab1−/− murine epidermis displayed lower levels of active Ras and MAPK with postnatal Gab1−/− epidermis, demonstrating the hypoplasia and enhanced differentiation seen previously with transgenic epidermal Ras blockade. These data provide support for a Ras role in promoting epidermal proliferation and opposing differentiation and indicate that Gab1 and SHP-2 promote the undifferentiated epidermal cell state by facilitating Ras/MAPK signaling.

Ras palmitoylation is necessary for N-Ras activation and signal propagation in growth factor signalling

2013 ◽  
Vol 454 (2) ◽  
pp. 323-332 ◽  
Author(s):  
Shu-Ping Song ◽  
Anne Hennig ◽  
Katja Schubert ◽  
Robby Markwart ◽  
Philipp Schmidt ◽  
...  
Keyword(s):  
Growth Factor ◽  
Signal Propagation ◽  
Dominant Negative ◽  
Post Translational Modifications ◽  
Ras Gtpases ◽  
Ras Activation ◽  
Growth Factor Signalling ◽  
Mitogenic Signalling ◽  
Epidermal Growth

Ras GTPases undergo post-translational modifications that govern their subcellular trafficking and localization. In particular, palmitoylation of the Golgi tags N-Ras and H-Ras for exocytotic transport and residency at the PM (plasma membrane). Following depalmitoylation, PM-Ras redistributes to all subcellular membranes causing an accumulation of palmitate-free Ras at endomembranes, including the Golgi and endoplasmic reticulum. Palmitoylation is unanimously regarded as a critical modification at the crossroads of Ras activity and trafficking control, but its precise relevance to native wild-type Ras function in growth factor signalling is unknown. We show in the present study by use of palmitoylation-deficient N-Ras mutants and via the analysis of palmitate content of agonist-activated GTP-loaded N-Ras that only palmitoylated N-Ras becomes activated by agonists. In line with an essential role of palmitoylation in Ras activation, dominant-negative RasS17N loses its blocking potency if rendered devoid of palmitoylation. Live-cell Ras–GTP imaging shows that N-Ras activation proceeds only at the PM, consistent with activated N-Ras–GTP being palmitoylated. Finally, palmitoylation-deficient N-Ras does not sustain EGF (epidermal growth factor) or serum-elicited mitogenic signalling, confirming that palmitoylation is essential for signal transduction by N-Ras. These findings document that N-Ras activation proceeds at the PM and suggest that depalmitoylation, by removing Ras from the PM, may contribute to the shutdown of Ras signalling.

scholarly journals Epidermal growth factor and Ras regulate gene expression in GH4 pituitary cells by separate, antagonistic signal transduction pathways.

1995 ◽  
Vol 15 (12) ◽  
pp. 6777-6784 ◽  
Author(s):  
C A Pickett ◽  
A Gutierrez-Hartmann
Keyword(s):  
Signal Transduction ◽  
Transcription Factors ◽  
Growth Factor ◽  
Map Kinase ◽  
Dominant Negative ◽  
Neuroendocrine Cells ◽  
Dependent Manner ◽  
Epidermal Growth ◽  
Dose Dependent

We have previously demonstrated that epidermal growth factor (EGF) produces activation of the rat prolactin (rPRL) promoter in GH4 neuroendocrine cells via a Ras-independent mechanism. This Ras independence of the EGF response appears to be cell rather than promoter specific. Oncogenic Ras also produces activation of the rPRL promoter when transfected into GH4 cells and requires the sequential activation of Raf kinase, mitogen-activated protein (MAP) kinase, and c-Ets-1/GHF-1 to mediate this response. In these studies, we have investigated the interaction between EGF and Ras in stimulating rPRL promoter activity and the role of Raf and MAP kinases in mediating the EGF response. We have also examined the role of several transcription factors and used various promoter mutants of the rPRL gene in order to better define the trans- and cis-acting components of the EGF response. EGF treatment of GH4 cells inhibits activation of the rPRL promoter produced by transfection of V12Ras from 24- to 4-fold in an EGF dose-dependent manner. This antagonistic effect of EGF and Ras is mutual in that transfection of V12Ras also blocks EGF-induced activation of the rPRL promoter in a Ras dose-dependent manner, from 5.5- to 1.6-fold. Transfection of a plasmid encoding the dominant-negative Raf C4 blocks Ras-induced activation by 66% but fails to inhibit EGF-mediated activation of the rPRL promoter. Similarly, transfection of a construct encoding an inhibitory form of MAP kinase decreases the Ras response by 50% but does not inhibit the EGF response. Previous studies have demonstrated that c-Ets-1 is necessary and that GHF-1 acts synergistically with c-Ets-1 in the Ras response of the rPRL promoter. In contrast, overexpression of neither c-Ets-1 nor GHF-1 enhanced EGF-mediated activation of the rPRL promoter, and dominant-negative forms of these transcription factors failed to inhibit the EGF response. Using 5' deletion and site-specific mutations, we have mapped the EGF response to two regions on the proximal rPRL promoter. One region maps between -255 and -212, near the Ras response element, and a second maps between -125 and -54. The latter region appears to involve footprint 2, a previously identified repressor site on the rPRL promoter. Neither footprint 1 nor 3, known GHF-1 binding sites, appears to be crucial to RGF-mediated rPRL promoter activation. The results of these studies indicate that in GH4 neuroendocrine cells, rPRL gene regulation by EGF is mediated by a signal transduction pathway that is separate and antagonistic to the Ras pathway. Hence, the functional role of the Ras/Raf/MAP kinase pathway in mediating transcriptional responses to EGF and other receptor tyrosine kinase may differ in highly specialized cell types.

Role of Epidermal Cell Thymocyte-Activating Factor in the Proliferation and Differentiation of Murine B Cells

1987 ◽  
Vol 42 (3) ◽  
pp. 222-229 ◽  
Author(s):  
P.S. Pillai ◽  
S.D. Reynolds ◽  
D.W. Scott ◽  
J. Gauldie ◽  
D.N. Sauder
Keyword(s):  
B Cells ◽  
Epidermal Cell ◽  
Proliferation And Differentiation

Role of cAMP inhibition of p44/p42 mitogen-activated protein kinase in potentiation of protein secretion in rat lacrimal gland

2007 ◽  
Vol 293 (5) ◽  
pp. C1551-C1560 ◽  
Author(s):  
Chika Funaki ◽  
Robin R. Hodges ◽  
Darlene A. Dartt
Keyword(s):  
Protein Kinase ◽  
Protein Secretion ◽  
Lacrimal Gland ◽  
Mitogen Activated Protein Kinase ◽  
Adrenergic Agonist ◽  
Mapk Activity ◽  
Mitogen Activated Protein ◽  
Epidermal Growth ◽  
Camp Levels

We previously found that addition of cAMP and a Ca2+/PKC-dependent agonist causes synergism or potentiation of protein secretion from rat lacrimal gland acini. In the present study we determined whether cAMP decreases p44/p42 mitogen-activated protein kinase (MAPK) activity in the lacrimal gland. Since we know that activation of MAPK attenuates protein secretion stimulated by Ca2+- and PKC-dependent agonists, we also determined whether this activation causes potentiation of secretion. Freshly prepared rat lacrimal gland acinar cells were incubated with dibutyryl cAMP (DBcAMP), carbachol (a cholinergic agonist), phenylephrine (an α1-adrenergic agonist), or epidermal growth factor (EGF). The latter three agonists are known to activate p44/p42 MAPK. p44/p42 MAPK activity and protein secretion were measured. As measured by Western blot analysis, DBcAMP inhibited both basal and agonist-stimulated p44/p42 MAPK activity. Cellular cAMP levels were increased by 1) using two different cell-permeant cAMP analogs, 2) activating adenylyl cyclase (L-858051), or 3) activation of Gs-coupled receptors (VIP). The cell-permeant cAMP analogs, L-858051, and VIP inhibited basal p44/p42 MAPK activity by 50, 40, and 40%, respectively. DBcAMP and VIP inhibited carbachol- and EGF-stimulated MAPK activity. cAMP, but not VIP, inhibited phenylephrine-stimulated MAPK activity. Potentiation of secretion was detected when carbachol, phenylephrine, or EGF was simultaneously added with DBcAMP. We conclude that increasing cellular cAMP levels inhibits p44/p42 MAPK activity and that this could account for potentiation of secretion obtained when cAMP was elevated and Ca2+ and PKC were increased by agonists.

scholarly journals Involvement of Prolonged Ras Activation in Thrombopoietin-Induced Megakaryocytic Differentiation of a Human Factor-Dependent Hematopoietic Cell Line

1998 ◽  
Vol 18 (7) ◽  
pp. 4282-4290 ◽  
Author(s):  
Itaru Matsumura ◽  
Koichi Nakajima ◽  
Hiroshi Wakao ◽  
Seisuke Hattori ◽  
Koji Hashimoto ◽  
...  
Keyword(s):  
Cell Line ◽  
Tyrosine Kinases ◽  
Intracellular Signaling ◽  
Time Course ◽  
Dominant Negative ◽  
High Rate ◽  
Ras Pathway ◽  
Megakaryocytic Differentiation ◽  
Ras Activation ◽  
Proliferation And Differentiation

ABSTRACT Thrombopoietin (TPO) is a hematopoietic growth factor that plays fundamental roles is both megakaryopoiesis and thrombopoiesis through binding to its receptor, c-mpl. Although TPO has been shown to activate various types of intracellular signaling molecules, such as the Janus family of protein tyrosine kinases, signal transducers and activators of transcription (STATs), and ras, the precise mechanisms underlying TPO-induced proliferation and differentiation remain unknown. In an effort to clarify the mechanisms of TPO-induced proliferation and differentiation, c-mpl was introduced into F-36P, a human interleukin-3 (IL-3)-dependent erythroleukemia cell line, and the effects of TPO on the c-mpl-transfected F-36P (F-36P-mpl) cells were investigated. F-36P-mpl cells were found to proliferate and differentiate at a high rate into mature megakaryocytes in response to TPO. Dominant-negative (dn) forms of STAT1, STAT3, STAT5, and ras were inducibly expressed in F-36P-mpl cells, and their effects on TPO-induced proliferation and megakaryocytic differentiation were analyzed. Among these dn molecules, both dn ras and dn STAT5 reduced TPO- or IL-3-induced proliferation of F-36P-mpl cells by ∼30%, and only dn ras could inhibit TPO-induced megakaryocytic differentiation. In accord with this result, overexpression of activated ras (H-rasG12V) for 5 days led to megakaryocytic differentiation of F-36P-mpl cells. In a time course analysis on H-rasG12V-induced differentiation, activation of the ras pathway for 24 to 28 h was required and sufficient to induce megakaryocytic differentiation. Consistent with this result, the treatment of F-36P-mpl cells with TPO was able to induce prolonged activation of ras for more than 24 h, whereas IL-3 had only a transient effect. These results suggest that prolonged ras activation may be involved in TPO-induced megakaryocytic differentiation.

Carbachol activates ERK2 in isolated gastric parietal cells via multiple signaling pathways

1999 ◽  
Vol 276 (6) ◽  
pp. G1484-G1492 ◽  
Author(s):  
Yoshiaki Takeuchi ◽  
Nonthalee Pausawasdi ◽  
Andrea Todisco
Keyword(s):  
Molecular Mechanisms ◽  
Parietal Cells ◽  
Dominant Negative ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Ras Activation ◽  
Gastric Parietal Cells ◽  
Tyrosyl Phosphorylation

We previously reported that both carbachol and epidermal growth factor (EGF) are potent inducers of the extracellular signal-regulated protein kinases (ERKs) in isolated gastric canine parietal cells and that induction of these kinases leads to acute inhibitory and chronic stimulatory effects on gastric acid secretion. In this study we investigated the molecular mechanisms responsible for these effects. Both carbachol (100 μM) and EGF (10 nM) induced Ras activation. The role of Ras in ERK2 induction was examined by transfecting parietal cells with a vector expressing hemoagglutinin (HA)-tagged ERK2 (HA-ERK2) together with a dominantly expressed mutant (inactive) ras gene. HA-ERK2 activity was quantitated by in-gel kinase assays. Dominant negative Ras reduced carbachol induction of HA-ERK2 activity by 60% and completely inhibited the stimulatory effect of EGF. Since Ras activation requires the assembly of a multiprotein complex, we examined the effect of carbachol and EGF on tyrosyl phosphorylation of Shc and its association with Grb2 and the guanine nucleotide exchange factor Sos. Western blot analysis of anti-Shc immunoprecipitates with an anti-phosphotyrosine antibody demonstrated that both carbachol and EGF induced tyrosyl phosphorylation of a major 52-kDa shc isoform. Grb2 association with Shc was demonstrated by blotting Grb2 immunoprecipitates with an anti-Shc antibody. Probing of anti-Sos immunoprecipitates with an anti-Grb2 antibody revealed that Sos was constitutively bound to Grb2. To examine the functional role of Sos in ERK2 activation, we transfected parietal cells with the HA-ERK2 vector together with a dominantly expressed mutant (inactive) sos gene. Dominant negative Sos did not affect carbachol stimulation of HA-ERK2 but inhibited the stimulatory effect of EGF by 60%. We then investigated the role of βγ-subunits in carbachol induction of HA-ERK2. Parietal cells were transfected with the HA-ERK2 vector together with a vector expressing the carboxy terminus of the β-adrenergic receptor kinase 1, known to block signaling mediated by βγ-subunits. In the presence of this vector, carbachol induction of HA-ERK2 was inhibited by 40%. Together these data suggest that, in the gastric parietal cells, carbachol activates the ERKs through Ras- and βγ-dependent mechanisms that require guanine nucleotide exchange factors other than Sos.

scholarly journals p50cdc37 Acting in Concert with Hsp90 Is Required for Raf-1 Function

1999 ◽  
Vol 19 (3) ◽  
pp. 1661-1672 ◽  
Author(s):  
Nicholas Grammatikakis ◽  
Jun-Hsiang Lin ◽  
Aliki Grammatikakis ◽  
Philip N. Tsichlis ◽  
Brent H. Cochran
Keyword(s):  
Mapk Pathway ◽  
Specific Inhibitor ◽  
Mapk Signaling ◽  
Dominant Negative ◽  
Sf9 Cells ◽  
Genetic Screens ◽  
Kinase Activation ◽  
Primary Determinant ◽  
Hsp90 Chaperone

ABSTRACT Genetic screens in Drosophila have identified p50 cdc37 to be an essential component of the sevenless receptor/mitogen-activated kinase protein (MAPK) signaling pathway, but neither the function nor the target of p50 cdc37 in this pathway has been defined. In this study, we examined the role of p50 cdc37 and its Hsp90 chaperone partner in Raf/Mek/MAPK signaling biochemically. We found that coexpression of wild-type p50 cdc37 with Raf-1 resulted in robust and dose-dependent activation of Raf-1 in Sf9 cells. In addition, p50 cdc37 greatly potentiated v-Src-mediated Raf-1 activation. Moreover, we found that p50 cdc37 is the primary determinant of Hsp90 recruitment to Raf-1. Overexpression of a p50 cdc37 mutant which is unable to recruit Hsp90 into the Raf-1 complex inhibited Raf-1 and MAPK activation by growth factors. Similarly, pretreatment with geldanamycin (GA), an Hsp90-specific inhibitor, prevented both the association of Raf-1 with the p50 cdc37 -Hsp90 heterodimer and Raf-1 kinase activation by serum. Activation of Raf-1 via baculovirus coexpression with oncogenic Src or Ras in Sf9 cells was also strongly inhibited by dominant negative p50 cdc37 or by GA. Thus, formation of a ternary Raf-1–p50 cdc37 –Hsp90 complex is crucial for Raf-1 activity and MAPK pathway signaling. These results provide the first biochemical evidence for the requirement of the p50 cdc37 -Hsp90 complex in protein kinase regulation and for Raf-1 function in particular.

scholarly journals Insulin increases the adrenergic stimulation of 5′ deiodinase activity and mRNA expression in rat brown adipocytes; role of MAPK and PI3K

2005 ◽  
Vol 34 (1) ◽  
pp. 139-151 ◽  
Author(s):  
Raquel Martinez-deMena ◽  
Maria-Jesus Obregón
Keyword(s):  
Mrna Expression ◽  
Primary Cultures ◽  
Mapk Signaling ◽  
Adrenergic Stimulation ◽  
Brown Adipocytes ◽  
Mapk Activity ◽  
Absolute Requirement ◽  
Brown Adipose ◽  
Stimulation Of

Type II 5′ deiodinase (D2) activity produces triiodothyronine (T3) from thyroxine (T4) and is induced by cold and norepinephrine (NE) in brown adipose tissue. T3 is required for and amplifies the adrenergic stimulation of D2 activity and mRNA in cultured brown adipocytes. D2 is upregulated by insulin and decrease in fasting. We now study the regulation by insulin of the adrenergically induced D2 activity and mRNA in primary cultures of rat brown adipocytes. Insulin alone does not increase D2 activity or mRNA. Insulin-depleted cells show a reduction in the adrenergically induced D2 activity, which is proportional to the length of insulin depletion and is restored after insulin addition. IGFs mimic this effect at higher doses. ERK 1/2 MAPK activity (p44/p42), stimulated by insulin, serum and NE, is an absolute requirement for the adrenergic stimulation of D2 activity and mRNA. PI3K is stimulated by insulin and serum, and NE increases the effect of insulin. The action of insulin on D2 is not due to changes in D2 half-life or in the proteasome-mediated degradation of D2, but it seems to modulate the transcriptional induction mediated by NE. D2 mRNA expression, induced by NE plus T3, is reduced when insulin is withdrawn at early differentiation stages. Insulin or IGF-I promotes increases in D2 mRNA. Insulin is required for the induction of D2 mRNA by T3. In conclusion, MAPK signaling is required for the adrenergic stimulation of D2 activity and mRNA, and insulin stimulates D2 activity via MAPK and PI3K and enhances the adrenergic pathways.

Role of Ras signaling in erythroid differentiation of mouse fetal liver cells: functional analysis by a flow cytometry–based novel culture system

Blood ◽  
2003 ◽  
Vol 102 (12) ◽  
pp. 3938-3946 ◽  
Author(s):  
Jing Zhang ◽  
Merav Socolovsky ◽  
Alec W. Gross ◽  
Harvey F. Lodish
Keyword(s):  
Flow Cytometry ◽  
Colony Formation ◽  
Culture System ◽  
Fetal Liver ◽  
Erythroid Differentiation ◽  
Dominant Negative ◽  
Ras Signaling ◽  
Proliferation And Differentiation

Abstract Ras signaling plays an important role in erythropoiesis. Its function has been extensively studied in erythroid and nonerythroid cell lines as well as in primary erythroblasts, but inconclusive results using conventional erythroid colony-forming unit (CFU-E) assays have been obtained concerning the role of Ras signaling in erythroid differentiation. Here we describe a novel culture system that supports terminal fetal liver erythroblast proliferation and differentiation and that closely recapitulates erythroid development in vivo. Erythroid differentiation is monitored step by step and quantitatively by a flow cytometry analysis; this analysis distinguishes CD71 and TER119 double-stained erythroblasts into different stages of differentiation. To study the role of Ras signaling in erythroid differentiation, different H-ras proteins were expressed in CFU-E progenitors and early erythroblasts with the use of a bicistronic retroviral system, and their effects on CFU-E colony formation and erythroid differentiation were analyzed. Only oncogenic H-ras, not dominant-negative H-ras, reduced CFU-E colony formation. Analysis of infected erythroblasts in our newly developed system showed that oncogenic H-ras blocks terminal erythroid differentiation, but not through promoting apoptosis of terminally differentiated erythroid cells. Rather, oncogenic H-ras promotes abnormal proliferation of CFU-E progenitors and early erythroblasts and supports their erythropoietin (Epo)–independent growth.

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