scholarly journals αvβ3 Integrins and Pyk2 Mediate Insulin-Like Growth Factor I Activation of Src and Mitogen-Activated Protein Kinase in 3T3-L1 Cells

2005 ◽  
Vol 19 (7) ◽  
pp. 1859-1867 ◽  
Author(s):  
Hiroko Sekimoto ◽  
Jodi Eipper-Mains ◽  
Sunthorn Pond-Tor ◽  
Charlotte M. Boney
Keyword(s):  
Binding Site ◽  
Mapk Pathway ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Immunofluorescent Staining ◽  
Calcium Dependent ◽  
Tyrosine Kinase 2 ◽  
Igf I ◽  
Αvβ3 Integrins ◽  
Inside Out

Abstract IGF-I stimulates cell growth through interaction of the IGF receptor with multiprotein signaling complexes. However, the mechanisms of IGF-I receptor-mediated signaling are not completely understood. We have previously shown that IGF-I-stimulated 3T3-L1 cell proliferation is dependent on Src activation of the ERK-1/2 MAPK pathway. We hypothesized that IGF-I activation of the MAPK pathway is mediated through integrin activation of Src-containing signaling complexes. The disintegrin echistatin decreased IGF-I phosphorylation of Src and MAPK, and blocking antibodies to αv and β3 integrin subunits inhibited IGF-I activation of MAPK, suggesting that αvβ3 integrins mediate IGF-I mitogenic signaling. IGF-I increased ligand binding to αvβ3 as detected by immunofluorescent staining of ligand-induced binding site antibody and stimulated phosphorylation of the β3 subunit, consistent with inside-out activation of αvβ3 integrins. IGF-I increased tyrosine phosphorylation of the focal adhesion kinase (FAK) Pyk2 (calcium-dependent proline-rich tyrosine kinase-2) to a much greater extent than FAK, and increased association of Src with Pyk2 but not FAK. The intracellular calcium chelator BAPTA prevented IGF-I phosphorylation of Pyk2, Src, and MAPK, suggesting that IGF-I activation of Pyk2 is calcium dependent. Transient transfection with a dominant-negative Pyk2, which lacks the autophosphorylation and Src binding site, decreased IGF-I activation of MAPK, but no inhibition was seen with transfected wild-type Pyk2. These results indicate that IGF-I signaling to MAPK is dependent on inside-out activation of αvβ3 integrins and integrin-facilitated multiprotein complex formation involving Pyk2 activation and association with Src.

scholarly journals Critical role of the extracellular signal–regulated kinase–MAPK pathway in osteoblast differentiation and skeletal development

2007 ◽  
Vol 176 (5) ◽  
pp. 709-718 ◽  
Author(s):  
Chunxi Ge ◽  
Guozhi Xiao ◽  
Di Jiang ◽  
Renny T. Franceschi
Keyword(s):  
Transcriptional Activity ◽  
Mapk Pathway ◽  
Skeletal Development ◽  
Critical Role ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal

The extracellular signal–regulated kinase (ERK)–mitogen-activated protein kinase (MAPK) pathway provides a major link between the cell surface and nucleus to control proliferation and differentiation. However, its in vivo role in skeletal development is unknown. A transgenic approach was used to establish a role for this pathway in bone. MAPK stimulation achieved by selective expression of constitutively active MAPK/ERK1 (MEK-SP) in osteoblasts accelerated in vitro differentiation of calvarial cells, as well as in vivo bone development, whereas dominant-negative MEK1 was inhibitory. The involvement of the RUNX2 transcription factor in this response was established in two ways: (a) RUNX2 phosphorylation and transcriptional activity were elevated in calvarial osteoblasts from TgMek-sp mice and reduced in cells from TgMek-dn mice, and (b) crossing TgMek-sp mice with Runx2+/− animals partially rescued the hypomorphic clavicles and undemineralized calvaria associated with Runx2 haploinsufficiency, whereas TgMek-dn; Runx2+/− mice had a more severe skeletal phenotype. This work establishes an important in vivo function for the ERK–MAPK pathway in bone that involves stimulation of RUNX2 phosphorylation and transcriptional activity.

Synergistic Activation of Mitogen-Activated Protein Kinase by Cyclic AMP and Myeloid Growth Factors Opposes Cyclic AMP’s Growth-Inhibitory Effects

Blood ◽  
1999 ◽  
Vol 93 (2) ◽  
pp. 537-553 ◽  
Author(s):  
Angel Wai-mun Lee
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Cell Line ◽  
Mapk Pathway ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Erk Activation ◽  
Mitogen Activated Protein ◽  
Erk Activity ◽  
Myeloid Progenitors

Abstract Colony-stimulating factors (CSFs) promote the proliferation, differentiation, commitment, and survival of myeloid progenitors, whereas cyclic AMP (cAMP)-mediated signals frequently induce their growth arrest and apoptosis. The ERK/mitogen-activated protein kinase (MAPK) pathway is a target for both CSFs and cAMP. We investigated how costimulation by cAMP and colony-stimulating factor-1 (CSF-1) or interleukin-3 (IL-3) modulates MAPK in the myeloid progenitor cell line, 32D. cAMP dramatically increased ERK activity in the presence of CSF-1 or IL-3. IL-3 also synergized with cAMP to activate ERK in another myeloid cell line, FDC-P1. The increase in ERK activity was transmitted to a downstream target, p90rsk. cAMP treatment of 32D cells transfected with oncogenic Ras was found to recapitulate the superactivation of ERK seen with cAMP and CSF-1 or IL-3. ERK activation in the presence of cAMP did not appear to involve any of the Raf isoforms and was blocked by expression of dominant-negative MEK1 or treatment with a MEK inhibitor, PD98059. Although cAMP had an overall inhibitory effect on CSF-1–mediated proliferation and survival, the inhibition was markedly increased if ERK activation was blocked by PD98059. These findings suggest that upregulation of the ERK pathway is one mechanism induced by CSF-1 and IL-3 to protect myeloid progenitors from the growth-suppressive and apoptosis-inducing effects of cAMP elevations.

scholarly journals Positive and negative selection invoke distinct signaling pathways.

1996 ◽  
Vol 184 (1) ◽  
pp. 9-18 ◽  
Author(s):  
J Alberola-Ila ◽  
K A Hogquist ◽  
K A Swan ◽  
M J Bevan ◽  
R M Perlmutter
Keyword(s):  
T Cell ◽  
Positive Selection ◽  
Negative Selection ◽  
Mapk Pathway ◽  
Cell Receptor ◽  
Dominant Negative ◽  
Mapk Cascade ◽  
Mitogen Activated Protein Kinase ◽  
Mitogen Activated Protein

During T cell development, interaction of the T cell receptor (TCR) with cognate ligands in the thymus may result in either maturation (positive selection) or death (negative selection). The intracellular pathways that control these opposed outcomes are not well characterized. We have generated mice expressing dominant-negative Ras (dnRas) and Mek-1 (dMek) transgenes simultaneously, either in otherwise normal animals, or in animals expressing a transgenic TCR, thereby permitting a comprehensive analysis of peptide-specific selection. In this system, thymocyte maturation beyond the CD4+8+ stage is blocked almost completely, whereas negative selection, assessed using an in vitro deletion protocol, is quantitatively intact. This suggests that activation of the mitogen-activated protein kinase (MAPK) cascade is necessary for positive selection, but irrelevant for negative selection. Generation of gamma/delta and of CD4-8- alpha/beta T cells proceeds normally despite blockade of the MAPK cascade. Hence, only cells that mature via conventional, TCR-mediated repertoire selection require activation of the MAPK pathway to complete their maturation.

IGF-I and insulin amplify IL-1β-induced nitric oxide and prostaglandin biosynthesis

1998 ◽  
Vol 274 (4) ◽  
pp. F673-F679 ◽  
Author(s):  
Zhonghong Guan ◽  
Shaavhree Y. Buckman ◽  
Lisa D. Baier ◽  
Aubrey R. Morrison
Keyword(s):  
Nitric Oxide ◽  
Mesangial Cells ◽  
Mapk Pathway ◽  
Mitogen Activated Protein Kinase ◽  
No Production ◽  
Glomerular Mesangial Cells ◽  
Igf I ◽  
Mitogen Activated Protein ◽  
Transduction Mechanisms ◽  
Cox 2

The inflammatory cytokine interleukin-1β (IL-1β) induces both cyclooxygenase-2 (Cox-2) and the inducible nitric oxide synthase (iNOS) with concomitant release of PGs and nitric oxide (NO) by glomerular mesangial cells. In our current studies, we determine whether insulin and IGF-I are involved in the signal transduction mechanisms resulting in IL-1β-induced NO and PGE2biosynthesis in renal mesangial cells. We demonstrate that both insulin and IGF-I increase IL-1β-induced Cox-2 and iNOS protein expression, which in turn enhance PGE2 and NO production. Our data also indicate that both insulin and IGF-I enhance IL-1β-induced p38 mitogen-activated protein kinase (MAPK) phosphorylation and SAPK activation. These findings implicate the possible role of the MAPK pathway in mediating the effects of insulin and IGF-I on the upregulation of cytokine-stimulated NO and PG biosynthesis. Together, our results indicate that IGF-I and insulin may function to modulate the renal inflammatory process.

Novel RAF Fusions in Pediatric Low-Grade Gliomas Demonstrate MAPK Pathway Activation

2021 ◽  
Author(s):  
Katherine T Lind ◽  
Hannah V Chatwin ◽  
John DeSisto ◽  
Philip Coleman ◽  
Bridget Sanford ◽  
...  
Keyword(s):  
Mapk Pathway ◽  
Enrichment Analysis ◽  
Mitogen Activated Protein Kinase ◽  
Immunofluorescent Staining ◽  
Low Grade ◽  
Normal Brain ◽  
Mapk Activation ◽  
Low Grade Gliomas ◽  
Pathway Activation ◽  
Mitogen Activated Protein

Abstract Brain tumors are the most common solid tumor in children, and low-grade gliomas (LGGs) are the most common childhood brain tumor. Here, we report on 3 patients with LGG harboring previously unreported or rarely reported RAF fusions: FYCO1-RAF1, CTTNBP2-BRAF, and SLC44A1-BRAF. We hypothesized that these tumors would show molecular similarity to the canonical KIAA1549-BRAF fusion that is the most widely seen alteration in pilocytic astrocytoma (PA), the most common pediatric LGG variant, and that this similarity would include mitogen-activated protein kinase (MAPK) pathway activation. To test our hypothesis, we utilized immunofluorescent imaging and RNA-sequencing in normal brain, KIAA1549-BRAF-harboring tumors, and our 3 tumors with novel fusions. We performed immunofluorescent staining of ERK and phosphorylated ERK (p-ERK), identifying increased p-ERK expression in KIAA1549-BRAF fused PA and the novel fusion samples, indicative of MAPK pathway activation. Geneset enrichment analysis further confirmed upregulated downstream MAPK activation. These results suggest that MAPK activation is the oncogenic mechanism in noncanonical RAF fusion-driven LGG. Similarity in the oncogenic mechanism suggests that LGGs with noncanonical RAF fusions are likely to respond to MEK inhibitors.

scholarly journals Activated Mutants of SHP-2 Preferentially Induce Elongation of Xenopus Animal Caps

2000 ◽  
Vol 20 (1) ◽  
pp. 299-311 ◽  
Author(s):  
Alana M. O'Reilly ◽  
Scott Pluskey ◽  
Steven E. Shoelson ◽  
Benjamin G. Neel
Keyword(s):  
Structural Information ◽  
Mapk Pathway ◽  
Tyrosine Phosphatase ◽  
Signal Transduction Pathway ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Dominant Negative Mutant ◽  
Mesoderm Induction ◽  
Fgf Receptor

ABSTRACT In Xenopus ectodermal explants (animal caps), fibroblast growth factor (FGF) evokes two major events: induction of ventrolateral mesodermal tissues and elongation. TheXenopus FGF receptor (XFGFR) and certain downstream components of the XFGFR signal transduction pathway (e.g., members of the Ras/Raf/MEK/mitogen-activated protein kinase [MAPK] cascade) are required for both of these processes. Likewise, activated versions of these signaling components induce mesoderm and promote animal cap elongation. Previously, using a dominant negative mutant approach, we showed that the protein-tyrosine phosphatase SHP-2 is necessary for FGF-induced MAPK activation, mesoderm induction, and elongation of animal caps. Taking advantage of recent structural information, we now have generated novel, activated mutants of SHP-2. Here, we show that expression of these mutants induces animal cap elongation to an extent comparable to that evoked by FGF. Surprisingly, however, activated mutant-induced elongation can occur without mesodermal cytodifferentiation and is accompanied by minimal activation of the MAPK pathway and mesodermal marker expression. Our results implicate SHP-2 in a pathway(s) directing cell movements in vivo and identify potential downstream components of this pathway. Our activated mutants also may be useful for determining the specific functions of SHP-2 in other signaling systems.

Endothelin signalling and regulation of protein kinases in glomerular mesangial cells

2002 ◽  
Vol 103 (s2002) ◽  
pp. 132S-136S ◽  
Author(s):  
Andrey SOROKIN ◽  
Marco FOSCHI ◽  
Michael J. DUNN
Keyword(s):  
Map Kinase ◽  
Map Kinases ◽  
Molecular Mechanisms ◽  
Mesangial Cells ◽  
Mapk Pathway ◽  
P38 Map Kinase ◽  
Dominant Negative ◽  
Glomerular Mesangial Cells ◽  
Calcium Dependent ◽  
Mitogen Activated Protein

The molecular mechanisms of endothelin (ET)-dependent activation of extracellular signal-regulated kinase (ERK)and p38 mitogen-activated protein (MAP) kinase were studied in rat and human renal glomerular mesangial cells. ET-1 induced a rapid and transient activation of Ras in renal mesangial cells, which was dependent upon the formation of the Shc/Grb2/Sos1 signalling complex and resulted in transient ERK activation. We have observed that Pyk2, a calcium-dependent cytoplasmic tyrosine kinase, was expressed in human renal mesangial cells and was tyrosine phosphorylated after ET-1 treatment. ET-1-induced activation of p38 MAPK pathway (but not ERK pathway) was inhibited in human and in rat glomerular mesangial cells expressing dominant-negative form of Pyk2, suggesting the engagement of Pyk2 in ET-1-mediated activation of p38 MAP kinase cascade. Contractive responsiveness of renal mesangial cells was shown to depend on activation of the p38 MAP kinases. Thus, p38 MAP kinase stimulation could perhaps partially account for ET-1 contractive properties, whereas ET-1-induced cell proliferation occurs primarily via Ras-dependent activation of the ERK.

Erythropoietin regulation of Raf-1 and MEK: evidence for a Ras-independent mechanism

Blood ◽  
2004 ◽  
Vol 104 (1) ◽  
pp. 73-80 ◽  
Author(s):  
Changmin Chen ◽  
Arthur J. Sytkowski
Keyword(s):  
Kinase Activity ◽  
Mapk Pathway ◽  
Pi3k Pathway ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Transcriptional Initiation ◽  
Epo Receptor ◽  
Independent Mechanism ◽  
Mitogen Activated Protein ◽  
Murine Erythroleukemia

Abstract Stimulation of the erythropoietin (EPO) receptor triggers a cascade of signaling events. We reported that EPO upregulates c-myc expression through 2 pathways in BaF3-EpoR cells—a phosphatidylinositol 3-kinase (PI3K) pathway operating on transcriptional initiation and a Raf-1–mitogen-activated protein kinase (MAPK) pathway affecting elongation. We now show that EPO induces phosphorylation of Raf-1 at serine 338 and within the carboxy-terminal domain, resulting in an electrophoretic mobility change (hyperphosphorylation). Importantly, MEK 1 inhibitor PD98059 blocked only the hyperphosphorylation of Raf-1 but not the phosphorylation at serine 338. This inhibition of Raf-1 hyperphosphorylation resulted in increased kinase activity of Raf-1 and increased phosphorylation of MEK, suggesting that the hyperphosphorylation of Raf-1 inhibits its MEK kinase activity. Deletion of the first 184 amino acids of Raf-1, which are involved in its interaction with Ras, had no effect on EPO-induced phosphorylation. Introducing the dominant-negative N17Ras or GAP had no effect on EPO-induced kinase activity of Raf-1 and ELK activation. N17Ras failed to inhibit ELK activation in another cell line—Rauscher murine erythroleukemia— which expresses the EPO receptor endogenously and differentiates in response to the hormone. These results indicate the presence of a Ras-independent mechanism for Raf-1 and MEK activation in these cells.

MAPK and JNK transduction pathways can phosphorylate Sp1 to activate the uPA minimal promoter element and endogenous gene transcription

Blood ◽  
2004 ◽  
Vol 104 (1) ◽  
pp. 256-262 ◽  
Author(s):  
Elisa Benasciutti ◽  
Gilles Pagès ◽  
Olga Kenzior ◽  
William Folk ◽  
Francesco Blasi ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Mapk Pathway ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Minimal Promoter ◽  
Jnk Pathway ◽  
Enhancer Element ◽  
Endogenous Gene ◽  
Mitogen Activated Protein ◽  
Pc3 Cells

Abstract Two upstream regions of the human urokinase (uPA) gene regulate its transcription: the minimal promoter (MP) and the enhancer element. The activity of the minimal promoter is essential for basal uPA transcription in prostate adenocarcinoma PC3 cells. Binding of a phosphorylated Sp1 transcription factor is, in turn, essential for the activity of the MP. Here we report that the Jun kinase (JNK) pathway is required for the basal activity of the MP and for the expression of the endogenous uPA gene in PC3 cells and for activated transcription in LNCaP cells. On the other hand, the p42/p44 mitogen-activated protein kinase (MAPK) pathway activates uPA gene expression through Sp1 phosphorylation in HeLa, LNCaP, and CCL39-derivative cells that do not typically express uPA in basal conditions. In HeLa cells the dominant-negative form of JNK interferes with the p42/p44 MAPK activation of the uPA-MP. The results suggest that the stress-activated protein kinase (SAPK)/JNK pathway plays an important role in the phosphorylation of Sp1, which, in turn, leads to basal or activated transcription from the uPA-MP element.

scholarly journals The mitogen-activated protein kinase pathway tonically inhibits both basal and IGF-I-stimulated IGF-binding protein-5 production in mammary epithelial cells

2007 ◽  
Vol 194 (2) ◽  
pp. 349-359 ◽  
Author(s):  
Jodie M Fleming ◽  
Jeffrey A Brandimarto ◽  
Wendie S Cohick
Keyword(s):  
Mammary Epithelial Cells ◽  
Mapk Pathway ◽  
Mammary Epithelial ◽  
Mitogen Activated Protein Kinase ◽  
Mapk Activity ◽  
Igf I ◽  
Mitogen Activated Protein ◽  
Igf Binding ◽  
Mapk Inhibitors ◽  
Igfbp 3

The IGF system plays a key role in mammary gland growth and development. Our lab previously reported that IGF-I primarily regulates IGF-binding protein (BP)-3 in bovine mammary epithelial cells (MEC) and IGFBP-5 in mammary fibroblasts (MF). Presently, we examined the signaling pathways used by IGF-I to elicit this distinct, cell-type specific regulation. The phosphatidylinositol-3 kinase pathway was required for IGF-I to increase IGFBP-3 and -5 in MF and IGFBP-3 in MEC. Surprisingly, inhibiting the mitogen-activated protein kinase (MAPK) pathway in MEC increased IGFBP-5 mRNA levels 2- to 4-fold under basal conditions and 8- to 12-fold in cells treated with IGF-I within 4 h. Similar patterns of IGFBP-3 and -5 regulation were observed in murine MEC. Cells treated with IGF-I in the presence of MAPK inhibitors secreted more IGFBP-5 protein into conditioned media relative to cells treated with IGF-I alone; however, IGFBP-5 protein was not detected in conditioned media of cells treated with only a MAPK inhibitor. The IGFBP-5 mRNA response to MAPK inhibitors was specific for MEC, as blocking MAPK activity decreased the ability of IGF-I to induce IGFBP-5 in MF. In addition, no other IGFBP was increased in either cell type when MAPK activity was inhibited. These increases in IGFBP-5 expression in response to inhibition of the MAPK pathway corresponded with the induction of apoptosis. In conclusion, we report the novel observation that the MAPK/extracellular signal regulated kinase (ERK) pathway specifically represses IGFBP-5 expression in MEC. The corresponding changes in apoptosis and IGFBP-5 expression support a role for this specific IGFBP in mammary gland involution.

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