scholarly journals Differential inhibition of signaling pathways by dominant-negative SH2/SH3 adapter proteins.

1995 ◽  
Vol 15 (12) ◽  
pp. 6829-6837 ◽  
Author(s):  
M Tanaka ◽  
R Gupta ◽  
B J Mayer
Keyword(s):  
Protein Kinase ◽  
Signaling Pathways ◽  
Tyrosine Kinases ◽  
Egf Receptor ◽  
Sh3 Domain ◽  
Sh2 Domain ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Erk Activation ◽  
Mitogen Activated Protein

SH2/SH3 adapters are thought to function in signal transduction pathways by coupling inputs from tyrosine kinases to downstream effectors such as Ras. Members of the mitogen-activated protein kinase family are known to be activated by a variety of mitogenic stimuli, including tyrosine kinases such as Abl and the epidermal growth factor (EGF) receptor. We have used activation of the mitogen-activated protein kinase Erk-1 as a model system with which to examine whether various dominant-negative SH2/SH3 adapters (Grb2, Crk, and Nck) could block signaling pathways leading to Erk activation. Activation of Erk-1 by oncogenic Abl was effectively inhibited by Grb2 with mutations in either its SH2 or SH3 domain or by Crk-1 with an SH3 domain mutation. The Crk-1 SH2 mutant was less effective, while Nck SH2 and SH3 mutants had little or no effect on Erk activation. These results suggest that both Crk and Grb2 may contribute to the activation of Erk by oncogenic Abl, whereas Nck is unlikely to participate in this pathway. Next we examined whether combinations of these dominant-negative adapters could inhibit Erk activation more effectively than each mutant alone. When combinations of Crk-1 and Grb2 mutants were analyzed, the combination of the Crk-1 SH3 mutant plus the Grb2 SH3 mutant gave a striking synergistic effect. This finding suggests that in Abl-transformed cells, more than one class of tyrosine-phosphorylated sites (those that bind the Grb2 SH2 domain and those that bind the Crk SH2 domain) can lead to Ras activation. In contrast to results with Abl, Erk activation by EGF was strongly inhibited only by Grb2 mutants; Crk and Nck mutants had little or no effect. This finding suggests that Grb2 is the only adapter involved in the activation of Erk by EGF. Dominant-negative adaptors provide a novel means to identify binding interactions important in vivo for signaling in response to a variety of stimuli.

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 Akt-Dependent Phosphorylation Specifically Regulates Cot Induction of NF-κB-Dependent Transcription

2002 ◽  
Vol 22 (16) ◽  
pp. 5962-5974 ◽  
Author(s):  
Lawrence P. Kane ◽  
Marianne N. Mollenauer ◽  
Zheng Xu ◽  
Christoph W. Turck ◽  
Arthur Weiss
Keyword(s):  
Protein Kinase ◽  
Signaling Pathways ◽  
Cellular Transformation ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Iκb Kinase ◽  
Ikk Complex ◽  
Mitogen Activated Protein ◽  
Tumor Necrosis Factor Induction ◽  
Necrosis Factor

ABSTRACT The Akt (or protein kinase B) and Cot (or Tpl-2) serine/threonine kinases are associated with cellular transformation. These kinases have also been implicated in the induction of NF-κB-dependent transcription. As a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, Cot can also activate MAP kinase signaling pathways that target AP-1 and NFAT family transcription factors. Here we show that Akt and Cot physically associate and functionally cooperate. Akt appears to function upstream of Cot, as Akt can enhance Cot induction of NF-κB-dependent transcription, and dominant-negative Cot blocks the activation of this element by Akt. Furthermore, deletion analysis shows that binding to Akt is critical for Cot function. The regulation of NF-κB-dependent transcription by Cot requires Akt-dependent phosphorylation of serine 400 (S400), near the carboxy terminus of Cot. However, phosphorylation at this site is not required for Cot kinase activity or AP-1 induction, suggesting it specifically regulates Cot effector function at the level of the NF-κB pathway. Mutation of S400 in Cot does indeed abolish its ability to activate IκB-kinase (IKK) complexes, but paradoxically it allows for increased Cot association with the IKK complex. This mutated form of Cot also acts as a dominant negative for T-cell antigen receptor/CD28- or Akt/phorbol myristate acetate-induced NF-κB induction, while having relatively little effect on tumor necrosis factor induction of NF-κB. These findings suggest that the activation of different signaling pathways by MAP3Ks may be regulated separately and may provide evidence for how such discrimination by one member of this kinase family occurs.

scholarly journals Rac-PAK Signaling Stimulates Extracellular Signal-Regulated Kinase (ERK) Activation by Regulating Formation of MEK1-ERK Complexes

2002 ◽  
Vol 22 (17) ◽  
pp. 6023-6033 ◽  
Author(s):  
Scott T. Eblen ◽  
Jill K. Slack ◽  
Michael J. Weber ◽  
Andrew D. Catling
Keyword(s):  
Protein Kinase ◽  
Signaling Pathway ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Wild Type ◽  
Erk Activation ◽  
Extracellular Signal Regulated Kinase ◽  
Signaling Complex ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

ABSTRACT Utilizing mutants of extracellular signal-regulated kinase 2 (ERK2) that are defective for intrinsic mitogen-activated protein kinase or ERK kinase (MEK) binding, we have identified a convergent signaling pathway that facilitates regulated MEK-ERK association and ERK activation. ERK2-Δ19-25 mutants defective in MEK binding could be phosphorylated in response to mitogens; however, signaling from the Raf-MEK pathway alone was insufficient to stimulate their phosphorylation in COS-1 cells. Phosphorylation of ERK2-Δ19-25 but not of wild-type ERK2 in response to Ras V12 was greatly inhibited by dominant-negative Rac. Activated forms of Rac and Cdc42 could enhance the association of wild-type ERK2 with MEK1 but not with MEK2 in serum-starved adherent cells. This effect was p21-activated kinase (PAK) dependent and required the putative PAK phosphorylation sites T292 and S298 of MEK1. In detached cells placed in suspension, ERK2 was complexed with MEK2 but not with MEK1. However, upon replating of cells onto a fibronectin matrix, there was a substantial induction of MEK1-ERK2 association and ERK activation, both of which could be inhibited by dominant-negative PAK1. These data show that Rac facilitates the assembly of a mitogen-activated protein kinase signaling complex required for ERK activation and that this facilitative signaling pathway is active during adhesion to the extracellular matrix. These findings reveal a novel mechanism by which adhesion and growth factor signals are integrated during ERK activation.

scholarly journals The Transcriptional Response to Raf Activation Is Almost Completely Dependent on Mitogen-activated Protein Kinase Kinase Activity and Shows a Major Autocrine Component

2004 ◽  
Vol 15 (7) ◽  
pp. 3450-3463 ◽  
Author(s):  
Almut Schulze ◽  
Barbara Nicke ◽  
Patricia H. Warne ◽  
Simon Tomlinson ◽  
Julian Downward
Keyword(s):  
Gene Expression ◽  
Growth Factor ◽  
Protein Kinase ◽  
Signaling Pathways ◽  
Egf Receptor ◽  
Transcriptional Response ◽  
Mitogen Activated Protein Kinase ◽  
Mitogen Activated Protein ◽  
Epidermal Growth ◽  
Protein Kinase Kinase

The Raf protein kinases are major effectors of Ras GTPases and key components of the transcriptional response to serum factors, acting at least in part through the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway. It has recently been suggested that Raf also may trigger other as yet uncharacterized signaling pathways. Here, we have used cDNA microarrays to dissect changes in gene expression induced by activation of inducible c-Raf-1 constructs in human mammary epithelial and ovarian epithelial cells. The majority of Raf-induced transcriptional responses are shown to be blocked by pharmacological inhibition of the Raf substrate mitogen-activated protein kinase kinase, indicating that potential mitogen-activated protein kinase kinase-independent Raf signaling pathways have no significant influence on gene expression. In addition, we used epidermal growth factor receptor inhibitory drugs to address the contribution of autocrine signaling by Raf-induced EGF family proteins to the Raf transcriptional response. At least one-half of the transcription induced by Raf activation requires epidermal growth factor (EGF) receptor function The EGF receptor-independent component of the Raf transcriptional response is entirely up-regulation of gene expression, whereas the EGF receptor-dependent component is an equal mixture of up- and down-regulation. The use of transcriptional profiling in this way allows detailed analysis of the architecture of signaling pathways to be undertaken.

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

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.

Leukotriene D4 activates MAPK through a Ras-independent but PKCϵ-dependent pathway in intestinal epithelial cells

2002 ◽  
Vol 115 (9) ◽  
pp. 1883-1893
Author(s):  
Sailaja Paruchuri ◽  
Bengt Hallberg ◽  
Maria Juhas ◽  
Christer Larsson ◽  
Anita Sjölander
Keyword(s):  
Protein Kinase ◽  
Epithelial Cells ◽  
Proliferative Response ◽  
Egf Receptor ◽  
Intestinal Epithelial Cells ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Intestinal Epithelial ◽  
Leukotriene D4 ◽  
Mitogen Activated Protein

We have recently shown that leukotriene D4 (LTD4)increases cell survival in intestinal epithelial cells. Here we report and explore the complementary finding that LTD4 also enhances proliferation in these cells. This proliferative response was approximately half of that induced by epidermal growth factor (EGF) and its required activation of protein kinase C (PKC), Ras and the mitogen-activated protein kinase (MAPK) Erk-1/2. EGF also activated Erk-1/2 in these cells; however the EGF-receptor inhibitor PD153035 did not affect the LTD4-induced activation of Erk-1/2. In addition, LTD4 did not induce phosphorylation of the EGF receptor, nor did pertussis toxin (PTX) block EGF-induced activation of Erk-1/2, thus refuting a possible crosstalk between the receptors. Furthermore, LTD4-induced, but not EGF-induced,activation of Erk-1/2 was sensitive to PTX, PKC inhibitors and downregulation of PKCϵ. A definite role for PKCϵ in LTD4-induced stimulation of Erk-1/2 was documented by the inability of LTD4 to activate Erk-1/2 in cells transfected with either the regulatory domain of PKCϵ (an isoform specific dominant-negative inhibitor) or a kinase-dead PKCϵ. Although Ras and Raf-1 were both transiently activated by LTD4, only Raf-1 activation was abolished by abrogation of the PKC signal. Furthermore, the LTD4-induced activation of Erk-1/2 was unaffected by transfection with dominant-negative N17 Ras but blocked by transfection with kinase-dead Raf-1. Consequently, LTD4 regulates the proliferative response by a distinct Ras-independent, PKCϵ-dependent activation of Erk-1/2 and a parallel Ras-dependent signaling pathway.

β-Adrenergic-responsive activation of extracellular signal-regulated protein kinases in salivary cells: role of epidermal growth factor receptor and cAMP

2005 ◽  
Vol 288 (6) ◽  
pp. C1357-C1366 ◽  
Author(s):  
Chih-Ko Yeh ◽  
Paramita M. Ghosh ◽  
Howard Dang ◽  
Qun Liu ◽  
Alan L. Lin ◽  
...  
Keyword(s):  
Egf Receptor ◽  
Growth Factor Receptor ◽  
Immunoblot Analysis ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Salivary Gland Cell ◽  
Erk Activation ◽  
Transient Activation ◽  
Erk Phosphorylation ◽  
Mitogen Activated Protein

The β-adrenergic receptor agonist isoproterenol exerts growth-promoting effects on salivary glands. In this study, activation of ERKs, members of the mitogen-activated protein kinase family, by isoproterenol was examined in a human salivary gland cell line (HSY). Immunoblot analysis indicated that isoproterenol (10−5 M) induced transient activation of ERK1/2 (4.4-fold relative to basal at 10 min) similar to that caused by EGF (6.7 fold). Isoproterenol, like EGF, also induced phosphorylation of the EGF receptor. However, inhibition of EGF receptor phosphorylation by the tyrphostin AG-1478 only partially attenuated isoproterenol-induced ERK phosphorylation, whereas EGF-responsive ERK activation was completely blocked. The Gi inhibitor pertussis toxin also caused partial inhibition of isoproterenol-stimulated ERK activation. The cAMP analog 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate (CPT-cAMP) and the cAMP-elevating agents IBMX and cholera toxin produced transient ERK1/2 activation, similar to the effect of isoproterenol, in HSY cells. The stimulatory effects of isoproterenol and cAMP on ERK phosphorylation were not reduced by the PKA inhibitor H-89, whereas the Src family inhibitor 4-amino-5-(4-chlorophenyl)-7-( t-butyl)pyrazolo[3,4- d]pyrimidase (PP2) and transfection of a dominant-negative Src construct diminished isoproterenol-induced ERK activation. Isoproterenol induced marked overexpression of the cell growth-related adhesion molecule CD44, and this effect of isoproterenol was abolished by the ERK pathway inhibitor PD-98059. In summary, we show a dual mechanism of isoproterenol-induced ERK phosphorylation in HSY cells—one pathway mediated by EGF receptor transactivation and the other by an EGF receptor-independent pathway possibly mediated by cAMP. Our results also suggest that isoproterenol-induced growth of salivary tissue may involve ERK-mediated CD44 expression.

scholarly journals Activation of tyrosine kinases by α1A-adrenergic and growth factor receptors in transfected PC12 cells

1999 ◽  
Vol 344 (3) ◽  
pp. 889-894 ◽  
Author(s):  
Hongying ZHONG ◽  
Kenneth P. MINNEMAN
Keyword(s):  
Growth Factor ◽  
Protein Kinase ◽  
Tyrosine Phosphorylation ◽  
Pc12 Cells ◽  
Tyrosine Kinases ◽  
Egf Receptor ◽  
Growth Factor Receptor ◽  
Mitogen Activated Protein Kinase ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

We compared the role of tyrosine kinases in α1A-adrenergic receptor (AR) and growth factor receptor stimulation of mitogen-activated protein kinase pathways in PC12 cells. Norepinephrine (NE) (noradrenaline), epidermal growth factor (EGF) and nerve growth factor (NGF) caused different patterns of tyrosine phosphorylation in PC12 cells stably expressing α1A-ARs. NE increased tyrosine phosphorylation of focal adhesion-related kinase Pyk2 and a 70 kDa protein, probably paxillin, whereas EGF strongly stimulated tyrosine phosphorylation of the EGF receptor and cytokine-activated kinase Jak2. The EGF receptor inhibitor AG1478 inhibited activation of extracellular signal-regulated kinases (ERKs) by EGF but not by NE. EGF and NGF strongly activated tyrosine phosphorylation of Shc and caused association of Src-homology collagen (Shc) with growth-factor-receptor-bound protein 2 (Grb2); however, neither NE nor UTP caused substantial activation of the Shc/Grb2 pathway. NE, UTP, EGF and NGF all increased tyrosine phosphorylation of Src, and this was inhibited by the Src inhibitor PP2. However, PP2 inhibited ERK activation in response to NE and UTP, but not in response to EGF or NGF. PP2 also completely blocked NE-induced PC12 cell differentiation, but had no measurable effect on NGF-induced differentiation. These studies show that activation of mitogen-activated protein kinase pathways by G-protein-coupled receptors and tyrosine kinase receptors proceed through distinct molecular pathways in PC12 cells, and support an obligatory role for Src activation in mitogenic responses to α1A-ARs in these cells.

scholarly journals Transcriptional Regulators of theSchizosaccharomyces pombe fbp1Gene Include Two Redundant Tup1p-like Corepressors and the CCAAT Binding Factor Activation Complex

Genetics ◽  
2001 ◽  
Vol 157 (3) ◽  
pp. 1205-1215 ◽  
Author(s):  
Rozmin T K Janoo ◽  
Lori A Neely ◽  
Burkhard R Braun ◽  
Simon K Whitehall ◽  
Charles S Hoffman
Keyword(s):  
Protein Kinase ◽  
Fold Increase ◽  
Transcriptional Regulators ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Lacz Expression ◽  
Protein Subunit ◽  
Plasmid Library ◽  
Binding Factor ◽  
Mitogen Activated Protein

AbstractThe Schizosaccharomyces pombe fbp1 gene, which encodes fructose-1,6-bis-phosphatase, is transcriptionally repressed by glucose through the activation of the cAMP-dependent protein kinase A (PKA) and transcriptionally activated by glucose starvation through the activation of a mitogen-activated protein kinase (MAPK). To identify transcriptional regulators acting downstream from or in parallel to PKA, we screened an adh-driven cDNA plasmid library for genes that increase fbp1 transcription in a strain with elevated PKA activity. Two such clones express amino-terminally truncated forms of the S. pombe tup12 protein that resembles the Saccharomyces cerevisiae Tup1p global corepressor. These clones appear to act as dominant negative alleles. Deletion of both tup12 and the closely related tup11 gene causes a 100-fold increase in fbp1-lacZ expression, indicating that tup11 and tup12 are redundant negative regulators of fbp1 transcription. In strains lacking tup11 and tup12, the atf1-pcr1 transcriptional activator continues to play a central role in fbp1-lacZ expression; however, spc1 MAPK phosphorylation of atf1 is no longer essential for its activation. We discuss possible models for the role of tup11- and tup12-mediated repression with respect to signaling from the MAPK and PKA pathways. A third clone identified in our screen expresses the php5 protein subunit of the CCAAT-binding factor (CBF). Deletion of php5 reduces fbp1 expression under both repressed and derepressed conditions. The CBF appears to act in parallel to atf1-pcr1, although it is unclear whether or not CBF activity is regulated by PKA.

Sign in / Sign up

Export Citation Format

Share Document