scholarly journals Differential activation of the Ras/extracellular-signal-regulated protein kinase pathway is responsible for the biological consequences induced by the Axl receptor tyrosine kinase.

1996 ◽  
Vol 16 (1) ◽  
pp. 135-145 ◽  
Author(s):  
Y W Fridell ◽  
Y Jin ◽  
L A Quilliam ◽  
A Burchert ◽  
P McCloskey ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Egf Receptor ◽  
Kinase Domain ◽  
Phosphatidylinositol 3 Kinase ◽  
Erk Activation ◽  
Interleukin 3 ◽  
Extracellular Signal ◽  
Axl Receptor Tyrosine Kinase ◽  
Epidermal Growth ◽  
Kinase Pathway

To understand the mechanism of Axl signaling, we have initiated studies to delineate downstream components in interleukin-3-dependent 32D cells by using a chimeric receptor containing the recombinant epidermal growth factor (EGF) receptor extracellular and transmembrane domains and the Axl kinase domain (EAK [for EGF receptor-Axl kinase]). We have previously shown that upon exogenous EGF stimulation, 32D-EAK cells are capable of proliferation in the absence of interleukin-3. With this system, we determined that EAK-induced cell survival and mitogenesis are dependent upon the Ras/extracellular-signal-regulated protein kinase (ERK) cascade. Although the phosphatidylinositol-3 kinase pathway is activated upon EAK signaling, it appears to be dispensable for the biological actions of the Axl kinase. Furthermore, we demonstrated that different threshold levels of Ras/ERK activation are needed to induce a block to apoptosis or proliferation in 32D cells. Recently, we have identified an Axl ligand, GAS6. Surprisingly, GAS6-stimulated 32D-Axl cells exhibited no blockage to apoptosis or mitogenic response which is correlated with the absence of Ras/ERK activation. Taken together, these data suggest that different extracellular domains dramatically alter the intracellular response of the Axl kinase. Furthermore, our data suggest that the GAS6-Axl interaction does not induce mitogenesis and that its exact role remains to be determined.

scholarly journals An Uncleavable uPAR Mutant Allows Dissection of Signaling Pathways in uPA-dependent Cell Migration

2006 ◽  
Vol 17 (1) ◽  
pp. 367-378 ◽  
Author(s):  
Roberta Mazzieri ◽  
Silvia D'Alessio ◽  
Richard Kamgang Kenmoe ◽  
Liliana Ossowski ◽  
Francesco Blasi
Keyword(s):  
Cell Migration ◽  
Signaling Pathways ◽  
Egf Receptor ◽  
Alternative Pathway ◽  
Erk Activation ◽  
Type Plasminogen Activator ◽  
Extracellular Signal ◽  
Urokinase Type Plasminogen Activator ◽  
Dependent Cell ◽  
Epidermal Growth

Urokinase-type plasminogen activator (uPA) binding to uPAR induces migration, adhesion, and proliferation through multiple interactions with G proteins-coupled receptor FPRL1, integrins, or the epidermal growth factor (EGF) receptor (EGFR). At least two forms of uPAR are present on the cell surface: full-length and cleaved uPAR, each specifically interacting with one or more transmembrane proteins. The connection between these interactions and the effects on the signaling pathways activation is not clear. We have exploited an uPAR mutant (hcr, human cleavage resistant) to dissect the pathways involved in uPA-induced cell migration. This mutant is not cleaved by proteases, is glycosylphosphatidylinositol anchored, and binds uPA with a normal Kd. Both wild-type (wt) and hcr-uPAR are able to mediate uPA-induced migration, are constitutively associated with the EGFR, and associate with α3β1 integrin upon uPA binding. However, they engage different pathways in response to uPA. wt-uPAR requires both integrins and FPRL1 to mediate uPA-induced migration, and association of wt-uPAR to α3β1 results in uPAR cleavage and extracellular signal-regulated kinase (ERK) activation. On the contrary, hcr-uPAR does not activate ERK and does not engage FPRL1 or any other G protein-coupled receptor, but it activates an alternative pathway initiated by the formation of a triple complex (uPAR-α3β1-EGFR) and resulting in the autotyrosine phosphorylation of EGFR.

scholarly journals Epidermal-growth-factor receptor and metalloproteinases mediate thromboxane A2-dependent extracellular-signal-regulated kinase activation

2003 ◽  
Vol 371 (3) ◽  
pp. 733-742 ◽  
Author(s):  
Carole GALLET ◽  
Stéphanie BLAIE ◽  
Sylviane LÉVY-TOLEDANO ◽  
Aïda HABIB
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Protein Kinase ◽  
Epidermal Growth Factor ◽  
Growth Factor Receptor ◽  
Egfr Transactivation ◽  
Erk Activation ◽  
Transfected Cells ◽  
Extracellular Signal ◽  
Epidermal Growth

The signalling pathways that link G-protein-coupled receptors to mitogen-activated protein kinases involve receptor and non-receptor tyrosine kinases and protein kinase C (PKC). We explored the pathways that are implicated in the thromboxane (TX) A2-dependent activation of extracellular-signal-regulated protein kinase (ERK) and the role of the two TX receptor (TP) isoforms, TPα and TPβ. ERK activation by IBOP, a TX analogue, was dependent on epidermal-growth-factor receptor (EGFR) in TPα- or TPβ-transfected cells and in human aortic smooth muscle cells (hASMCs), since AG1478, a selective inhibitor of tyrosine phosphorylation of the EGFR, strongly blocked ERK and EGFR phosphorylation. In addition, EGFR transactivation leading to ERK activation involved matrix metalloproteinases (MMPs), since BB2516, an inhibitor of MMP, decreased ERK and EGFR phosphorylation in TPα- or TPβ-transfected cells. Moreover, we showed that both isoforms activate ERK phosphorylation in an Src-kinase-dependent manner, whereas PKC was mainly implicated in ERK activation and EGFR phosphorylation by TPβ. In hASMCs, we showed that ERK activation depended on both pertussis-sensitive and -insensitive Gα-proteins. We demonstrated further that EGFRs, PKC, Src kinase and MMPs are involved in ERK activation by TX. The results of the present study highlight a role for MMPs and PKC in EGFR transactivation triggered by the TPs and demonstrate this mechanism for the first time in primary cells, i.e. hASMCs.

scholarly journals Photoaffinity labelling of the ATP-binding site of the epidermal growth factor-dependent protein kinase

1984 ◽  
Vol 220 (3) ◽  
pp. 677-683 ◽  
Author(s):  
J E Kudlow ◽  
Y Leung
Keyword(s):  
Protein Kinase ◽  
Binding Site ◽  
Egf Receptor ◽  
Light Exposure ◽  
Atp Binding ◽  
Receptor Kinase ◽  
Dependent Protein Kinase ◽  
Atp Binding Site ◽  
Dependent Protein ◽  
Epidermal Growth

Epidermal growth factor (EGF), after binding to its receptor, activates a tyrosine-specific protein kinase which phosphorylates several substrates, including the EGF receptor itself. The effects of a photoaffinity analogue of ATP, 3′-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)adenosine 5′-triphosphate (arylazido-beta-alanyl-ATP) on the EGF-dependent protein kinase in A431 human tumour cell plasma membrane vesicles was investigated. This analogue was capable of inactivating the EGF-receptor kinase in a photodependent manner. Partial inactivation occurred at an analogue concentration of 1 microM and complete inactivation occurred at 10 microM when a 2 min light exposure was used. Arylazido-beta-alanine at 100 microM and ATP at 100 microM were incapable of inactivating the enzyme with 2 min of light exposure. The photodependent inactivation of the enzyme by the analogue could be partially blocked by 20 mM-ATP and more effectively blocked by either 20 mM-adenosine 5′-[beta gamma-imido]triphosphate or 20 mM-guanosine 5′-[beta gamma-imido]triphosphate, indicating nucleotide-binding site specificity. Arylazido-beta-alanyl-[alpha-32P]ATP was capable of labelling membrane proteins in a photodependent manner. Numerous proteins were labelled, the most prominent of which ran with an apparent Mr of 53000 on polyacrylamide-gel electrophoresis. A band of minor intensity was seen of Mr corresponding to the EGF receptor (170000). Immunoprecipitation of affinity-labelled and solubilized membranes with an anti-(EGF receptor) monoclonal antibody demonstrated that the Mr 170000 receptor protein was photoaffinity labelled by the analogue. The Mr 53000 peptide was not specifically bound by the anti-receptor antibody. The affinity labelling of the receptor was not enhanced by EGF, suggesting that EGF stimulation of the kinase activity does not result from changes in the affinity of the kinase for ATP. These studies demonstrate that arylazido-beta-alanyl-ATP interacts with the ATP-binding site of the EGF-receptor kinase with apparent high affinity and that this analogue is an effective photoaffinity label for the kinase. Furthermore, these studies demonstrate that the EGF receptor, identified by using monoclonal antibodies, contains an ATP-binding site, providing further confirmation that the EGF receptor and EGF-dependent protein kinase are domains of the Mr 170000 protein.

scholarly journals Epidermal growth factor (EGF) stimulates association and kinase activity of Raf-1 with the EGF receptor.

1991 ◽  
Vol 11 (2) ◽  
pp. 913-919 ◽  
Author(s):  
H App ◽  
R Hazan ◽  
A Zilberstein ◽  
A Ullrich ◽  
J Schlessinger ◽  
...  
Keyword(s):  
Growth Factor ◽  
Protein Kinase ◽  
Epidermal Growth Factor ◽  
Kinase Activity ◽  
Egf Receptor ◽  
Sodium Dodecyl ◽  
Specific Protein ◽  
Kinase Assay ◽  
Downstream Effector ◽  
Epidermal Growth

Raf-1 serine- and threonine-specific protein kinase is transiently activated in cells expressing the epidermal growth factor (EGF) receptor upon treatment with EGF. The stimulated EGF receptor coimmunoprecipitates with Raf-1 kinase and mediates protein kinase C-independent phosphorylation of Raf-1 on serine residues. Hyperphosphorylated Raf-1 has lower mobility on sodium dodecyl sulfate gels and has sixfold-increased activity in immunocomplex kinase assay with histone H1 or Raf-1 sequence-derived peptide as a substrate. Raf-1 activation requires kinase-active EGF receptor; a point mutant lacking tyrosine kinase activity in inactive in Raf-1 coupling and association. It is noteworthy that tyrosine phosphorylation of c-Raf-1 induced by EGF was not detected in these cells. These observations suggest that Raf-1 kinase may act as an important downstream effector of EGF signal transduction.

Epidermal growth factor and angiotensin II regulation of extracellular signal-regulated protein kinase in rat liver epithelial WB cells

1999 ◽  
Vol 57 (4) ◽  
pp. 425-432 ◽  
Author(s):  
Li-Jun Yang ◽  
Yan-Lin Guo ◽  
Oxana Trygankova ◽  
Qiu-Yang Li ◽  
Judith A. Maloney ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Growth Factor ◽  
Protein Kinase ◽  
Epidermal Growth Factor ◽  
Rat Liver ◽  
Extracellular Signal ◽  
Epidermal Growth
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