Mitogen-activated protein kinase-dependent and -independent routes control shedding of transmembrane growth factors through multiple secretases

2002 ◽  
Vol 363 (2) ◽  
pp. 211-221 ◽  
Author(s):  
Juan Carlos MONTERO ◽  
Laura YUSTE ◽  
Elena DÍAZ-RODRÍGUEZ ◽  
Azucena ESPARÍS-OGANDO ◽  
Atanasio PANDIELLA
Keyword(s):  
Growth Factors ◽  
Protein Kinase ◽  
Uv Light ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Mitogen Activated Protein Kinase ◽  
Ovary Cells ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
Factor Α

Solubilization of a number of membrane proteins occurs by the action of cell-surface proteases, termed secretases. Recently, the activity of these secretases has been reported to be controlled by the extracellular signal-regulated kinases 1 and 2 (ERK1/ERK2) and the p38 mitogen-activated protein kinase (MAPK) routes. In the present paper, we show that shedding of membrane-anchored growth factors (MAGFs) may also occur through MAPK-independent routes. In Chinese-hamster ovary cells, cleavage induced by protein kinase C (PKC) stimulation was largely insensitive to inhibitors of the ERK1/ERK2 and p38 routes. Other reagents such as sorbitol or UV light stimulated MAGF cleavage independent of PKC. The action of sorbitol on cleavage was only partially prevented by the combined action of inhibitors of the p38 and ERK1/ERK2 routes, indicating that sorbitol can also stimulate shedding by MAPK-dependent and -independent routes. Studies in cells devoid of activity of the secretase tumour necrosis factor-α-converting enzyme (TACE) indicated that this protease had an essential role in PKC- and ERK1/ERK2-mediated shedding. However, secretases other than TACE may also cleave MAGFs since sorbitol could still induce shedding in these cells. These observations suggest that cleavage of MAGFs is a complex process in which multiple secretases, activated through different MAPK-dependent and -independent routes, are involved.

scholarly journals Activation of B-Raf and Regulation of the Mitogen-activated Protein Kinase Pathway by the Go α chain

2000 ◽  
Vol 11 (4) ◽  
pp. 1129-1142 ◽  
Author(s):  
Valeria Antonelli ◽  
Francesca Bernasconi ◽  
Yung H. Wong ◽  
Lucia Vallar
Keyword(s):  
Protein Kinase ◽  
Egf Receptor ◽  
Mapk Pathway ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Mitogen Activated Protein Kinase ◽  
Phosphatidylinositol 3 Kinase ◽  
Ovary Cells ◽  
Mapk Activity ◽  
Mitogen Activated Protein

Many receptors coupled to the pertussis toxin-sensitive Gi/o proteins stimulate the mitogen-activated protein kinase (MAPK) pathway. The role of the α chains of these G proteins in MAPK activation is poorly understood. We investigated the ability of Gαo to regulate MAPK activity by transient expression of the activated mutant Gαo-Q205L in Chinese hamster ovary cells. Gαo-Q205L was not sufficient to activate MAPK but greatly enhanced the response to the epidermal growth factor (EGF) receptor. This effect was not associated with changes in the state of tyrosine phosphorylation of the EGF receptor. Gαo-Q205L also potentiated MAPK stimulation by activated Ras. In Chinese hamster ovary cells, EGF receptors activate B-Raf but not Raf-1 or A-Raf. We found that expression of activated Gαo stimulated B-Raf activity independently of the activation of the EGF receptor or Ras. Inactivation of protein kinase C and inhibition of phosphatidylinositol-3 kinase abolished both B-Raf activation and EGF receptor-dependent MAPK stimulation by Gαo. Moreover, Gαo-Q205L failed to affect MAPK activation by fibroblast growth factor receptors, which stimulate Raf-1 and A-Raf but not B-Raf activity. These results suggest that Gαo can regulate the MAPK pathway by activating B-Raf through a mechanism that requires a concomitant signal from tyrosine kinase receptors or Ras to efficiently stimulate MAPK activity. Further experiments showed that receptor-mediated activation of Gαo caused a B-Raf response similar to that observed after expression of the mutant subunit. The finding that Gαo induces Ras-independent and protein kinase C- and phosphatidylinositol-3 kinase-dependent activation of B-Raf and conditionally stimulates MAPK activity provides direct evidence for intracellular signals connecting this G protein subunit to the MAPK pathway.

scholarly journals Mitogen-activated protein kinase/extracellular signal-regulated kinase 2 regulates cytoskeletal organization and chemotaxis via catalytic and microtubule-specific interactions.

1997 ◽  
Vol 8 (7) ◽  
pp. 1219-1232 ◽  
Author(s):  
A A Reszka ◽  
J C Bulinski ◽  
E G Krebs ◽  
E H Fischer
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Mitogen Activated Protein Kinase ◽  
Ovary Cells ◽  
Morphological Alterations ◽  
Cytoskeletal Regulation ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

The extracellular signal-regulated kinases (ERKs) 1 and 2 are mitogen-activated protein kinases that act as key components in a signaling cascade linking growth factor receptors to the cytoskeleton and the nucleus. ERK2 mutants have been used to alter cytoskeletal regulation in Chinese hamster ovary cells without affecting cell growth or feedback signaling. Mutation of the unique loop L6 (residues 91-95), which is in a portion of the molecule that is cryptic upon the binding of ERK2 to the microtubules (MTs), generated significant morphological alterations. Most notable phenotypes were observed after expression of a combined mutant incorporating changes to both L6 and the TEY phosphorylation lip, including a 70% increase in cell spreading. Actin stress fibers in these cells, which normally formed a single broad parallel array, were arranged in three or more orientations or in fan-like arrays. MTs, which ordinarily extend longitudinally from the centrosome, spread radially, covering a larger surface area. Single, but not the double, mutations of the Thr and Tyr residues of the TEY phosphorylation lip caused a ca. 25% increase in cell spreading, accompanied by a threefold increase in chemotactic cell migration. Mutation of Lys-52 triggered a 48% increase in cell spreading but no alteration to chemotaxis. These findings suggest that wild-type ERK2 inhibits the organization of the cytoskeleton, the spreading of the cell, and chemotactic migration. This involves control of the orientation of actin and MTs and the positioning of focal adhesions via regulatory interactions that may occur on the MTs.

Endothelins stimulate mitogen-activated protein kinase cascade through either ETA or ETB

1994 ◽  
Vol 267 (4) ◽  
pp. C1130-C1135 ◽  
Author(s):  
Y. Wang ◽  
P. M. Rose ◽  
M. L. Webb ◽  
M. J. Dunn
Keyword(s):  
Protein Kinase ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Mapk Cascade ◽  
Mitogen Activated Protein Kinase ◽  
Thymidine Uptake ◽  
Mapk Activation ◽  
Transfected Cells ◽  
Gel Retardation ◽  
Mitogen Activated Protein

Endothelin (ET) has been shown to activate mitogen-activated protein kinase (MAPK). However, it has been unclear which of the ET receptors is coupled to MAPK activation. In the present study, we conducted experiments to determine which ET receptor is linked to MAPK activation. We found that both human ETA and ETB were coupled to the MAPK cascade in ETA or ETB cDNA-transfected Chinese hamster ovary cells. ET-1 was more potent than ET-3 in the activation of p42 MAPK, induction of MAPK kinase (MAPKK) gel retardation and uptake of [3H]thymidine in ETA-transfected cells, whereas sarafotoxin (S6c) showed no stimulatory effect on the kinases and [3H]thymidine uptake. ET-1, ET-3, and S6c had approximately the same potency to activate p42 MAPK, MAPKK gel retardation, and [3H]thymidine uptake in ETB-transfected cells. These data suggest that 1) ET isopeptides, through either ETA or ETB receptors, induce the MAPK cascade as well as cell proliferation; and 2) the different potencies of ET isopeptides for activation of the MAPK cascade and induction of cell growth are mainly due to their different affinities toward ETA and ETB.

scholarly journals Insulin-stimulated disassociation of the SOS-Grb2 complex.

1995 ◽  
Vol 15 (5) ◽  
pp. 2791-2799 ◽  
Author(s):  
S B Waters ◽  
K Yamauchi ◽  
J E Pessin
Keyword(s):  
Protein Kinase ◽  
Electrophoretic Mobility ◽  
Chinese Hamster Ovary Cells ◽  
Sodium Dodecyl ◽  
Chinese Hamster ◽  
Mitogen Activated Protein Kinase ◽  
Insulin Stimulation ◽  
Cell Extracts ◽  
Mitogen Activated Protein

Insulin stimulation of differentiated 3T3-L1 adipocytes or Chinese hamster ovary cells expressing high levels of the insulin receptor resulted in a time-dependent decrease in the electrophoretic mobility of SOS on sodium dodecyl sulfate-polyacrylamide gels. The reduction in SOS mobility was completely reversed by alkaline phosphatase treatment, and the in vitro phosphorylation of SOS by mitogen-activated protein kinase resulted in a decrease of electrophoretic mobility identical to that following in vivo insulin stimulation. Immunoprecipitation of Grb2 followed by SOS immunoblotting demonstrated a disassociation of the SOS-Grb2 complex that paralleled the decrease in SOS electrophoretic mobility. Similarly, SOS immunoprecipitation followed by Grb2 immunoblotting also indicated an uncoupling of the SOS-Grb2 complex. Further, incubation of whole-cell extracts with glutathione-S-transferase-Grb2 fusion proteins demonstrated that insulin stimulation resulted in a decreased affinity of SOS for Grb2. In contrast, the dissociation of SOS from Grb2 did not affect the interactions between Grb2 and tyrosine-phosphorylated Shc. In addition to insulin, several other agents which activate the mitogen-activated protein kinase pathway (platelet-derived growth factor, serum, and phorbol ester) also resulted in the uncoupling of the SOS-Grb2 complex. Consistent with these results, expression of v-ras and v-raf resulted in a constitutive decrease in the association between SOS and Grb2. Together, these data suggest a molecular mechanism accounting for the transient activation of ras due to the uncoupling of the SOS-Grb2 complex following SOS phosphorylation.

Growth Factors and Extracellular Signal-Regulated Kinases (Mitogen-Activated Protein Kinase) in the Rat Pineal Gland

1994 ◽  
Vol 59 (2) ◽  
pp. 152-155 ◽  
Author(s):  
Hiroshi Kiyama ◽  
Akio Wanaka ◽  
Hidemasa Kato ◽  
Hiroshi Maeno ◽  
Kazumasa Matsumoto ◽  
...  
Keyword(s):  
Growth Factors ◽  
Protein Kinase ◽  
Pineal Gland ◽  
Mitogen Activated Protein Kinase ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
Rat Pineal Gland
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