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Epidermal growth factor induces phosphorylation of extracellular signal-regulated kinase 2 via multiple pathways
B M Burgering1, A M de Vries-Smits, R H Medema
1Laboratory of Physiological Chemistry, Utrecht University, The Netherlands.
Abstract:
Expression of p21rasAsn-17, a dominant negative mutant of p21ras that blocks p21ras activation by growth factors, inhibits activation of extracellular signal-regulated kinase 2 (ERK2) by insulin and platelet-derived growth factor in rat-1 cells [A. M. M. de Vries-Smits, B. M. T. Burgering, S. J. Leevers, C. J. Marshall, and J. L. Bos, Nature (London) 357:602-604, 1992]. Here we report that expression of p21rasAsn-17 does not abolish epidermal growth factor (EGF)-induced phosphorylation of ERK2 in fibroblasts. Since EGF activates p21ras in these cells, this indicates that EGF induces a p21ras-independent pathway for the phosphorylation of ERK2 as well. We investigated whether activation of protein kinase C (PKC) or increase in intracellular calcium could be involved in p21ras-independent signaling. In rat-1 cells, inhibition of either PKC, by prolonged 12-O-tetradecanoylphorbol-13-acetate (TPA) pretreatment, or calcium influx, by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) pretreatment, did not abolish EGF-induced ERK2 phosphorylation. However, a combined inhibition of both p21ras and calcium influx, but not PKC, resulted in a complete inhibition of EGF-induced ERK2 phosphorylation. In contrast, in Swiss 3T3 cells, inhibition of both p21ras activation and TPA-sensitive PKC, but not calcium influx, inhibited EGF-induced ERK2 phosphorylation. These results demonstrate that in fibroblasts, EGF induces alternative pathways of ERK2 phosphorylation in a cell-type-specific manner.
Insights
Epidermal Growth Factor (EGF) activates a p21ras-independent pathway for ERK2 phosphorylation in fibroblasts. This pathway involves calcium influx in rat-1 cells but protein kinase C (PKC) in Swiss 3T3 cells.
Area of Science:
- Cell signaling pathways
- Molecular biology
- Signal transduction
Background:
- Growth factors like EGF activate signaling pathways crucial for cell growth and differentiation.
- Extracellular signal-regulated kinase 2 (ERK2) is a key mediator in these pathways.
- The role of p21ras in EGF-induced ERK2 activation has been previously established, but alternative pathways are being explored.
Purpose of the Study:
- To investigate the involvement of p21ras-independent signaling in epidermal growth factor (EGF)-induced ERK2 phosphorylation.
- To determine the roles of protein kinase C (PKC) and intracellular calcium in EGF-mediated ERK2 activation.
- To elucidate cell-type-specific differences in EGF signaling pathways.
Main Methods:
- Expression of a dominant-negative p21ras mutant (p21rasAsn-17) to block p21ras activation.
- Inhibition of protein kinase C (PKC) using 12-O-tetradecanoylphorbol-13-acetate (TPA).
- Inhibition of calcium influx using ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).
- Analysis of ERK2 phosphorylation in response to EGF in rat-1 and Swiss 3T3 cells under various inhibition conditions.
Main Results:
- EGF-induced ERK2 phosphorylation was not abolished by p21rasAsn-17 alone, indicating a p21ras-independent pathway.
- In rat-1 cells, combined inhibition of p21ras and calcium influx, but not PKC, completely inhibited EGF-induced ERK2 phosphorylation.
- In Swiss 3T3 cells, combined inhibition of p21ras and TPA-sensitive PKC, but not calcium influx, inhibited EGF-induced ERK2 phosphorylation.
Conclusions:
- EGF utilizes alternative signaling pathways for ERK2 phosphorylation in fibroblasts.
- These alternative pathways are cell-type-specific, involving calcium in rat-1 cells and PKC in Swiss 3T3 cells.
- Understanding these divergent pathways is crucial for comprehending EGF-mediated cellular responses.