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Mitogenic signaling from the egf receptor is attenuated by a phospholipase C-gamma/protein kinase C feedback
1Department of Pathology, University of Alabama at Birmingham 35294-0007, USA.
Abstract:
We recently demonstrated that epidermal growth factor receptor (EGFR)-mediated signaling of cell motility and mitogenesis diverge at the immediate post-receptor level. How these two mutually exclusive cell responses cross-communicate is not known. We investigated a possible role for a phospholipase C (PLC)-dependent feedback mechanism that attenuates EGF-induced mitogenesis. Inhibition of PLC gamma activation by U73122 (1 microM) augmented the EGF-induced [3H]thymidine incorporation by 23-55% in two transduced NR6 fibroblast lines expressing motility-responsive EGFR; increased cell division and mitosis was observed in parallel. The time dependence of this increase revealed that it was due to an increase in maximal incorporation and not a foreshortened cell cycle. Motility-responsive cell lines expressing a dominant-negative PLC gamma fragment (PLCz) also demonstrated augmented mitogenic responses by 25-68% when compared with control cells. PLCz- or U73122-augmented mitogenesis was not observed in three non-PLC gamma activating, nonmotility-responsive EGFR-expressing cell lines. Protein kinase C (PKC), which may be activated by PLC-generated second messengers, has been proposed as mediating feedback attenuation due to its capacity to phosphorylate EGFR and inhibit the receptor's tyrosine kinase activity. Inhibition of PKC by Calphostin C (0.05 microM) resulted in a 57% augmentation in the fold of EGF-induced thymidine incorporation. To further establish PKC's role in this feedback attenuation mechanism, an EGFR point mutation, in which the PKC target threonine654 was replaced by alanine, was expressed. Cells expressing these PKC-resistant EGFR constructs demonstrated EGF-induced motility comparable to cells expressing the threonine-containing EGFR. However, when these cells were treated with U73122 or Calphostin C, the mitogenic responses are not enhanced. These findings suggest a model in which PKC activation subsequent to triggering of motility-associated PLC gamma activity attenuates the EGFR mitogenic response.
Insights
A phospholipase C (PLC)-dependent feedback mechanism involving Protein Kinase C (PKC) attenuates epidermal growth factor receptor (EGFR) mitogenesis. Inhibiting PLC or PKC enhances EGF-induced cell division, suggesting a novel cross-talk pathway.
Area of Science:
- Cell Biology
- Molecular Signaling
- Cancer Research
Background:
- Epidermal growth factor receptor (EGFR) signaling controls cell motility and mitogenesis.
- The cross-communication between these distinct EGFR-mediated cellular responses remains unclear.
- A potential feedback mechanism attenuating mitogenesis warrants investigation.
Purpose of the Study:
- To investigate a phospholipase C (PLC)-dependent feedback mechanism that attenuates EGF-induced mitogenesis.
- To elucidate the role of Protein Kinase C (PKC) in this feedback attenuation.
- To understand the cross-talk between EGFR-mediated cell motility and mitogenesis.
Main Methods:
- Utilized chemical inhibitors (U73122 for PLC, Calphostin C for PKC) in NR6 fibroblast lines.
- Employed dominant-negative PLC gamma fragment (PLCz) and site-directed mutagenesis of EGFR (T654A).
- Assessed EGF-induced [3H]thymidine incorporation, cell division, and mitosis.
Main Results:
- Inhibition of PLC gamma activation significantly augmented EGF-induced mitogenesis (23-55%) in motility-responsive cells.
- Expression of dominant-negative PLC gamma also enhanced mitogenic responses (25-68%).
- Inhibition of PKC augmented EGF-induced thymidine incorporation by 57%; PKC-resistant EGFR mutants abrogated this augmentation.
Conclusions:
- A model is proposed where PKC activation, downstream of motility-associated PLC gamma activity, attenuates EGFR mitogenic signaling.
- This feedback loop provides a mechanism for cross-communication between EGFR-driven motility and mitogenesis.
- Findings highlight a novel regulatory pathway in EGFR signaling relevant to cell proliferation.