A negative feedback loop attenuates EGF-induced morphological changes.

Abstract

Abstract. Activation of the EGF receptor tyrosine kinase by ligand indirectly activates a series of other cellular enzymes, including protein kinase C . To test the hypothesis that phosphorylation of the EGF receptor by protein kinase C provides an intracellular negative feedback loop to attenuate EGF receptor signaling, we used scanning EM to follow the characteristic EGFinduced retraction of lamellipodia and concomitant cell shape changes . Wild type and mutant EGF receptors were expressed in receptor-deficient NR6 cells. The mutant receptors were prepared by truncation at C' terminal residue 973 (c'973) to provide resistance to ligand-induced down regulation that strongly attenuates receptor signaling and by replacement of threonine 654 (T654) with alanine (A654) to remove the site of phosphorylation by protein kinase C. Cells expresscomplex network of growth stimulatory and growth inhibitory factors provides environmental information that regulates cell proliferation . Sufficient signal strength is required to elicit cell proliferation while mechanisms for attenuating signaling arenecessary to limit inappropriate cell division . Posttranscriptional mechanisms that attenuate signaling by ligand-activated EGF receptors include down regulation and transmodulation . On ligand binding, EGF receptors, which are diffusely distributed on the cell surface, cluster in coated pits (Haigler et al ., 1978), undergo internalization via a high affinity saturable endocytic process , and are ultimately degraded (Stoscheck and Carpenter, 1984) . Mutant EGF receptors that fail to down regulate in response to ligand activation display increased mitogenesis and transformation at low concentrations of EGF (Chen et al ., 1989 ;Wells et al ., 1990), emphasizing the consequences of loss of this attenuation mechanism .Other signaling pathways can decrease the ability of EGF to act . The most extensively characterized mechanism for transmodulation of the EGF receptor involves protein kinase C. Activated protein kinase C catalyzes phosphorylation of ing WT and c'973 EGF receptors demonstrated characteristic lamellipodial retraction after exposure to EGF, with the non-down regulating c'973 EGF receptors responding more rapidly. Exposure of cells to TPA blocked this response . Replacement of T614 by alanine resulted in EGF receptors that were resistant to TPA. Cells expressing the A654 mutation underwent more rapid and more extensive morphologic changes than cells with the corresponding T614 EGF receptor. In cells expressing T654 EGF receptors, down regulation of protein kinase C resulted in more rapid and extensive EGF-induced changes similar to those seen in cells expressing A614 EGF receptors . These data indicate that activation of protein kinase C and subsequent phosphorylation of the EGF receptor at T 654 lead to rapid physiological attenuation of EGF receptor signaling .

Luteinising hormone-releasing hormone analogue reverses the cell adhesion profile of EGFR overexpressing DU-145 human prostate carcinoma subline

“…Other LHRH analogues have been shown to limit prostate carcinoma cell growth secondary to downregulation of EGFR (Moretti et al, 1996;Jungwirth et al, 1997a, b) or through interference with signalling pathways initiated by the EGFR (Wells et al, 2002). This occurred via PKC-mediated crossattenuation (Wells et al, 2002) secondary to phosphorylation on threonine 654 of EGFR (Lin et al, 1986;Welsh et al, 1991). In this study, we show direct activation of PKC substrate MARCKS by LHRH receptors in a time-dependent manner (Figure 3).…”

“…This subline is identical to DU-145 WT except that it contains a full-length EGFR in which the target site for PKC phosphorylation, amino-acid threonine 654 (T654), has been replaced with alanine (DU-145 A654) by site-directed mutagenesis; this construct is resistant to PKC phosphorylation and negative transmodulation (Welsh et al, 1991;Chen et al, 1996).…”

Co-culturing human prostate carcinoma cells with hepatocytes leads to increased expression of E-cadherin

“…As this was reminiscent of the situation with LHRH receptor transmodulation of autocrine EGFR signalling in DU-145 cells (Yates et al, 2005), we asked whether a similar situation might be functioning in this setting. We expressed the PKC transmodulation-resistant EGFR A654 variant (Welsh et al, 1991) in the DU-145 cells. Co-culturing these cells with hepatocytes did not lead to decreases in EGFR or increases in E-cadherin and cytokeratin 18 expression (Figure 4).…”

Label-Free and Real-Time Cell-Based Kinase Assay for Screening Selective and Potent Receptor Tyrosine Kinase Inhibitors Using Microelectronic Sensor Array

“…[14][15][16][17] To demonstrate the utility of impedance, which quantitatively measures changes in cell morphology, as a readout to identify potent inhibitors of EGFR or its activated downstream signaling pathways, Sigma's enzyme inhibitor Ligand Set was screened. This is a collection of 80 diverse small organic compounds that have drug-like properties.…”

“…10,11 Using scanning electron microscopy (SEM), time-lapse imaging, or FRET-based imaging, several studies have assessed cell morphology as a sensitive assay for the integrated biological response elicited by EGF. [14][15][16][17] However, the use of cell morphology as an output for a screen to identify inhibitors of growth factor receptors or of its downstream signaling pathways is hampered by the intractability of existing techniques used to measure morphological changes.…”