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Mitogenic Signaling from Theegf Receptor Is Attenuated by A Molecular Biology of the Cell Vol. 7, 871-881, June 1996 Mitogenic Signaling from the EGF Receptor Is Attenuated by a Phospholipase C-y/Protein Kinase C Feedback Mechanism Philip Chen, Heng Xie, and Alan Wells* Department of Pathology, University of Alabama at Birmingham, and Veterans Administration Medical Center, Birmingham, Alabama 35294-0007 Submitted December 29, 1995; Accepted March 28, 1996 Monitoring Editor: Joseph Schlessinger We recently demonstrated that epidermal growth factor receptor (EGFR)-mediated sig- naling 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 inves- tigated a possible role for a phospholipase C (PLC)-dependent feedback mechanism that attenuates EGF-induced mitogenesis. Inhibition of PLCy activation by U73122 (1 JIM) augmented the EGF-induced [ Hithymidine 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 PLCy 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- PLCOy 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 JIM) 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 mo- tility 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 PLCy activity attenuates the EGFR mitogenic response. INTRODUCTION Marikovsky et al., 1993; Miettinen et al., 1995; Sibilia Activation of the epidermal growth factor receptor and Wagner, 1995). Many reports have demonstrated (EGFR) elicits multiple cellular and biological re- that growth factor-induced cell motility and mitogenic sponses in vitro and in vivo, including mitogenesis responses can be signaled independently (Nister et al., and cell movement. Both cell proliferation and move- 1988; Hartmann et al., 1992; Faletto et al., 1993; Chen et mentment~are requiredetule~~for important~ ~1993).an~cellWel~~~~~~~ptwyfunctionsrecentlyssuch ashavesal., 1994a;t Manske(Rynldand Bade,a!.,1994), via distinct signal- development and wound repair (Adamson, 1990; ing pathways (Reynolds et al., 1993). We recently have demonstrated that the divergence of the pathways re- Corresponding author: LHRB 531, Department of Pathology leading to EGFR-elicited motility and mitogenic University of Alabama at Birmingham, Birmingham, AL 35294- sponses occurs at the immediate post-receptor level 0007. (Chen et al., 1994a,b). How the balance of signaling ( 1996 by The American Society for Cell Biology 871 P. Chen et al. between these two pathways is resolved remains to be MATERIALS AND METHODS elucidated. Functional tyrosine kinase activity is required for Generation of NR6 Cells Expressing both mitogenic and motility responses elicited by EGFR Constructs EGFR (Chen et al., 1987, 1994a). Receptor autophos- Construction of the EGFR and stable expression in NR6 cells were by standard methods, and have been described previously (Wells, phorylation and subsequent activation of phospho- 1990; Welsh et al., 1991; Chen et al., 1994a). Briefly, WT EGFR is a lipase Cy (PLCy) are required for EGF-induced cell full-length cDNA derived from a human placental cDNA library. movement but not for mitogenesis (Wells et al., 1990; c'973, c'991, and c'1000 represent EGFR in which stop codons are Decker, 1993; Chen et al., 1994a,b). PLCy activation by introduced just distal to the amino acid number indicated. c'1000F992 was created from c'1000 by replacing the sole remaining EGFR (Margolis et al., 1990a) produces diacylglycerol autophosphorylation site at y992 with a phenylalanine (F92). (DAG) and inositol trisphosphate, which activate the WTA61 is a full length EGFR in which the target site of PKC ser/thr kinase protein kinase C (PKC). PKC represents phosphorylation T5 was replaced with alanine (A654) by site-di- a large gene family of at least 12 isoforms differing in rected mutagenesis (Welsh et al., 1991). c'1000A654 was created from c'1000 with A654 replacing T6'. These EGFR are shown schemati- their structure, tissue distribution, subcellular local- cally in Table 1. ization, mode of activation, and substrate specificity The constructs were expressed on NR6 cells, 3T3-derivatives that (Dekker and Parker, 1994). PKCs phosphorylate a lack endogenous receptors (Pruss and Herschman, 1977). This was wide variety of substrates including proteins involved accomplished by retroviral-mediated transduction as previously described (Chen et al., 1994a). Polyclonal lines were established by in signal transduction (including Ras, GAP, and Raf) selection in 300 jig/ml G418 (Life Technologies, Gaithersburg, MD). (Hug and Sarre, 1993) as well as motility-associated The infectant cell lines presented physiologic levels of receptors cytoskeletal modulators (including fak, profilin, and (60,000-250,000 EGF binding sites per cell) with similar dissociation MARCKS) (Hansson et al., 1988; Aderem, 1992). These constants (Kd were 0.2 nM to 0.7 nM). All of the EGFR possessed kinase activity; the cells that presented the kinase-active EGFR all characteristics suggest a role for PKC isoforms in me- demonstrated a mitogenic response to EGF (Table diating specificity in intracellular signal transduction 1). pathways. Thymidine Incorporation Assay PKCs also attenuate signaling from growth factor EGF-induced mitogenesis was assessed by incorporation of [3H]thy- receptors including EGFR (Whitely and Glaser, 1986; midine in the target cells (Chen et al., 1994a). Cells were plated on Bowen et al., 1991; Welsh et al., 1991). PKC phos- plastic and grown to confluence in MEMa with 7.5% fetal bovine phorylates the threonine654 residue (T654) on EGFR serum (FBS). The cells were then switched to media containing 1% (Welsh et al., 1991), decreasing binding affinity of the dialyzed FBS for 24 h. The cells were subsequently treated with or without EGF (25 nM) and incubated at 37°C for 16 h. [3H]thymidine receptor for ligands (Jimenez-deAsua and Goin, 1992; (5,uCi/ml) was added and incubation continued for another 10 h. In Morrison et al., 1993) and diminishing tyrosine kinase the time-course experiments, [3H]thymidine was added at the times activity of the receptor (Lund et al., 1990). Phosphor- indicated to determine incorporation over the subsequent 3-h inter- ylation at this site on the receptor by PMA-activated val. The cells were then washed with ice-cold phosphate-buffered saline (PBS) twice and then treated with 5% trichloroacetic acid at PKC has been shown to block EGF-induced lamelli- 40C for 30 min. After two more washes with PBS the cells' incorpo- podia retraction (Welsh et al., 1991). In addition, serine rated [3H]thymidine was solubilized with 0.2N NaOH and mea- (S654) substitution causes constitutive phosphoryla- sured by scintillation counter. tion at this site with a concomitant decrease in thymi- The pharmacological agent, U73122 (1-(6-((17,3-3-methoxyestra- 1,3,5(10)-trien-17-yl)amino)hexyl)-lH-pyrrole-2,5-dione) (BIOMOL) dine incorporation (Bowen et al., 1991). These obser- was added to the cells to inhibit EGF-induced PLC activity. It was vations suggest a linear, physiological feedback introduced at 1 ,uM into the media in the [3H]thymidine incorpora- attenuation mechanism in which the activation of the tion assays 15 min prior to the addition of EGF. The inhibitory motility-associated PLCy and subsequent PKC lead to effects on EGF-induced PLC activity by this compound were deter- mined previously (Chen et al., 1994b); U73122 inhibits PLC activities attenuation of the mitogenic response elicited by but not phospholipase A2 or phospholipase D activities (Bleas- EGFR. However, the physiological importance of this dale et al., 1990; Powis et al., 1992; our unpublished observations). biochemical connection is yet to be demonstrated. Dis- The inactive congener of U73122, U73343 (1-(6((17,3-3-methoxy- section of this putative pathway will determine estra-1,3,5(10)-trien-17-yl)amino)hexyl)-2,5-pyrrolidine-dione), whether this serves as a decision point by which a cell was added at the same concentration in parallel and served as a control. The pharmacological agent Calphostin C (BIOMOL) was can direct a pluripotential extracellular stimulus into a added to the cells to inhibit PKC activity; Calphostin C was intro- specific biological response. In this study, we utilized duced at 0.05 ,tM into the media in the [3H]thymidine incorporation signaling-restricted EGFR expressed on receptor-de- assays 15 min prior to the addition
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