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 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 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 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 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 determined 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 of EGF. void NR6 fibroblast cells to differentially activate the EGF-induced motility pathway and this putative Cell Proliferation Assays downstream mitogenesis-attenuation mechanism. We Two alternative methods to assess EGF-induced mitogenesis were also used pharmacological as well as molecular agents used to correlate thymidine incorporation with cell division. Cell to disrupt this motility-associated pathway, and proliferation determinations by counting were described previously we (Chen et al., 1994a). Briefly, NR6 cells (-5,000 cells/well) were assessed the affects on EGFR-mediated mitogenic re- plated in a 12-well tissue culture plate. Immediately after attach- sponse. ment on the plastic, the cells were switched to media containing 1%

872 Molecular Biology of the Cell EGF Receptor Feedback Inhibition by PLC/PKC

Table 1. EGF-induced PLCy activity and cell migration in NR6 cells expressing EGFR constructs

Construct Schematic of EGF receptor Migrationa PLCy activityb Parental no EGF receptor 101±4 97±2 068 1148 1173 T654 9 5 8 y992 y1 WT 225±41 337±74 c' 1 000 -1 EI-E 170±11 335±55 c'1000F9 9 2 121±14 121±4 c'991 105±18 110±10 _ c'973 0I/IMZI 114±4 101±11 FPMFIMF!tl c' 1186 F3 177±25 240±75 WTA654 215±8 n.d. c'1 000A654 213±28 n.d.

- Extracellular ligand-binding domain O - Transmembrane domain - Tyrosine kinase domain - Carboxy-terminal regulatory domain - Domain around autophosphorylatable tyrosine y992 - Domain around autophosphorylatable tyrosine y1068 - Domain around autophosphorylatable tyrosine y 1148 - Domain around autophosphorylatable tyrosine y1173

F, phenylalanine replacing the autophosphorylated tyrosines at the indicated sites; A654, alanine replacing the PKC phosphorylating site T654. a EGF-induced migration after 24 h of 25 nM EGF treatment; expressed as percentage of non-EGF treated cells, mean + SEM, n = 4-12. b EGF-induced PLCy activity measured as 3H-phosphotidylinositol turnover after 30 min treatment of 25 nM EGF; expressed as percentage of non-EGF treated cells, mean + SEM, n = 3-7.

dialyzed FBS for 24 h. The cells were subsequently treated with or control of an SV40 promoter serving as a selectable marker. These without EGF (10 nM) and incubated at 37°C for 3 days (Chen et al., constructs were transfected into WT and c'1000 EGFR-expressing 1994a; Reddy et al., 1994). At the end of incubation, cells were NR6 cells and selected with 400 nM methotrexate (Chen et al., trypsinized and counted by Coulter counter. Alternatively, the frac- 1994b). Induced expression of PLCz in the pDexMTX transfectants tion of cells entering mitosis was assessed by incorporation of was accomplished by introducing 2 ,uM dexamethasone into the bromo-deoxyuridine utilizing commercially available reagents media 16 h before the thymidine incorporation assay. Dexametha- (BrdU Staining Kit, Oncogene Sciences). BrdU incorporation was sone remained in the media throughout the assay period. The determined from 14-24 h post EGF stimulation as with thymidine expression of constitutive and dexamethasone-induced PLCz ex- incorporation. pression was verified by immuno-blot analyses described below. The effect of PLC inhibition on cell proliferation was demonstrated by introduction of the selective PLC inhibitor U73122. This Cell Motility Assay pharmacological agent was added at 1 ,AM 15 min prior to the addition of EGF; U73343 served as the control. EGF-induced migration was assessed by the ability of the cells to move into an acellular area as previously described (Chen et al., Cloning and Expression of Dominant Negative 1994a). U73122 (and U73343) or Calphostin C was introduced at 1 p.M or 0.05 ,uM, respectively, into the media 15 min prior to the PLCy-1 in NR6 Cells addition of EGF and remained throughout the assays. A dominant-negative PLCy-1 gene fragment that encodes the Z region SH2 and SH3 domains (amino acids 517-901, designated as Western Blot Analysis on Whole Cell Lysate PLCz; Homma and Takenawa, 1992) of this enzyme was cloned into the pXf vector (Chen et al., 1994b). The PLCz fragment also was Effects of TPA (12-O-tetradecanoylphorbol 13-acetate) on the ty- cloned into pDexMTX, the pXf vector with the 1.2-kb MMTV pro- rosine kinase activity of EGFR were demonstrated by immuno-blot moter in place of the SV40 early promoter, for inducible expression. analyses. Cells (4 x 106) were treated with 10 nM TPA 10 min prior These PLCz-containing vectors contain a DHFR gene under the to EGF (10 nM) treatment and total cell lysates were loaded on each

Vol. 7, June 1996 873 P. Chen et al. lane. Calphostin C was introduced 15 min prior to TPA treatment to porting an increased percentage of cells being re- inhibit PKC activation. The immuno-blot was probed with a mono- cruited into the proliferative response rather than a clonal anti-phosphotyrosine antibody (PY-20, Transduction Labora- tory, Lexington, KY) and visualized by probing with AP-conjugated foreshortened cell cycle. secondary antibodies followed by development with a colorimetric To demonstrate that this phenomenon is PLC'y spe- method (Promega, Madison, WI). cific and motility-associated, we examined the EGF- The expression of dexamethasone-induced, MMTV-driven PLCz induced thymidine incorporation response in cells ex- was verified by Western blot analyses. Cells (4 x 106) were treated with or without 2 ,uM dexamethasone for 16 h before cell lysis. pressing c'973, c'991, and c'100OF992 EGFR that do not Whole cell lysates were separated on 7.5% SDS-PAGE. The blot was activate PLCy nor elicit motility response upon ligand probed by mixed monoclonal anti-PLCy-1 antibody (05-163; UBI, stimulation (Table 1). In these cells, treatment with 1 Lake Placid, NY) and visualized as described above. ,uM U73122 did not result in increased EGF-induced thymidine incorporation (Figure 2), although basal RESULTS thymidine incorporation was increased in two of three cell lines. These data suggest that basal PLC activity Inhibition of PLCy Augments EGF-induced suppresses low level mitogenic stimuli. Thymidine Incorporation We further demonstrated the specificity of PLC/s We previously have demonstrated that EGF-induced involvement in this mitogenic attenuation mechanism cell motility requires PLCy activity. The selective by introducing a dominant negative PLCy-1 fragment pharmacologic inhibitor U73122 decreased both EGF- (PLCz) in two motility-responsive cell lines. PLCz en- induced PLC activity (but not EGF-induced PLD ac- codes the SH2- and SH3-containing Z region of tivity) and the motility response (Chen et al., 1994b). PLC-y-1 and overexpression of this peptide inhibits To assess the effects on mitogenesis by inhibiting PLC activation of the endogenous PLCy by EGFR (Chen activation, we introduced this agent at 1 ,uM in thy- et al., 1994b) and other receptor tyrosine kinases midine incorporation assays and observed an aug- (Homma and Takenawa, 1992). Expression of PLCz in mentation of 23-55% in EGF-induced incorporation NR6 cells expressing two motility-responsive EGFR, response in two transduced NR6 cell lines expressing c'1000 and c'l 186F3, augmented both basal and EGF WT and c'1000 EGFR (Figure 1A), which are motility- mitogenic responses (Figure 3). Because the consti- responsive and activate PLCy upon EGF stimulation tutive expression of PLCz may preferentially select (Table 1). The basal level of thymidine incorporation a subpopulation of fast growing cells, we expressed was increased in the presence of U73122, but to a PLCz acutely under the control of an MMTV pro- lesser extent than the EGF-induced increase. Although moter. WT EGFR-expressing NR6 cells were treated the absolute level of EGF-induced thymidine incorpo- with or without 2 ,uM dexamethasone to induce ration varied somewhat between the infectant cell PLCz expression, as verified by immuno-blot (Fig- lines, there was no correlation with EGFR level. To ure 4A). Concomitantly, the mitogenic response of correlate thymidine incorporation response with cell this expression was tested; treatment with dexa- division, we performed cell proliferation assays as an methasone augmented the EGF-induced thymidine alternative method to assess mitogenesis. Similar to incorporation by 58%, while decreasing the basal what is observed in thymidine incorporation re- rate of thymidine incorporation (Figure 4B). Con- sponse, EGF-induced cell proliferation in WT EGFR- versely, treatment of non-PLCz-transfectant WT expressing NR6 cells is also augmented by 31% in the EGFR-expressing NR6 cells with dexamethasone (2 presence of 1 ,tM U73122 (Figure 1B). As assessed by ,uM) decreased basal thymidine incorporation by BrdU incorporation, the percentage of cells stimulated 35-50%, although the EGF-induced thymidine in- to enter mitosis increased 23% in the face of U73122 corporation was not affected. Together, these data (60 ± 1% versus 74 ± 5% in U73343). Interestingly, suggest that a physiological feedback attenuation U73122 also increased basal thymidine incorporation mechanism on EGF-induced mitogenic response is and cell proliferation (Figure 1, A and B), although the mediated by receptor-activated, motility-associated augmentation in the absence of EGF was less than that PLCy. in the presence of EGF. The observed augmentation in both basal and EGF-induced mitogenesis suggests that a physiologically active inhibitory mechanism is atten- Pharmacological Inhibition of PKC Augments uated by U73122 treatment. EGF-induced Thymidine Incorporation To determine whether this augmentation is due to Activation of PKC by nonphysiological phorbol esters an increase in maximal incorporation or altered kinet- has been shown to inhibit the tyrosine kinase activity ics of mitogenesis, we determined thymidine incorpo- of the EGFR (Downward et al., 1985; Bowen et al., 1991) ration at various times. In cells expressing WT and as well as the biological responses it mediates (Welsh c'1000 EGFR, U73122 resulted in increased thymidine et al., 1991). To determine whether PKC participates in incorporation throughout the entire period of the mi- the physiological feedback attenuation mechanism de- togenic burst elicited by EGF (Figure 1C), further sup- scribed, we employed the selective PKC inhibitor Cal-

874 Molecular Biology of the Cell EGF Receptor Feedback Inhibition by PLC/PKC

A 4000 B WT EGFR-expressing NR6 Cells 6 o c 0

0 5 0

. N.0 c 0 o 4 0 o E a 0s L. o) 3 0 0 = o)

CE 0 0

.0 2 0 I-- E

1 0 -

WT c'1 000 EGFR Construct Expressed in NR6 Cells 0. control U73122

Treatment

Figure 1. Effect of the PLC-spe- C cific inhibitor U73122 on EGF-in- WT EGFR-expressing NR6 Cells c'1000 EGFR-expressing NR6 Cells duced [3Hlthymidine incorpora- 1000000 tion (A), cell proliferation (B), and E E I0owo the time course of [3Hlthymidine Q incorporation (C) in infectant 800000 .X NR6 cell lines expressing motili- X ty-responsive EGFR. EGF-in- L_ duced [Hithymidine incorpora- 60/00l tion was determined for each cell o line tested in the presence of c 4/00 4000/0 U73122 (1 ,aM) or its inactive con- e l O gener U73343 (1 ,uM). Equivalent X numbers of cells (-100,000 cells) E 200000 E > were tested. The incorporated l0000 [ Hithymidine is expressed as actual counts. *, basal incorpora- , tion without EGF; (III), basal in- 9 1 2 15 1 8 21 2 4 2 7 3 0 9 1 2 1 5 1 8 21 2 4 2 7 3 0 corporation in the presence of U73122; (1), EGF-stimulated in- Time (hrs) Time (hrs) corporation (25 nM EGF); and (D), EGF-stimulated incorporation in the presence of U73122. Shown are mean SD for three determinations; p < 0.01 between U73122-treated and U73343-treated cells. Cell proliferation were expressed as cell number after 3 days of 10 nM EGF exposure. (-), basal proliferation; (3), EGF-stimulated proliferation (25 nM EGF); Shown are mean + SD for three determinations. phostin C and assessed its effects on EGF-induced have reported that replacing this residue with serine mitogenic response. Treatment with TPA (10 nM) dra- (S654) has resulted in constitutive phosphorylation on matically decreased the EGF-induced tyrosine phos- this site and has dramatically decreased EGFR's sig- phorylation on EGFR as demonstrated by immuno- naling and the mitogenic response it mediates (Bowen blot analysis. The presence of Calphostin C (as low et al., 1991). On the other side, replacing this residue as 0.01 ,uM) reversed this inhibition (Figure 5). with alanine (A654) makes this site resistant to phos- Treatment with Calphostin C (0.05 ,uM) resulted in a phorylation by activated PKC; this has resulted in 57% augmentation in the fold of EGF-induced mi- augmented EGFR-mediated biological responses such togenic response in WT EGFR-expressing cells (Fig- as lamellipodia retraction and mitogenesis (Bowen et ure 6). Calphostin C treatment decreased the abso- al., 1991; Welsh et al., 1991). To determine whether lute level of both the basal and EGF-induced phosphorylation on T654 is secondary to PLC-medi- thymidine incorporation. ated feedback attenuation mechanism, we expressed on NR6 cells, WT, and c'1000 EGFR in which T654 was Replacing the PKC Phosphorylation Site P54 on replaced by alanine, WTA654, and c'1000A654, respec- EGFR with A654 Abrogates the PLCy and tively. We examined the EGF-induced mitogenic re- PKC-mediated Attenuation Mechanism sponse in the presence of the selective PLC inhibitor Activated PKC inhibits EGFR's tyrosine kinase activ- U73122 and assessed the attenuation response within ity by phosphorylating T654 on the receptor. Others each line independently. Although U73122 caused an

Vol. 7, June 1996 875 P. Chen et al.

experiments demonstrated that thymidine incorporation occurred at similar levels throughout the entire 0 mitogenic burst period regardless of the presence of '~3000 U73122 (Figure 7C). 0 The alanine replacement did not affect the upstream 00 PLC-mediated motility response (Figure 8 and Table 000- 1). To further establish this site in the feedback path- CL way, we examined the mitogenic response in the presence of PKC inhibitor Calphostin C. In the presence of 0.05 ,uM Calphostin C, NR6 cells expressing WTA654 0E00X 0 no longer exhibit the augmented fold-increase in mitogenic response as observed in WT EGFR-expressing cells (Figure 6). Together, these observations placed the T654 phosphorylation site in EGFR downstream in c'l00OF992 c'991 c'973 the motility-associated, PLC-mediated feedback atten- EGFR Construct Expressed in NR6 Cells uation mechanism on EGF-induced mitogenesis. Figure 2. Effect of the PLC-specific inhibitor U73122 on EGF-induced [3H]thymidine incorporation in infectant NR6 cell lines expressing nonmotility-responsive EGFR. EGF-induced [3Hithymi- DISCUSSION dine incorporation was determined for each cell line tested in the presence of U73122 (1 ,M) or its inactive congener U73343 (1 ,uM). Mitogenesis and cell movement are assumed to be Equivalent numbers of cells (--100,000 cells) were tested. The incor- mutually exclusive biological responses. However, porated [3Hlthymidine is expressed as actual counts. *, basal incor- many growth factors are capable of triggering both. poration without EGF; (WI), basal incorporation in the presence of The mechanism by which a cell chooses one response U73122; (0), EGF-stimulated incorporation (25 nM EGF); (O), EGF- is unknown. Signaling to stimulated incorporation in the presence of U73122. Shown are pathways leading EGFR- mean ± SD for three determinations. mediated mitogenic and motility responses diverge at the immediate post-receptor level. EGFR-activated PLC-y activity is required for the motility response but is not necessary for mitogenesis (Chen et al., 1994b). augmentation in thymidine incorporation in WT and MAP kinase activity, which has been implicated in c'1000 EGFR-expressing cells (Figure 1A), the muta- mediating the mitogenic response, is not sufficient tion to A654 totally abrogated this augmentation (Fig- to elicit the motility response (Chen et al., 1994b). ure 7A). Similar to thymidine incorporation, U73122- Whether the two signaling pathways leading to dis- enhanced cell proliferation also is abolished by alanine tinct biological responses communicate downstream replacement (Figures 1B and 7B). The time course has not been determined previously. Herein we de- lineated a motility-associated feedback attenuation mechanism affecting EGF-induced mitogenic re- 4000 -+ 668% + 25% sponse. 0 Inhibition of the PLCy activation by a selective h. 3000- pharmacological agent, U73122, or the expression of 0 a dominant-negative molecular inhibitor, PLCz, augmented the EGF-induced mitogenic response. C0 By using both a pharmacologic agent, x 2000 'Z~ which selec- E tively blocks PLC-mediated PIP2 hydrolysis, and a molecular approach to prevent EGFR-signaled activation of PLCy, we can definitively implicate a I-C 1000 physiological mitogenic attenuation mechanism mediated by motility-associated PLCy. Downstream of PLCy, second messengers DAG and inositol c'l 000 c'lOOOPLCz c'1186F3 c'1186F3PLCz trisphosphate elicit various intracellular biochemical processes including the activation of PKC. PKC EGFR and PLCz Constructs Expressed In NR6 Cells was a candidate for mediating this attenuation Figure 3. Effect of constitutively expressed dominant-negative mechanism because its activation by phorbol esters PLCz on EGF-induced [3H]thymidine incorporation in two motility- has been shown to inhibit EGFR's intrinsic tyrosine responsive EGFR NR6 cell lines with or without the expression of kinase activity secondary to phosphorylation of PLCz. Equivalent numbers of cells (-100,000 cells) were tested. The incorporated [3Hlthymidine is expressed as actual counts. *, basal EGFR (Whitely and Glaser, 1986; Lund et al., 1990; incorporation with no treatment; (1, EGF-stimulated incorporation Morrison et al., 1993). Inhibition of PKC by Calphos- (25 nM EGF). Shown are mean ± SD for six determinations. tin C augmented the EGF-induced mitogenic re-

876 Molecular Biology of the Cell EGF Receptor Feedback Inhibition by PLC/PKC

Figure 4. (A) Expression of A B dexamethasone-inducible PLCz Construct: WT/mmtv-PLCz and (B) its effect on EGF-induced [3Hlthymidine incorpo- Treatment: cdr dex c 1500 ration in WT EGFR-expressing : 200 NR6 cells. WT/mmtv-PLCz in- PLCg-1 dicates WT EGFR-expressing NR6 cells transfected with the 00 pDexMTX-PLCz construct (see text). Cells were exposed to 97 x dexamethasone (2 ,uM) for 16 h 0 before whole cell immuno-blot ._ E for PLCy; the band at -130 kDa E 500 is the endogenous PLCy and the 69 > band at -50 kDa represents the I. PLCz fragment. The incorporated [3H]thymidine is ex- PLCz pressed as actual counts. Shown 46 are mean + SD for three deter- Unstimulated Dexamethasone minations. Treatment of WT EGFR NR6 cells Expressing pDexMTX-PLCz

sponse. Replacement of the PKC target site on EGFR lating the EGFR at T654 (Lund et al., 1990; Bowen et abrogated U73122 and Calphostin C augmentation al., 1991; Welsh et al., 1991; Seedorf et al., 1995). This in mitogenic response. These data place PKC down- PLC,y/PKC feedback inhibition of signaling may be stream of PLCy in mediating this feedback mecha- common for other members of the receptor tyrosine nism. Together, an intracellular molecular mecha- kinase (RPTK) family because PKC has also been nism is demonstrated for the EGFR-activated observed to inhibit insulin receptor signaling (Seed- motility pathway to inhibit the mitogenic response. orf et al., 1995) as well as to attenuate Grb2 phos- PKC exerts pleiotropic effects on multiple intra- phorylation by PDGF receptor (Benjamin et al., cellular biochemical pathways as well as cellular 1994). Furthermore, these pathways may enable var- biological responses. Activated PKC has been dem- ious RPTKs to cross communicate; PDGF, probably onstrated to inhibit EGFR signaling by phosphory-

c 0 _ c 8 25nM EGF + + + + + + + lOnM TPA _ _ + + + + + + 00 Calphostin C (uM) - - - 0V1 0o 0o 10.s 0. 7- 0_ a. EGFR -> 00

L . 3- ILl

C 1-

WT WTA654 EGFR Construct Figure 5. Calphostin C reversal of TPA-induced inhibition of EGFR tyrosine kinase activity. Cells were exposed to EGF (25 nM) in Figure 6. Effect of Calphostin C on EGF-induced [3Hlthymidine the presence of TPA (10 nM) for 5 min to demonstrate PKC-medi- incorporation in WT and WTA654 EGFR-expressing NR6 cells. EGF- ated inhibition of EGFR kinase activity. Various concentrations of induced [3H]thymidine incorporation was determined in the pres- Calphostin C were added to the cells, concurrent with TPA and EGF ence or absence of 0.05 ,uM Calphostin C. Equivalent numbers of administration, to determine the concentration of Calphostin C that cells (-100,000 cells) were tested. The incorporated [3H]thymidine would prevent TPA inhibition of EGF-induced kinase. EGFR kinase is expressed as percent incorporation in the absence of EGF treat- activity was determined by autophosphorylation and phosphoryla- ment, with the basal (100%) being depicted as a horizontal line. (u), tion of other cellular substrates. Equal amounts of whole cell lysates EGF-stimulated incorporation (25 nM EGF); and (Ol), EGF-stimu- were loaded on each lane. The immuno-blot was probed with an lated incorporation in the presence of Calphostin C. Shown are anti-phosphotyrosine antibody. mean ± SD for three determinations.

Vol. 7, June 1996 877 P. Chen et al.

WTA654 EGFR-expressing NR6 Cells A 300 B 70-

0 6 0-

0co r 0 a.-. 2000- 010 50- 0 -w ._ 4 0- x 3 0- 01000 = C :6 0b- E 0 20 ai U 1 0-

0- WTA654 c'1 00OA654 control U73122 Construct Expressed in NR6 Cells EGFR Treatment C WTA654 EGFR-expressing NR6 Cells c'1OOOA654 EGFR-expressing NR6 Cells Figure 7. Effect of the PLC- specific inhibitor U73122 on 800000- E EGF-induced [3Hlthymidine in- a o corporation (A), cell proliferation (B), and time course of 600000- I0I\X /) ff [3Hlthymidine incorporation a. :7 '>L " q // t(C) in infectant NR6 cell lines 0 6000 expressing motility-response cNn..Nffi0L S \^I EGFR in which the PKC phos- 065 400000- site T654 was re- m phorylation 400000 \'K /placed. with A654. EGF-induced [3H]thymidine incorporation E ff '\\ s: s \9 was determined for each cell line m 200000 tested in the presence of U73122 (1 ,uM) or its inactive congener Xl* _ U73343. _d_ uM).Equivalent numbers of cells (-100,000 cells) were 0- 1 2 15 1 8 21 24 27 3 0 0- tested. The incorporated [3H]thy- 3091 2 1 5 18 21 24 27 30 midine is expressed as actual Time (hrs) Time (hrs) counts. N, basal incorporation without EGF; (Ill), basal incorporation in the presence of U73122; (0), EGF-stimulated incorporation (25 nM EGF); and 0, EGF-stimulated incorporation in the presence of U73122. Shown are mean ± SD for three determinations. Cell proliferation (B) was expressed as cell number after 3 days of 10 nM EGF exposure. Shown are mean ± SD for three determinations. via PLCy activation (Margolis et al., 1990b), has been basal and EGF-induced mitogenic responses. How- shown to induce the T654 phosphorylation on EGFR ever, in cells expressing EGFR that do not activate by PKC (Davis and Czech, 1987). The physiological PLC-y (Figure 2), only the basal mitogenic rate is importance of these putative biochemical processes altered, implying that EGFR-mediated mitogenesis is not clear. However, one phenomenon observed is already maximally induced. This model is further here may be attributed to such crosstalk. When the buttressed by the lack of effect of U73122 on the feedback attenuation pathway described herein was basal or EGF-induced mitogenic rate in cells ex- disrupted at the level of PLCy, we observed an pressing EGFR resistant to PKC transmodulation increase in basal mitogenic response (Figure 1, A (Figure 7). These findings suggest that low level and B, and Figure 2). This may be due to a back- PLCy activity may play an important physiologic ground signaling from various RPTKs in addition to role in preventing mitogenesis in cells either ex- EGFR to activate PLCy in cells in the basal state; this posed to suboptimal levels of growth factors or increment in basal proliferation is noted in cells secondary to nonspecific activation of RPTKs by expressing both motility-responsive and nonmotil- oligomerization (Lemmon and Schlessinger, 1994). ity-responsive EGFR. As PLCy is inhibited by TPA-activated PKC has been shown to decrease the U73122 or PLCz (Figures 3 and 4), the downstream high affinity binding between EGFR and its ligands. feedback inhibition on mitogenesis is relieved, thus This transmodulation may contribute to the mitogenic cells expressing WT and c'1000 exhibit augmented attenuation. However, replacement of the PKC target

878 Molecular Biology of the Cell EGF Receptor Feedback Inhibition by PLC/PKC

al., 1996). Blocking PKC activation with Calphostin most whose activation 0 C inhibits isoforms depends 1- . u ] T on DAG. Although this treatment augmented the 0 fold-increase in EGF-induced mitogenic response '=200 co (Figure 6), it decreased the absolute basal mitogenic E responses by 50-60%. This indicates that in addi- 0; tion to the isoform(s) involved in the feedback at- 150 tenuation pathway, other PKC isoforms may medi- =°i0 ate at least part of the mitogenic response. This is consistent with previous findings demonstrating La- CD some PKC isoforms have the capacity to induce w mitogenesis (Ullrich et al., 1986; Hug and Sarre, 50 1993). Furthermore, the A654 point mutation that WT WTA654 replaced the PKC target site in EGFR established the EGFR Construct Expressed in NR6 Cells specific effects on mitogenesis of PKC isoform(s) Figure 8. EGF-induced cell motility in the face of disruption of the involved in the feedback pathway (Figure 7, A-C). PKC-mediated attenuation signal by replacing the PKC phosphor- Identification of which specific isoform(s) will in- ylation site T654 on EGFR with A654 and treatment with Calphostin volve specific inhibitors (i.e., anti-sense treatment) C (0.05 ,kM). EGF-induced cell migration response is expressed as percent response in the absence of EGF treatment. *, basal cell and will clarify and differentiate the mixed effects migration with no treatment; (I1), basal migration in the presence of PKC activity exerts on mitogenesis. Calphostin C; (12), EGF-stimulated migration (25 nM EGF); and El, Phosphorylation of T654 on EGFR can serve as a EGF-stimulated incorporation in the presence of Calphostin C. general feedback mechanism in EGF-induced biologi- Shown are mean ± SD for three determinations. cal responses. It has been demonstrated that lamellipodia retraction response, like mitogenesis, is sub- site T654 with acidic glutamate did not alter the EGF jected to this feedback attenuation mechanism (Welsh binding properties (Morrison et al., 1993). Bowen et al. et al., 1991). In keratinocytes, activation of PKC by has also reported that constitutive phosphorylation on nonphysiologic phorbol esters resulted in inhibition of this site by S654 replacement did not affect TPA-acti- EGF-induced cell migration, and inhibition of PKC by vated transmodulation on EGFR, nor did replacement H7 led to a slight increase in the migration response by the nonphosphorylatable A654 (Bowen et al., 1991). (Ando et al., 1993). In our system, however, replacing These observations suggest that the PKC-mediated T654 with A654 or treating cells with Calphostin C feedback attenuation mechanism is probably not due (Figure 8) to disrupt the feedback attenuation loop did to decreased ligand binding but rather by altering the not significantly affect the EGF-induced motility re- intrinsic tyrosine kinase activity of the receptor (Fig- sponse. These observations suggest that different bio- ure 5). The exact mechanism of how the phosphory- logical responses elicited by EGFR in different cells/ lation on this site blocks EGFR signaling still remains tissues may have differential sensitivity to this elusive. physiological feedback attenuation mechanism. PKC is a family of ser/thr kinases with a large Again, the tissue/cell type-specific and isoform-spe- number of isotypes, each differs greatly in structure, cific characteristics of PKC actions most likely play a function, subcellular localization, tissue distribu- role as do the subcellular localizations of PKC and tion, and substrate specificity (Dekker and Parker, 1994). Activation of PKC has been shown to mediate PLC'y. These findings also suggest that the motility numerous biological responses (Gladhaug et al., response is not sensitive to intermittent disruption of 1992; Hug and Sarre, 1993; Dekker and Parker, EGFR signaling by feedback attenuation, while the 1994). These responses likely result from the diver- mitogenic response may require prolonged, constant sity of these isotypes and often demonstrate isotype- EGFR activity. and tissue/cell-type specificity. The isoform(s) in- As more signaling pathways are dissected, commu- volved in this feedback attenuation pathway has nications among pathways become more apparent been determined to be a classical, calcium-depen- and complex (Pawson, 1995). Often multiple path- dent isotype(s) (Welsh et al., 1991). We further spec- ways converge to control one biological response. It is ulate that it is a caveolae-associated isoform, such as likely that subtly altering the balance of the signaling PKCa, because EGFR and PKC colocalize within strength among these pathways dictates which re- caveolae (Mineo et al., 1995). This attenuation mech- sponse predominates. We propose that a specific PKC anism likely acts locally in a submembrane localiza- isoform or subcellular localization of PKC serves this tion, in conjunction with the EGFR-mediated PIP2 role in determining whether a cell moves or prolifer- hydrolysis that actuates the motility signal (Chen et ates upon EGF stimulation.

Vol. 7, June 1996 879 P. Chen et al.

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