©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Rapid and Long Term Effects of Protein Kinase C on Receptor Tyrosine Kinase Phosphorylation and Degradation (*)

(Received for publication, November 21, 1994; and in revised form, May 10, 1995)

Klaus Seedorf (§) Mark Shearman (¶) Axel Ullrich (**)

From theDepartment of Molecular Biology, Max-Planck-Institut für Biochemie, Am Klopferspitz 18A, 82152 Martinsried, Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Rapid and long term effects of protein kinase Calpha activation on receptor tyrosine kinase signaling parameters were investigated in human 293 embryonic fibroblasts and mouse NIH 3T3 cells. Within minutes of phorbol 12-myristate 13-acetate treatment, epidermal growth factor receptor and HER2 tyrosine phosphorylation was decreased, while platelet-derived growth factor receptor and insulin receptor autophosphorylation was up-regulated. These effects are not mediated by protein kinase C-dependent receptor tyrosine kinase phosphorylation but apparently by activation or inactivation of receptor tyrosine kinase-specific phosphatases, as indicated by neutralization of these phenomena upon treatment of cells with sodium orthovanadate. In contrast to these short term effects, sustained activation of protein kinase Calpha by phorbol 12-myristate 13-acetate results in translocation of protein kinase C from the cytosol to the membrane fraction where it forms stable complexes with all receptor tyrosine kinases investigated. Ligand-induced receptor tyrosine kinase/protein kinase C association in NIH 3T3 fibroblasts is accompanied by a mobility shift of the receptor, indicating phosphorylation by activated protein kinase C. This phenomenon correlates with the disappearance of receptor tyrosine kinases from the cell surface, implying that this interaction plays a role in the process of receptor internalization and degradation. Interestingly, ligand-stimulated receptor down-regulation is also enhanced by overexpression of phospholipase C, which strongly indicates a role for this common receptor tyrosine kinase substrate in negative regulation of growth factor signals.


INTRODUCTION

Reversible protein phosphorylation on tyrosine residues represents a regulatory mechanism that governs a wide spectrum of fundamental processes in cells of higher eukaryotes. Among the cellular enzymes that catalyze these reactions are tyrosine kinases with a covalently connected extracellular ligand binding function, which play a key role in the intercellular communication network that is crucial for the regulation of growth, development, and metabolic homeostasis of multicellular organisms. The tyrosine-specific phosphorylation function of these receptor tyrosine kinases (RTKs) (^1)is indispensable for the activation of signaling pathways that promote cellular responses such as cell division, differentiation, and migration. Engagement of such cellular effector systems occurs through phosphorylated tyrosine residues of RTKs, which function as high affinity binding sites for proteins containing Src homology 2 (SH2) domains. These occur either in combination with a catalytic function such as phospholipase C (PLC), the GTPase-activating protein (GAP), the Src tyrosine kinase, the tyrosine phosphatase PTP1D/Syp, and the GDP/GTP exchanger protein Vav or as adapter proteins such as the p85 subunit of phosphatidylinositol 3`-kinase (PI 3`-kinase), CRK, NCK, SHC, and GRB2 (reviewed in (1, 2, 3) ).

Despite extensive information concerning molecular interactions in the signaling cascade triggered by receptor-ligand interaction on the cell surface, the events that eventually activate specific gene transcription and define the ligand-characteristic response of the cell remain poorly understood. It is clear, however, that downstream from tyrosine-phosphorylated primary substrates the activation of serine/threonine kinases is of major significance for signal transmission and definition. These include the Raf-1 kinase(4, 5) , the S6 kinases(6, 7, 8) , mitogen-activated protein kinase kinase(9) , mitogen-activated protein kinase(10, 11, 12) , casein-2 kinases(13) , and the members of the protein kinase C (PKC) family.

The PKC serine/threonine kinases, a family of at least 12 isoenzymes with distinct tissue distribution characteristics, have been subdivided on the basis of different primary structures and enzymatic properties into Ca-dependent or conventional PKCs and the Ca-independent or novel PKCs (reviewed in ref. 14), and extensive evidence is available for their involvement in control of cell proliferation, differentiation, and motility(15) . While certain PKC isotypes are activated by phorbol esters such as phorbol 12-myristate 13-acetate (PMA), their physiological ligand is the second messenger diacylglycerol (DAG)(16, 17) , a product of the phosphoinositide-specific PLC, whose subtype was shown to be activated by mitogens such as epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) through receptor-mediated tyrosine phosphorylation(18, 19, 20, 21, 22) . In addition to DAG, PLC-catalyzed hydrolysis of phosphatidylinositol 4,5-biphosphate yields inositol 1,4,5-triphosphate, a regulator of intracellular [Ca], which together with DAG activates PKC.

Among its diverse effects on the physiology of cells (reviewed in (16) ), PKC was shown to phosphorylate and activate the serine/threonine kinase Raf-1(23, 24) , which triggers the mitogen-activated protein kinase signaling pathway and ultimately leads to transcriptional activation of specific genes(25) . In addition to this positive influence on growth factor signals, PKC activation is known to down-modulate the signaling potential of the receptors for EGF and insulin. Phosphorylation of the EGF-R at Thr-654 attenuates high affinity EGF binding (26) and causes a decrease in EGF-stimulated tyrosine kinase activity and DNA synthesis(27, 28) . Similarly, the signaling activity of the insulin receptor, which appears to be primarily phosphorylated on carboxyl-terminal serine and threonine residues(29, 30) , is impaired after PMA treatment of some cell types due to a reduction of its tyrosine kinase activity(31, 32, 33) , while in other cells an activation of RTK activity was observed(34, 35, 36) . Moreover, as recently shown, phorbol ester treatment of Chinese hamster ovary cells overexpressing PKCalpha and the insulin receptor resulted in phosphorylation of receptor threonine and serine residues without an effect on tyrosine phosphorylation but caused inhibition of insulin-stimulated PI 3`-kinase activity(37) . Interestingly, although the EGF-R and PDGF-R share common signaling pathways, they differ with respect to PKC-dependent negative feedback mechanisms. While PMA treatment of A431 cells blocks EGF-induced PI turnover(38, 39) , it does not inhibit that induced by PDGF(40, 41) , suggesting differential regulation of RTKs by the PKC system within the same cell.

In addition to its differential effects on receptor-ligand interactions and signal transduction, PKC has been implicated in receptor internalization and degradation. Chen et al.(42) identified a distinct region within the EGF-R COOH terminus that appears to be required for ligand-dependent down-regulation and degradation. Interestingly, the high affinity binding site for PLC, Tyr-992, is located within this region(43) , which suggests that PKC activation via PLC-generated second messengers is involved in receptor down-regulation. Nevertheless, the molecular signals that are involved are still poorly understood, and their interpretation is a matter of controversy.

To further elucidate the complex role of PKC in the cellular signaling network, we investigated its short and long term effects on RTK function. While short term stimulation of PKC affects the EGF and HER1-2 receptors differently than those for PDGF and insulin, the functional consequences for these receptors are identical after prolonged PKC activation. PKC-mediated signal modulation involves ligand-induced, high affinity complex formation with receptors. Moreover, our findings strongly support a role for PLC in PKC-mediated down-regulation of RTK signals.


MATERIALS AND METHODS

Cells

Transient expression was performed in human embryonic kidney fibroblast 293 cells, while stable expression of RTK cDNAs was performed in NIH 3T3 fibroblasts. All cells were cultured in DMEM containing 4 g/liter of glucose, 10% fetal calf serum, and 2 mML-glutamine.

Expression Plasmids and Antibodies

cDNAs coding for the human EGF-R, EGF-R/HER2 chimera (HER1-2)(44) , EGF-R/PDGFbeta-R chimera (EP-R)(45) , EGF-R/insulin receptor chimera (EI-R)(46) , and human PLC (47) were cloned into a cytomegalovirus-driven expression vector (48) for all transient overexpression experiments. For stable expression in NIH 3T3 fibroblasts, the RTK cDNAs were introduced into an expression plasmid containing SV40 early promoter elements and 3`-untranslated sequences of the hepatitis virus surface antigen gene.

The following monoclonal antibodies and polyclonal antisera were used: 108.1 (49) mouse monoclonal antibody against the extracellular domain of the EGF-R; 5E2 mouse monoclonal antibody against phosphotyrosine (50) ; CT-PLC, rabbit polyclonal antiserum against a peptide corresponding to amino acid residues 1255-1274 of human PLC (DFRISQEHLADHFDSRERR) (provided by Reiner Lammers); and PKC-specific polyclonal antibodies (RIOS) (provided by Etta Livneh).

Transfection and Transient Expression

High efficiency transformation of human embryonic kidney fibroblast 293 cells was performed essentially as described by Chen and Okayama (51) and Gorman et al.(52) . Briefly, 293 cells were seeded into 6-well dishes (1.5 10^5 cells/well) or 10-cm dishes (8 10^5 cells/dish) and incubated overnight in DMEM containing 10% fetal calf serum (Life Technologies, Inc.). A total of 6 and 20 µg, respectively, of expression plasmid DNA was mixed with 0.15 or 0.5 ml of 0.25 M CaCl(2), 0.15 or 0.5 ml of 2 BBS (280 mM NaCl, 1.5 mM Na(2)HPO(4), 50 mM BES, pH 6.95). The mixture was incubated for 15 min at room temperature, added to the cells, swirled gently, and incubated for 18 h at 35 °C under 3% CO(2). The medium was replaced by fresh DMEM containing 10% fetal calf serum, incubated for 24 h at 37 °C under 5% CO(2), and subsequently lysed. In experiments in which cells were metabolically labeled with [S]methionine (Amersham Corp.), the cells were transfected as described. The next morning, medium was replaced by DMEM containing 10% fetal calf serum and in the evening replaced again by methionine-free DMEM containing 3% fetal calf serum and 60 µCi of [S]methionine/ml. Cell labeling was carried out overnight. Immunoprecipitation and immunoblotting procedures were carried out according to standard protocols(45) .

PI 3`-Kinase Assay

Cells overexpressing HER1-2 and control cells were cultured overnight in DMEM (Life Technologies, Inc.) containing 0.5% fetal calf serum and subsequently treated with EGF, EGF and PMA, or vehicle for 10 min. Cells were lysed as described above. After a series of washes with HNTG buffer containing 0.1% Triton X-100, the presence of PI 3`-kinase activity in the precipitates was determined by incubating the precipitates with kinase buffer containing 30 mM Hepes (pH 7.4), 30 mM MgCl(2), 0.2 mM adenosine, 40 µM ATP, 0.2 mg/ml sonicated phosphatidylinositol and phosphatidylserine, and 10 µCi of [-P]ATP (3000 Ci/mmol) for 10 min at 30 °C. The phospholipids were extracted with chloroform/methanol (1:1) and washed twice with methanol, 1 N HCl (1:1) and finally spotted onto thin layer chromatography (TLC) plates. After one-dimensional chromatography, the plates were exposed to x-ray film. Radioactive lipids corresponding to authentic phosphatidylinositol monophosphate (PIP) standards were scraped from the plates and quantitated by Cerenkov counting.

PKC Immunokinase Assay

The PKC activity associated with the HER1-2 receptor following ligand stimulation was assayed as follows. Immunoprecipitated HER1-2 receptor complexes (10-µl packed protein A-Sepharose beads) were resuspended in reaction mixture (final volume, 50 µl) containing 20 mM Tris-Cl (pH 7.5), 5 mM magnesium acetate, 10 µM synthetic substrate peptide, 40 µg/ml phosphatidylserine, 4 µg/ml diolein, 0.1 mM calcium chloride, 40 µM EGTA, and 40 µM EDTA. For the assay of non-PKC kinase activity, the lipid mixture was replaced with 20 mM Tris-Cl (pH 7.5), and the calcium chloride/EGTA/EDTA solution was replaced with 0.5 mM EGTA. The samples were incubated at 30 °C for 25 min, followed by brief cooling on ice and centrifugation, and then a 20-µl aliquot of each sample was spotted onto a 1.5 1.5-cm square of phosphocellulose paper (P81, Whatman). The paper squares were washed in 75 mM phosphoric acid, with four changes of solution, and the bound radiolabeled peptide was quantitated by Cerenkov radiation.

Ligand- and PMA-induced Receptor Degradation

293 cells transfected with receptor, receptor and PLC, receptor and PKC, or receptor, PLC, and PKC expression plasmids were labeled for 18 h with [S]methionine, and receptor degradation was subsequently measured by a chase in normal culture medium in the presence of EGF (50 ng/ml) or PMA (1 µM) for various times. Receptors were immunoprecipitated with a monoclonal antibody directed against the extracellular domain of the EGF-R (mAb 108.1) and analyzed by 7.5% SDS-PAGE. The gel was treated with Amplify (Amersham) and exposed to x-ray film.


RESULTS

To analyze the functional relationship of PKC and various RTKs in intact cells, we employed chimeric receptors consisting of the human EGF-R extracellular domain fused to the cytoplasmic domains of the murine PDGF-beta-R (EP-R), human insulin receptor (EI-R), and the EGF-R isozyme HER2 (HER1-2). As previously shown, all of these chimeric receptors are functional and generate a cytoplasmic domain-characteristic cellular signal upon stimulation with EGF (44, 45, 46) . The chimeric receptor cDNAs and those of EGF-R, PLC, GAP, p85 subunit of PI 3`-kinase, and PKCalpha were used in the form of cytomegalovirus early promoter-driven expression vector (CMV) constructs for transient expression experiments in human 293 embryonic fibroblasts. To confirm results obtained in transient overexpression experiments, we employed NIH 3T3 cell lines, which had been transfected with RTK cDNA expression plasmids and were established as stable lines.

Differential Effects of PKC Activation and Phosphatase Inhibition on RTK Autophosphorylation

Short term effects of activated PKC on the autophosphorylation of different RTKs under comparable conditions were investigated by transfection of 293 cells with pCMV expression plasmids containing EP-R, HER2, HER1-2, EI-R cDNAs, and the insertless vector as a control either alone or in combination with pCMV-PKCalpha. As shown in Fig.1, tyrosine phosphorylation of all receptors was induced upon EGF stimulation of intact cells for 10 min (lanes1 and 2). When EGF was added together with the PKC activator PMA (shown as TPA) (lane9), the phosphotyrosine content of EGF-R and HER1-2 was significantly reduced, while EP-R and EI-R autophosphorylation was enhanced about 2-fold. Incubation with sodium orthovanadate, a potent inhibitor of phosphotyrosine-specific phosphatases, did not stimulate receptor phosphorylation in the absence of EGF (lane5) and did not further increase EGF-induced tyrosine phosphorylation (lane6); however, it caused full recovery of PMA-mediated suppression of EGF-R and HER1-2 phosphorylation (compare lanes9 and 11). Cotransfection with PKCalpha expression plasmid (lanes3, 4, 7, 8, 10, and 12) suppressed autophosphorylation of EGF-R, HER1-2, and EI-R in the presence of EGF (lanes3 and 4), and PMA-mediated activation of overexpressed PKCalpha caused complete abolishment of EGF-R and HER1-2 tyrosine phosphorylation while EI-R showed a slight recovery (lane10). Inhibition of phosphotyrosine-specific phosphatases in these cells by sodium orthovanadate had a positive effect on EGF-R, HER1-2, and EI-R autophosphorylation (compare lanes4 and 8, and 10 and 12) but was not able to fully reverse the PKCalpha-mediated suppression on receptor autophosphorylation. In all cases, EP-R tyrosine phosphorylation remained relatively unaffected. Expression levels of receptor and PKC were the same under all experimental conditions (not shown). These data suggest that EGF-R and HER1-2 phosphorylation on tyrosine residues may be down-regulated through PMA-mediated activation of a kinase inhibitory mechanism involving vanadate ion-sensitive phosphotyrosine phosphatases, which appear not to participate under PKC overexpression conditions. The EI-R and EP-R were positively affected by PMA treatment, indicating that these receptors are differently regulated by phosphatases; however, PKCalpha overexpression also inhibited EI-R autophosphorylation, suggesting that the tyrosine kinase was also inhibited.


Figure 1: Effect of EGF, PMA, and sodium orthovanadate on receptor tyrosine phosphorylation. 293 cells overexpressing the receptor cDNA alone (lanes1, 2, 5, 6, 9, 11) or together with PKCalpha (lanes3, 4, 7, 8, 10, 12) were treated with vehicle, 50 ng/ml EGF, EGF + PMA (1 µM), sodium orthovanadate (1 mM), or EGF + PMA + orthovanadate for 10 min as indicated. Receptors were immunoprecipitated using mAb 108.1, separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antiphosphotyrosine antibody 5E2. TPA, phorbol 12-myristate 13-acetate.



Physical Association of PKC with RTKs

Phosphorylation of RTKs such as EGF-R and I-R by PMA-activated PKC has been reported previously (reviewed in (53, 54, 55) ). The significance of this modification is not fully understood and appears to differ for individual RTKs and different cell types. In view of the differential effects on autophosphorylation of four chimeric receptors (Fig.1), we investigated the parameters that regulate RTK-PKC interaction.

Transfected 293 cells transiently overexpressing either RTKs alone or with PKCalpha were metabolically labeled with [S]methionine for 18 h and treated with PMA for 60 min. As shown in Fig.2, in the absence of PKCalpha overexpression (lanes1, 2, 4, 5, 7, 8, 10, 11), PMA treatment (lanes2, 5, 8, 11) caused no mobility shift of RTKs in SDS-PAGE, suggesting that the differential effects on RTK phosphorylation shown in Fig.1were not due to covalent, PMA-inducible modifications. In contrast, in cells coexpressing PKCalpha, PMA treatment resulted in a mobility shift of all RTKs. Interestingly, under these conditions immunoprecipitation with the EGF-R extracellular domain-specific mAb 108.1 led to coprecipitation of PKCalpha, suggesting that even in the absence of ligand, the RTKs used in this experiment bound PMA-activated PKC, but only when both proteins were overexpressed. This observation was confirmed with PKC-specific antibodies, which coimmunoprecipitated all receptors from samples that contained overexpressed PKCalpha (data not shown). Moreover, PMA-activated PKC associated with the kinase-deficient EGF-R mutant HERK721A (data not shown), further supporting our observation that receptor autophosphorylation is not required for PKC binding.


Figure 2: Immunoprecipitation of radiolabeled receptor tyrosine kinases after PMA treatment. 293 cells were transfected with EP-R, EGF-R, HER1-2, EI-R expression plasmid alone (lanes1, 2, 4, 5, 7, 8, 10, 11) or together with PKCalpha expression plasmid (lanes3, 6, 9, 12). Cells were metabolically labeled with [S]methionine and treated with PMA (lanes2, 3, 5, 6, 8, 9, 11, 12) for 60 min. Subsequently, the receptors were immunoprecipitated using mAb 108.1 (alphaEGF-R) and analyzed by SDS-PAGE.



To investigate whether complex formation of RTKs with PKC was an artifact of overexpression, we examined NIH 3T3 cell lines expressing the various receptors at levels of 1.5-2.5 10^5 per cell. As shown in Fig.3, immunoprecipitation of EP-R, EGF-R, HER1-2, and EI-R led to coimmunoprecipitation of endogenous PKC already in the absence of EGF. The amounts of RTK-bound PKC in starved cells varied, especially in the case of HER1-2, between experiments from barely detectable to somewhat further inducible by EGF treatment. Coimmunoprecipitation was further increased by ligand addition for all cell lines, indicating that receptor activation leads to enhanced PKC binding.


Figure 3: Coimmunoprecipitation of PKC after immunoprecipitation of RTKs stably expressed in NIH 3T3 cells. NIH 3T3 cells stably expressing the various receptors were cultured in low serum overnight and subsequently treated with EGF (100 ng/ml) for 30 min as indicated. Cells were lysed and receptors were immunoprecipitated using mAb 108.1 (alphaEGF-R) and separated by SDS-PAGE. After transfer to nitrocellulose, RTKs were detected using a polyclonal antiserum against the human EGF-R extracellular domain (antisera DIII, upperpanel), and coimmunoprecipitation of endogenous PKCalpha was determined by using PKC-specific antisera (lowerpanel).



Having demonstrated that PKC associates with the receptors for PDGF, EGF, HER2, and insulin in a ligand-dependent manner, we next investigated the growth factor effect on coimmunoprecipitating PKC activity. HER1-2-expressing NIH 3T3 cells were cultured overnight in low serum medium and then stimulated with EGF or vehicle prior to lysis and immunoprecipitation with mAb 108.1. The immunoprecipitates were washed and subsequently incubated in the presence of [- P]ATP, lipids, Ca, and AC-MBP, a PKC-specific substrate peptide (see ``Materials and Methods''). As shown in Fig.4, PKC that was coupled to unstimulated HER1-2 was activated by Ca and lipids and further enhanced by EGF addition in a time-dependent manner. These data confirm the results shown in Fig.2and Fig. 3and indicate that receptor autophosphorylation, which is completed seconds after ligand addition, does not directly lead to PKC binding but that receptor-activated signaling pathway(s) indirectly promote PKC activation, translocation to the membrane, and binding to RTKs.


Figure 4: Determination of receptor-bound PKC activity. HER1-2-expressing cells were left untreated or treated with EGF as indicated. HER1-2 receptor immunoprecipitates were assayed for PKC activity as described under ``Materials and Methods.'' The data given are the mean values from three independent experiments and are expressed relative to the protein kinase activity obtained in the presence of Ca and phospholipids from untreated cells. 100% activity approximates to the incorporation of 30 fmol -PO(4)bulletminbulletbulletmg cell lysate, under the standard assay conditions.



To further investigate the time course of EGF- versus PMA-induced RTKbulletPKC complex formation, HER1-2 NIH 3T3 cells were cultured for 24 h under low serum conditions and treated for various times with EGF or PMA. Coimmunoprecipitation of PKC with HER1-2 was then investigated by immunoblot analysis using PKCalpha-specific antibodies. EGF treatment led to a transient increase of PKC binding to HER1-2, reaching its maximum level after 30 min, while PMA treatment led to a more rapid and pronounced binding of PKC to the receptor, which continued to increase for 3 h (data not shown). Apparently, the experimental PKC activator PMA induces constitutive association with RTKs, while endogenous, presumably RTK activation-dependent signals are transient and subject to negative feedback mechanisms.

Effect of PKCalpha on HER1-2-bound PI 3`-Kinase Activity and Substrate Binding

Initial experiments with stable NIH 3T3 cell transfectants had shown that HER1-2, like the closely related EGF-R, interacts with PLC, GAP, PI 3`-kinase, SHC, and GRB2. (^2)To determine the effect of PKCalpha on HER1-2-substrate interaction, we measured the association of PI 3`-kinase activity with immunoprecipitated receptor from EGF- and/or PMA-stimulated cells. 293 cells were transfected with HER1-2 cDNA or HER1-2 and PKC cDNA expression plasmids or left untransfected. 24 h later, cells were metabolically labeled for 18 h and, prior to lysis and immunoprecipitation of the receptors, treated for 10 min with EGF, EGF and PMA, or vehicle. PI 3`-kinase activity was subsequently measured in receptor immunoprecipitates, as described under ``Materials and Methods.''

As indicated by the amount of PIP formed (Fig.5), almost no PI 3`-kinase activity coimmunoprecipitated with the endogenous receptors in untransfected cells under any of the stimulation conditions (lanes1-3). EGF stimulation of HER1-2-expressing cells (lane5), however, led to a strong increase in PIP levels, which was reduced by simultaneous addition of PMA (lane6), as measured by Cerenkov counting of the radiolabeled phospholipids. When PKCalpha was co-overexpressed with HER1-2, EGF stimulation resulted in PI 3`-kinase activity association with the receptor that was about 20% of that measured in the absence of PKC overexpression (lane5). PMA addition further reduced this effect (lane9), indicating that PKC activation or overexpression alone results in uncoupling of receptor-regulated signal transduction pathways.


Figure 5: HER1-2-bound PI 3-kinase activity. Untransfected (lanes1-3), HER1-2 (lanes4-6), and HER1-2 and PKCalpha (lanes7-9) expressing cells were metabolically labeled with [S]methionine overnight and subsequently left untreated, treated with EGF (50 ng/ml), or simultaneously with EGF and PMA (1 µM) as indicated. After immunoprecipitation with mAb 108.1 (alphaEGF-R), the samples were divided into three aliquots. One aliquot was used to determine receptor-bound PI 3`-kinase activity (lanes1-9), the second aliquot to determine receptor expression and PKC coimmunoprecipitation (lanes10-12), and the third to determine receptor autophosphorylation (not shown). Formation of PIP (lanes1-9) was determined by separation on TLC plates and exposure to film. The PIP spots were subsequently scraped from the plate and quantitated by Cerenkov counting. Expression of HER1-2 and coimmunoprecipitation of PKC was determined after receptor immunoprecipitation with 108.1 antibodies, separation by SDS-PAGE, and exposure to film (lanes10-12). TPA, phorbol 12-myristate 13-acetate.



To support this observation, we investigated the interaction of HER1-2 with additional substrates, including PLC, GAP, and p85, in the absence and presence of PKCalpha overexpression. Without PKCalpha cotransfection, EGF treatment resulted in coimmunoprecipitation of PLC, GAP, and p85 from transiently overexpressing 293 cells. Coincubation with PMA reduced receptor tyrosine phosphorylation similar to the effect shown in Fig.1(lane9) and concomitantly coimmunoprecipitation of all substrates investigated. Co-overexpression of PKCalpha reduced HER1-2 substrate interaction already in the absence of PMA, while activation of overexpressed PKCalpha by PMA almost completely abolished coprecipitation of PLC, GAP, and p85 (data not shown). These results demonstrate that PKCalpha is very potent in regulating the interaction of HER1-2 with subsequent signaling mediators.

Activated PKC Promotes RTK Internalization and Degradation

Upon ligand binding, RTKs that are thought to be diffusely distributed on the cell surface undergo rapid lateral movements, cluster in coated pits, and are internalized and degraded, a process for which PKC involvement has been postulated(56) . To test this hypothesis, we transiently overexpressed in 293 cells HER1-2 either alone or with PKC, PLC, or both PLC and PKCalpha and metabolically labeled with [S]methionine overnight. After washing the []methionine away, the cells were cultured in regular serum-free medium and treated with EGF or PMA for 4 h. Receptor degradation was determined by immunoprecipitation with mAb 108.1, separation by SDS-PAGE, and autoradiography. As shown in Fig.6(lanes1-3), no significant receptor degradation was detected after either EGF or PMA addition for 4 h in HER1-2-overexpressing 293 cells. Since it is possible that the amount of endogenous 293-cell PKC was insufficient to measurably affect the overexpressed HER1-2 receptor chimera, we repeated the experiment with cells co-overexpressing PKCalpha (Fig.6, lanes4-6). While EGF still had no significant effect on receptor degradation within the examined time period, PMA treatment led to a substantial loss of radiolabeled HER1-2, indicating that PKC activation plays a role in receptor down-regulation. To examine the function of PLC, which upon activation by RTK-mediated tyrosine phosphorylation catalyzes the production of the cellular PKC ligand DAG and IP3, a stimulator of Ca from intracellular stores, we included this RTK substrate in our co-overexpression experiment. As shown in Fig.6, PLC overexpression caused the HER1-2 receptor to be partially down-regulated in response to EGF stimulation (Fig.6, lane8), an effect that was enhanced by PKC co-overexpression (Fig.6, lane11). In the latter case, EGF- and PMA-induced receptor down-regulation were indistinguishable, demonstrating that activation of PKC via receptor-activated PLC and the corresponding second messengers was equivalent to direct activation of PKC by the artificial ligand PMA.


Figure 6: EGF- and PMA-induced degradation of radiolabeled HER1-2. 293 cells were transfected with HER1-2 expression plasmid alone (lanes1-3), together with PKC expression plasmid (lanes4-6), together with PLC expression plasmid (lanes7-9), and together with PKC and PLC expression plasmids (lanes10-12). Cells were metabolically labeled with [S]methionine overnight, and after washing away the radiolabel, HER1-2 degradation was determined in the absence or presence of EGF or PMA. After 4 h of incubation, cells were lysed, and HER1-2 was immunoprecipitated using mAb 108.1. After separation by SDS-PAGE, the gel was dried and exposed to film. TPA, phorbol 12-myristate 13-acetate.



The difference between PMA- and PLC-mediated receptor degradation in the absence of overexpressed PKC indicates that both DAG and elevated Ca levels are required to fully activate PKC isotypes involved in receptor down-regulation. PMA, which acts like DAG, may not be sufficient under low intracellular Ca concentrations to fully activate the respective isotypes, a deficiency that is compensated under PKC overexpression conditions.

To further investigate whether the time course of RTKbulletPKC complex formation correlated with the disappearance of radiolabeled receptors, 293 cells overexpressing HER1-2 and PKC were treated for various times with EGF, PMA, EGF together with PMA, or left untreated. The receptors were immunoprecipitated, separated by SDS-PAGE, and exposed to film. Fig.7shows that for 6 h, EGF had no effect on receptor degradation (compare untreated and EGF-treated lanes) and did not lead to enhanced binding of PKC to the receptor. Treatment with PMA alone or in combination with EGF induced, with increasing incubation time, the formation of a HER1-2bulletPKC complex and at the same time HER1-2 degradation. While the kinetics observed are influenced by de novo synthesis transport of receptors and PKC to the cell surface in the overexpression cell system, the information obtained by this analysis was not significantly affected. Identical results were obtained with EP-R, EGF-R, and EI-R (not shown), indicating that binding of activated PKC, leading to elimination of receptors from the cell surface by internalization and subsequent degradation, represents a general mechanism.


Figure 7: Time course of HER1-2bulletPKC complex formation and HER1-2 degradation. HER1-2-PKCalpha-overexpressing 293 cells were metabolically labeled for 18 h using [S]methionine. The labeling medium was replaced by normal DMEM, and the cells were then treated for various times with EGF, PMA, EGF + PMA, or left untreated as indicated. After immunoprecipitation of HER1-2 and separation by SDS-PAGE, the radiolabeled receptors and coimmunoprecipitated PKCalpha were visualized by exposure to x-ray film. TPA, phorbol 12-myristate 13-acetate.




DISCUSSION

In this study, we provide evidence that PKC may mediate rapid and delayed effects on RTKs. While the immediate consequences that influence receptor phosphorylation on tyrosine residues are diverse and RTK-specific, the delayed effects involve direct RTK-PKC interaction and appear to be common for different RTK subtypes.

EGF activates autophosphorylation of EP-R, EGF-R, HER1-2, and EI-R overexpressed in 293 fibroblasts. Simultaneous treatment with EGF and PMA reduced the autophosphorylation of EGF-R and HER1-2 but clearly enhanced EP-R and EI-R autophosphorylation. The PMA effect was very rapid and could be detected within a few minutes. Phorbol ester treatment of these cells did not lead to a mobility shift of RTKs in the absence of cotransfected PKCalpha, suggesting that the effects on RTK phosphorylation on tyrosine residues were not due to endogenous PKC-mediated RTK phosphorylation on serine and threonine residues. How phosphorylation of overexpressed RTKs is regulated by PMA is not clear, but one likely possibility is the involvement of phosphotyrosine-specific phosphatases. This is supported by the observation that orthovanadate, a potent inhibitor of this type of enzyme, is able to neutralize the PMA-mediated effects in the absence of overexpressed PKCalpha. Additionally, the PMA-mediated effects on EGF-R and HER1-2 tyrosine phosphorylation were dominant in experiments in which EGF and PMA were added simultaneously in cells that were treated first with EGF for 5 min and subsequently with PMA for 5 min or vice versa. (^3)These data indicate that PMA-induced receptor dephosphorylation can be reversed by orthovanadate treatment and is not due to covalent receptor modifications that, by inducing conformational changes, inhibit the intrinsic tyrosine kinase activity, as speculated previously. This effect might be regulated by PMA-inducible endogenous PKC isotypes, which phosphorylate and thereby activate certain tyrosine-specific phosphatases. Such PMA- and protein kinase C-dependent phosphorylation of tyrosine-specific phosphatases has been demonstrated in vivo and in vitro; however, no up-regulation of the enzymatic activity was detected (reviewed in (57) ). Alternatively, PMA might directly activate PTPases, but up to now, no data exist to support this possibility.

In the presence of overexpressed PKCalpha, all investigated RTKs formed a stable complex with PKCalpha upon PMA treatment, which at the same time resulted in a mobility shift of the receptors. Under these conditions, the PMA-inducible effects on tyrosine kinase activity were not reversible by orthovanadate, indicating that receptor phosphorylation on tyrosine residues is regulated by PKC-dependent phosphorylation on serine and threonine residues. Alternatively, binding of activated PKC to the receptor directly results in receptor inactivation. An EGF-R composed of the extracellular ligand binding domain, the transmembrane region, and 8 adjacent cytoplasmic amino acids (Na8) failed to interact with activated PKCalpha, while deletion of the entire carboxyl-terminal tail had no influence on the interaction, suggesting that the tyrosine kinase or juxtamembrane domains contain the binding site for this SH2 domain-less signal regulator protein. In contrast to the EGF-R, HER1-2, and EI-R, the PDGF-R kinase was only slightly affected by PKC binding, which might be due to the unique kinase structure of type III receptors.

Whether the inhibition of EGF-R, HER1-2, and EI-R autophosphorylation is indeed due to PKC-dependent transmodulation remains controversial. EGF-R phosphorylation at Thr-654 by activated PKC has been suggested to account for inhibition of tyrosine activity. This is supported by the observation that PMA fails to modulate receptor autophosphorylation in cells expressing EGF-receptors lacking Thr-654 (58, 59, 60, 61) . Activators of PKC also stimulate the phosphorylation of Thr-669 in vivo(62, 63) ; however, there is no evidence to date that phosphorylation of Thr-669 is required for inhibition of receptor autophosphorylation. In contrast, Morrison et al.(64) showed phorbol ester-stimulated EGF-R tyrosine kinase inhibition of a Thr-654 mutant expressed in Chinese hamster ovary cells and concluded that this residue is not sufficient to negatively regulate the EGF-R.

A second pathway of regulation of the EGF-R tyrosine kinase activity is represented by the phosphorylation of the receptor at Ser-1046/1047 in response to PMA treatment. Increased phosphorylation at this site is associated with an inhibition of RTK activity, and site-directed mutagenesis at Ser-1046/1047 has indicated that these residues are also important for receptor down-regulation(59, 65) . PKC itself is not able to phosphorylate Ser-1046/1047; however, CAM kinase II has been shown in vitro to phosphorylate these residues and cause an inhibition of protein kinase activity(65) . Thr-654 is conserved within the closely related RTK HER2 (Thr-686), as is Ser-1047 (Ser-1113 in HER2). If these residues are indeed important for regulating receptor tyrosine kinase activity in vivo, this could explain why HER2 is similarly affected by PMA and overexpressed PKCalpha.

While PKC-dependent transmodulation of the PDGF-R has not yet been described, it is well established that the insulin receptor is a PKC substrate. PKC-mediated insulin receptor phosphorylation can lead to a marked increase in the phosphorylation of the beta-subunit (34, 35, 36, 66) but can also inhibit autophosphorylation in certain cell types (31, 32, 33) . Additionally, it was recently demonstrated that in response to phorbol esters, Chinese hamster ovary cells overexpressing PKC isotypes alpha, beta1, and , but not , exhibit about 4-fold higher levels of insulin receptor phosphorylation. This increased phosphorylation occurred exclusively on serine and threonine (serine 1305/1306, threonine 1348, and other unidentified sites) without inhibition of receptor tyrosine kinase activity. However, activated PKCalpha inhibited the insulin-stimulated increase of phosphatidyl 3-kinase activity(37) .

Simultaneous treatment of HER1-2-expressing cells with EGF and PMA inhibited not only receptor autophosphorylation but also substrate binding, as investigated by coimmunoprecipitation of coexpressed PLC, GAP, and p85. In cells that also overexpress PKCalpha, the inhibitory effect on receptor autophosphorylation and substrate coimmunoprecipitation was even more dramatic. Activated PKC also abolished receptor-bound PI 3-kinase activity. These data are in agreement with the observation that phosphorylated tyrosine residues within the receptor are binding sites for SH2 domain-containing proteins such as PLC, GAP, and p85 PI 3-kinase subunit. Previously reported evidence indicates that PKC inhibits EGF-R-dependent tyrosine phosphorylation of PLC and activation of phosphoinositide hydrolysis(67) . Moreover, a link between GAP and PKC-mediated signaling pathways has been reported by several laboratories(68, 69, 70, 71) . As recently demonstrated, PMA treatment of T cells increases the amount of active GTP-bound Ras in cell membranes(68) , and furthermore, this increase is caused by PKC-mediated reduction of GAP activity, resulting in a positive response of the cell. In this context, it is important to note that direct activation of PKC by phorbol esters or indirect activation by mitogens such as PDGF, thrombin, and bombesin stimulates the proliferation of many cell types(72, 73, 74, 75) . In addition, PKC has been implicated in mediating crucial events in neoplastic transformation (76) .

The data indicate that the short term effect of activated PKC uncouples the EGF-R and HER2 receptors (subclass II RTKs) from their signal transduction pathways by inducing a reduction of receptor autophosphorylation, resulting in inhibition of EGF-induced mitogenicity. On the other hand, these data suggest that activated PKC, which enhances autophosphorylation of PDGF-R and insulin receptor in these cells, couples these receptors more tightly to their signal transduction pathways. However, whether PDGF and PMA, and insulin and PMA are additive in these cells with respect to mitogenicity remains to be investigated.

RTKbulletPKC complex formation was time dependent and must be regarded as a long term effect. EGF-induced complex formation in NIH 3T3 cells stably expressing HER1-2 reached its maximum after about 30 min and was undetectable after 180 min, while PMA induced a more rapid complex formation, which was still increasing after 180 min. This difference between EGF- and PMA-induced complex formation might be explained by the finding that growth factors transiently activate PLC (reviewed in (77) ), which then leads to transient activation of PKC, while PMA directly and constitutively activates PKC.

The time course of PKC binding to the receptors correlated extremely well with the time course of translocation of PKC from the cytosolic to the membrane fraction upon PMA treatment (data not shown). An involvement of PKC in receptor internalization and degradation has been proposed for several years. Exposing cells to phorbol esters results in decreased EGF binding and can be observed in most cell types. Immunofluorescent and electron microscopic localization of the EGF-R after phorbol ester treatment showed that a significant percentage of receptors become internalized(78) .

Deletion mutations within the EGF-R COOH terminus have been shown to cause an inhibition of EGF-R endocytosis(79) . The importance of PKC-mediated endocytosis and degradation is supported by the observation that Tyr-992, the major PLC interaction site, is located within this subdomain, suggesting that EGF receptors lacking this region are unable to activate PKC via PLC-activated polyphosphoinositide hydrolysis.

PKC mediation of EP-R, EGF-R, HER1-2, and EI-R internalization and degradation could be clearly demonstrated in 293 cells. Overexpression of the RTK alone and subsequent treatment with EGF or PMA had no effect on receptor degradation, while coexpression of PKCalpha resulted in a marked loss of radiolabeled receptors upon PMA treatment. Determination of the time course of receptor degradation in 293 cells overexpressing HER1-2 and PKC showed that EGF was not able to induce receptor degradation and PKC complex formation, whereas PMA induced receptor/PKC complex formation, receptor phosphorylation, and, simultaneously, receptor degradation. It is interesting to note that increased PKC binding clearly correlated with the loss of receptors. As mentioned earlier, EGF and PMA do not mediate receptor down-regulation when the receptor is overexpressed alone, suggesting that the endogenous down-regulating pathways are not sufficiently stimulated to activate degradation of these elevated amounts of receptors. Coexpression of PKC and PMA treatment can restore this pathway, whereas EGF still has no effect. If the assumption is correct that the ligand-activated receptor activates PLC, which leads to activation of PKC, then PLC should be the limiting factor in the latter case. Simultaneous overexpression of the receptor, PLC, and PKC should therefore restore the entire pathway and render it EGF-inducible. This is indeed the case, indicating that PLC is an important regulator of the receptor down-regulation pathway and supports the assumption that receptor internalization and degradation are dependent on PKC-mediated receptor phosphorylation.

In conclusion, the studies reported here show that PKC mediates short and long term effects on RTKs. The immediate effects, which appear to involve PMA-responsive tyrosine-specific phosphatases, are RTK-specific and regulate the phosphorylation state of the receptor on tyrosine residues, i.e. its ability to interact with SH2 domain-containing cellular signal transducers. In contrast, the PKC long term effects seem to be general in nature and are induced by translocation from the cytosol to the plasma membrane and formation of stable complexes with the receptor, concomitant with phosphorylation followed by internalization and degradation. To fully understand these mechanisms, characterization of specific phosphatases and an evaluation of the importance of PKC-mediated RTK phosphorylation in the triggering of receptor degradation are important goals for future research.


FOOTNOTES

*
This work was supported by a grant from Sugen, Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Hagedorn Research Institute, Niels Steensens Vej 6, 2820 Gentofte, Denmark.

Present address: Merck Sharpe & Dohme Research Laboratories, Neuroscience Research Centre, Terlings Park, Harlow, Essex CM20 2QR United Kingdom.

**
To whom all correspondence should be addressed. Tel.: 49-89-8578-2513; Fax: 49-89-857-7866.

^1
The abbreviations used are: RTK, receptor tyrosine kinase; PLC, phospholipase C; GAP, GTPase-activating protein; PI 3`-kinase, phosphatidylinositol 3`-kinase; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; DAG, diacylglycerol; PAGE, polyacrylamide gel electrophoresis; EGF, epidermal growth factor; EGF-R, epidermal growth factor receptor; PDGF, platelet-derived growth factor; PDGF-R, platelet-derived growth factor receptor; mAb, monoclonal antibodies; DMEM, Dulbecco's modified Eagle's medium; CMV, cytomegalovirus; PIP, phosphatidylinositol monophosphate; BES, 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid.

^2
K. Seedorf, M. Shearman, and A. Ullrich, unpublished results.

^3
K. Seedorf, M. Shearman, and A. Ullrich, unpublished observation.


ACKNOWLEDGEMENTS

We thank Jeanne Arch for expert preparation of the manuscript and figures.


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