©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
An Incomplete Program of Cellular Tyrosine Phosphorylations Induced by Kinase-defective Epidermal Growth Factor Receptors (*)

Jacqueline D. Wright (2), Christoph W. M. Reuter (1)(§), Michael J. Weber (1)(¶)

From the (1) Department of Microbiology and (2) Pharmacology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Although signaling by the epidermal growth factor (EGF) receptor is thought to be dependent on receptor tyrosine kinase activity, it is clear that mitogen-activated protein (MAP) kinase can be activated by receptors lacking kinase activity. Since analysis of the signaling pathways used by kinase-defective receptors could reveal otherwise masked capabilities, we examined in detail the tyrosine phosphorylations and enzymes of the MAP kinase pathway induced by kinase-defective EGF receptors. Following EGF stimulation of B82L cells expressing a kinase-defective EGF receptor mutant (K721M), we found that ERK2 and ERK1 MAP kinases, as well as MEK1 and MEK2 were all activated, and SHC became prominently tyrosine-phosphorylated. By contrast, kinase-defective receptors failed to induce detectable phosphorylations of GAP (GTPase-activating protein), p62, JAK1, or p91STAT1, all of which were robustly phosphorylated by wild-type receptors. These data demonstrate that kinase-defective receptors induce several protein tyrosine phosphorylations, but that these represent only a subset of those seen with wild-type receptors. This suggests that kinase-defective receptors activate a heterologous tyrosine kinase with a specificity different from the EGF receptor. We found that kinase-defective receptors induced ErbB2/c-Neu enzymatic activation and ErbB2/c-Neu binding to SHC at a level even greater than that induced by wild-type receptors. Thus, heterodimerization with and activation of endogenous ErbB2/c-Neu is a possible mechanism by which kinase-defective receptors stimulate the MAP kinase pathway.


INTRODUCTION

The epidermal growth factor receptor (EGF-R)() is a 170-kDa transmembrane receptor tyrosine kinase (1, 2) . EGF-R ligands, such as EGF or transforming growth factor-, bind to the extracellular domain of the receptor and induce receptor dimerization and clustering (3, 4, 5, 6) ; this in turn results in activation of the receptor's intracellular tyrosine kinase activity (7) . The activated, clustered receptors transphosphorylate other co-clustered receptors, creating binding sites for cellular proteins containing SH2 domains, and become activated to continue the signal transduction cascade. Well characterized substrates for the receptor include phospholipase C- (8, 9) , the GTPase-activating protein of p21 (GAP) (10) , and the SH2 containing adapter protein SHC (11, 12) . The nonreceptor tyrosine kinase JAK1 and transcription factors of the STAT family also become tyrosine-phosphorylated following EGF stimulation (13) .

The MAP kinase pathway is stimulated by activation of the EGF-R. It is believed that upon EGF stimulation, both the receptor and SHC become tyrosine-phosphorylated and bind to each other and the Grb2-Sos complex (12, 14, 15). This brings Sos to the plasma membrane thus enabling it to catalyze exchange of GTP for GDP on p21(16, 17, 18, 19) . The activated p21 -GTP is then able to bind and facilitate activation either of Raf or other serine/threonine protein kinases, which in turn stimulate the activity of MEK (MAPK or Erk kinase) (20-26). MEK acts as a dual specificity kinase which phosphorylates MAP kinase on both a threonine and a tyrosine, thereby activating MAP kinase (27, 28, 29, 30) .

Early reports indicated that the kinase activity of the EGF-R is necessary for all EGF-induced signaling. For example, it has been shown that kinase-defective (K721A or K721M) EGF-Rs can no longer generate EGF-induced mitogenesis or DNA synthesis, phospholipase C- phosphorylation or activation, fos or myc gene expression, cell surface transport processes, or increases in intracellular calcium concentration (31, 32, 33, 34) . More recently, however, Campos-Gonzalez and Glenney (35) , Selva et al.(34) , and Coker et al.(36) have reported that EGF stimulation of kinase-defective EGF-R mutants can still induce activation of MAP kinase.

Because of the surprising nature of this finding, and the fact that it runs counter to the normal paradigm explaining EGF receptor signaling, we have investigated the biochemical basis for signaling by a kinase-defective receptor. We have considered four possible mechanisms by which MAP kinase might be activated by kinase-defective EGF-Rs: 1) the kinase-defective receptors possess a low level of residual kinase activity, but a level which is sufficient to activate the MAP kinase pathway; 2) there are endogenous wild-type EGF-Rs present in the parental cells that can dimerize with the kinase-defective receptors, allowing amplification of endogenous wild-type receptor signaling; 3) a novel kinase-independent signaling pathway is stimulated by EGF binding and/or receptor dimerization; 4) there is a heterologous tyrosine kinase that is activated upon EGF binding and/or receptor dimerization, and it is the heterologous kinase which is responsible for activating the MAP kinase pathway. We failed to find evidence for the first three possibilities, but found substantial EGF-dependent tyrosine phosphorylation and activation of ErbB2/c-Neu in cells expressing kinase-defective EGF-Rs. We suggest that transphosphorylation and activation of ErbB2/c-Neu or other related receptor kinases is sufficient to explain signaling by kinase-defective EGF receptors.


EXPERIMENTAL PROCEDURES

Cell Culture

The B82L parental cells and the B82L cells expressing the human wild-type (Lys) EGF receptor and the mutated, kinase-defective (Met) EGF receptor were a generous gift from Dr. G. N. Gill (University of California, San Diego, CA) and have been described previously (32) . A mutant dihydrofolate reductase gene provides a dominant selectable marker for the B82L cells overexpressing the EGF receptor (37) . These cells were maintained in -minimum essential medium (Life Technologies, Inc.) containing 10% dialyzed fetal bovine serum and 2 µM methotrexate. Before experiments, the cells were kept in the absence of methotrexate (in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum) for at least 24 h. The B82L parental cells were maintained in DMEM containing 10% fetal bovine serum. Dishes of the confluent cells (1-2 10 cells/100-mm dish) were then incubated overnight (8-12 h) in serum-free DMEM before stimulation with 100 ng/ml EGF (Upstate Biotechnology, Inc. Catalog number 01-107).

Immunoprecipitation and Immunoblotting

Cells were left untreated(-) or exposed to EGF (+) for 3 min. The cells were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed in lysis buffer containing 50 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM EDTA, 1 mM NaVO, 10 µg/ml pepstatin A, 50 µg/ml leupeptin, 0.02 TIU/ml of aprotinin, 1 mM phenylmethylsulfonyl fluoride, 40 mM 4-nitrophenyl phosphate Na salt. Insoluble material was removed by centrifugation at 4 °C for 15 min at 14,000 rpm. Supernatants containing equal amounts of protein (500 µg) were incubated for 2 h at 4 °C with either: 1) 4 mg of washed protein A-Sepharose (Pharmacia Biotech Inc.) and a specific rabbit polyclonal antibody or rabbit nonimmune serum (as a control, data not shown) or 2) 100 µl of washed protein G-agarose (Boehringer Mannheim) and a specific mouse monoclonal antibody. After washing the immunoprecipitates three times with lysis buffer (or two washes with lysis buffer and 2 washes with Tris-buffered saline), Laemmli sample buffer was added, and the samples were boiled for 2 min. The samples were analyzed by SDS-PAGE and transferred to nitrocellulose filters for Western blot analysis.

The following antibodies were kindly provided: 3A and 4A, anti-human EGF-R monoclonal antibodies for immunoblotting (D. McCarley, Syntex Research, Palo Alto, CA); 108, an anti-human EGF-R monoclonal antibody for immunoprecipitation (Dr. J. Schlessinger, New York University Medical Center, New York); anti-p125 GAP monoclonal (6F2) and polyclonal (P23) antibodies for immunoprecipitation and immunoblotting (Dr. S. Parsons, University of Virginia, Charlottesville, VA); anti-p91 polyclonal antibodies for immunoprecipitation and immunoblotting (Dr. A. Larner, National Institutes of Health, Bethesda, MD); and an anti-Jak1 polyclonal antibody for immunoprecipitation and immunoblotting (Dr. A. Ziemiecki, University of Berne, Berne, Switzerland). The following antibodies were also used: an anti-phosphotyrosine rabbit polyclonal antibody and a rabbit nonimmune serum generated in our laboratory; an anti-SHC rabbit polyclonal and an anti-Jak2 rabbit polyclonal for immunoprecipitation and immunoblotting (Upstate Biotechnology, Inc.); an anti-c-Neu monoclonal antibody for immunoprecipitation and immunoblotting (Ab-3 from Oncogene Science); an anti-Grb2 monoclonal antibody and RC20H, a horseradish peroxidase-linked anti-phosphotyrosine antibody, for immunoblotting (Transduction Laboratories). All immunoblots (except RC20H, which is already linked to horseradish peroxidase) were probed with secondary antibodies (anti-rabbit or anti-mouse) conjugated to horseradish peroxidase and immunoreactive bands were visualized by enhanced chemiluminescence (ECL reagents and horseradish peroxidase-linked antibodies were purchased from Amersham Corp.).

MAP Kinase and MEK Assays

The B82L parental cells, as well as the B82L cells overexpressing the kinase-defective (K-) and wild-type (K+) EGF receptors were left untreated(-) or exposed to EGF (+) for 5 min. The activities of ERK2 (p42) and ERK1 (p44) MAP kinases were determined by immunoprecipitation using TR2 and R1798 rabbit polyclonal antibodies and performing in vitro kinase reactions using myelin basic protein as a substrate, as described (38) . The activities of MEK1 and MEK2 were determined by immunoprecipitation using rabbit polyclonal antibodies (raised against either a COOH-terminal peptide from MEK1 or a NH-terminal peptide from MEK2) and performing in vitro kinase reactions using recombinant kinase-defective MAP kinase as a substrate, as described (38). Nonimmune serum controls were done (data not shown) to ensure that the kinase activities were specific. Western blot analysis using anti-MAP kinase and anti-MEK antibodies were also done to ensure that equal amounts of MAP kinase or MEK proteins were present in the immunoprecipitations (data not shown).

Immunoprecipitation and Immunoblotting of Endogenous Murine EGF-Rs

Cells were grown in large scale in DMEM containing 10% fetal bovine serum (without methotrexate) for at least 24 h before cell lysis. For each immunoprecipitation, 4-10 150-mm confluent dishes were washed twice with ice-cold PBS. The plates were then incubated with 2 ml of ice-cold PBS containing 1 mM EDTA, and the cells were scraped into 15-ml conical tubes (8 ml/tube). The cells were pelleted by centrifugation and the PBS-EDTA was decanted. The pelleted cells were lysed in 1 ml radioimmune precipitation lysis buffer (containing 50 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM NaVO, 10 µg/ml pepstatin A, 50 µg/ml leupeptin, 0.02 TIU/ml of aprotinin, 1 mM phenylmethylsulfonyl fluoride, 40 mM 4-nitrophenyl phosphate Na salt) and thoroughly vortexed. The lysed cells were incubated on ice for 10 min, and insoluble material was removed by centrifugation at 4 °C for 15 min (twice) at 14,000 rpm. Supernatants containing equal amounts of protein (1-24 mg) were incubated for 2 h at 4 °C with 4 mg of washed protein A-Sepharose (Pharmacia) and a rabbit nonimmune serum. The samples were pelleted by centrifugation and the precleared supernatants were incubated with either: 1) 4 mg of washed protein A-Sepharose and a specific anti-EGF-R rabbit polyclonal antibody 1382 (which recognizes murine or human EGF-Rs) or 2) 4 mg of washed protein A-Sepharose and a rabbit nonimmune serum. After washing the immunoprecipitates three times with lysis buffer, Laemmli sample buffer was added, and the samples were boiled for 2 min. The samples were analyzed by SDS-PAGE and transferred to nitrocellulose filters. Western blot analysis was done using either an anti-EGF-R-specific rabbit polyclonal antibody 22 (which recognizes murine or human EGF-Rs) or RC20H, a horseradish peroxidase linked anti-phosphotyrosine antibody (data not shown). Antibodies 1382 and 22 were generously provided by Dr. H. S. Earp, University of North Carolina, Chapel Hill, NC.

RESULTS

In Vivo Tyrosine Phosphorylation and In Vitro Kinase Activity of Wild-type and Kinase-defective EGF-Rs

We have examined EGF-R signaling by using a mouse cell line (B82L) transfected with and overexpressing either the wild-type human EGF-R (K+) or a kinase-defective human EGF-R (K-) that contains a point mutation in which lysine 721 is changed to methionine. Expression and activity of the kinase-negative and wild-type receptors isolated from K+, K-, and B82L parental cells were analyzed by Western blot analysis and by measuring in vitro autokinase activity. The B82L parental cells contained no detectable EGF-Rs (Fig. 1A), and therefore no tyrosine phosphorylation or autokinase activity was detected in the EGF-R immunoprecipitates from these cells (Fig. 1, B and C). The kinase-defective EGF-R showed very little induced in vivo tyrosine phosphorylation following EGF stimulation. However, after prolonged overexposure of the phosphotyrosine immunoblot (Fig. 1B), EGF-induced in vivo phosphorylation of kinase-defective EGF-Rs was detectable, at a level less than 10% that of the wild-type EGF-Rs. This small amount of in vivo phosphorylation occurred in spite of the fact that the receptors were almost devoid of intrinsic kinase activity, as assayed by an in vitro autokinase reaction (Fig. 1C). We suspect that an endogenous (murine) tyrosine kinase is responsible for the low level in vivo tyrosine phosphorylation taking place on the kinase-defective EGF-Rs, in agreement with the suggestion of Selva et al.(34) .


Figure 1: In vivo tyrosine phosphorylation and in vitro kinase activity of wild-type and kinase-defective EGF-Rs. Confluent B82L mouse parental cells or B82L cells overexpressing either the kinase-defective (B82L K-) or wild-type (B82L K+) human EGF receptor were serum-starved overnight and treated with (+) or without (-) 100 ng/ml of EGF for 2 min before being lysed. EGF receptors were immunoprecipitated from the cell lysates using an EGF receptor-specific monoclonal antibody (108) and separated on a 7.5% polyacrylamide gel. The gel was transferred to nitrocellulose, and Western blot analysis was done using either EGF receptor specific monoclonal antibodies (3A and 4A) (A) or a phosphotyrosine-specific antibody (RC20H) (B). The immunoprecipitated EGF receptors were also used to perform in vitro kinase assays, which were analyzed by SDS-PAGE and exposed to film for 17 h (C). We found that immunoprecipitated wild-type EGF-Rs were constitutively active; there was no difference in the in vitro autokinase enzymatic activity in the presence or absence of in vivo EGF stimulation. The reduction of P incorporation into EGF-Rs from B82L K+ cells (+EGF) compared with B82L K+ (-EGF) is likely due to in vivo phosphorylation on the receptor (before the cells are lysed), which reduces the number of sites available for autophosphorylation by [-P]ATP in the in vitro kinase assay.



Activation of the MAP Kinase Pathway following EGF Stimulation of Wild-type or Kinase-defective EGF-Rs

The activities of p42 and p44 MAP kinases (ERK2 and ERK1) following EGF stimulation were determined using antibodies specific for p42 and p44 MAP kinases (Fig. 2, A and B). As expected, there was no significant EGF-inducible p42 or p44 MAP kinase activation in the B82L parental cells, which lack EGF-Rs. In the B82L cells which overexpress wild-type EGF-Rs, there was a clear activation of both p42 and p44 MAP kinase activity after EGF stimulation. Moreover, there was also a significant activation (25-50% of what is seen in the K+ cells) of both p42 and p44 MAP kinase upon EGF stimulation of kinase-defective EGF-Rs. Since kinase-defective EGF-Rs can signal to MAP kinase family members, we examined whether the normal upstream activators of MAP kinase, MEK1 and MEK2, were also stimulated by EGF treatment of kinase-defective EGF-Rs. EGF-induced activation of MEK1 or MEK2 occurred in a manner which was very similar to p42 and p44 MAP kinase. MEK1 and MEK2 were clearly activated following EGF stimulation of either the wild-type or (to a lesser degree) the kinase-defective receptors (Fig. 2, C and D). Therefore, ERK1, ERK2, MEK1, and MEK2 are all activated by kinase-defective EGF-Rs in similar relative proportions (e.g. 25-50% of the level induced by wild-type receptors), and thus, kinase-defective EGF-Rs do not selectively activate a subset of MEK or MAP kinase isoforms. As discussed below, this suggests that similar pathways are used by wild-type and kinase-defective EGF-Rs in activating MAP kinase.


Figure 2: Activation of ERK1 and ERK2 MAP kinases, MEK1, and MEK2 following EGF stimulation of wild-type or kinase-defective EGF-Rs. Confluent B82L mouse parental cells or B82L cells overexpressing either the wild-type (B82L K+) or kinase-defective (B82L K-) human EGF receptor were serum-starved overnight and treated with (+) or without (-) 100 ng/ml of EGF for 5 min before being lysed. Either ERK2 (A) or ERK1 MAP kinase (B) or MEK1 (C) or MEK2 (D) was immunoprecipitated and assayed for enzyme activity as described under ``Experimental Procedures.''



Tyrosine Phosphorylations Induced by EGF Stimulation of Kinase-defective EGF-Rs

Since the data in Fig. 1suggested stimulation of kinase-defective EGF-Rs can activate an endogenous tyrosine kinase, we determined whether other proteins (besides MAP kinase) might become tyrosine-phosphorylated following addition of EGF to cells expressing kinase-defective receptors (Fig. 3). In addition to MAP kinase, EGF stimulation of kinase-defective receptors induced the phosphorylation of proteins in the 40-60 kDa range, as well as at 90 kDa, and (faintly) at 120 kDa.


Figure 3: Tyrosine phosphorylation events induced by EGF stimulation of wild-type or kinase-defective EGF-Rs. B82L mouse parental cells or B82L cells overexpressing either the kinase-defective (B82L K-) or wild-type (B82L K+) human EGF receptor were grown to confluence. The cells were then serum-starved overnight and treated with (+) or without (-) 100 ng/ml of EGF for 3 min before being lysed. Equivalent amounts of protein were used to make whole cell lysates. These whole cell lysates were then electrophoresed on a 7.5% polyacrylamide gel and transferred to nitrocellulose. Phosphotyrosine levels were measured by doing Western blot analysis using an anti-phosphotyrosine rabbit polyclonal antibody. The molecular weights of prestained protein markers are indicated on the left.



Two of the phosphorylated bands seen in Fig. 3(p46 and p52) correspond in molecular weight to isoforms of SHC, an SH2-containing adapter protein which is a well characterized substrate for the EGF-R. In order to determine whether SHC becomes tyrosine-phosphorylated following EGF stimulation of kinase-defective EGF-Rs, SHC was immunoprecipitated from the same cell lysates used in Fig. 3. The immunoprecipitates were separated by SDS-PAGE and blotted with anti-phosphotyrosine antibodies. Fig. 4A demonstrates that three isoforms of SHC clearly became tyrosine phosphorylated after EGF stimulation of the kinase-defective EGF-R.


Figure 4: SHC is tyrosine-phosphorylated and associates with the EGF-R following EGF stimulation of wild-type or kinase-defective EGF-Rs. B82L mouse parental cells or B82L cells overexpressing either the kinase-defective (B82L K-) or wild-type (B82L K+) human EGF receptor were grown to confluence. The cells were then serum starved overnight and treated with (+) or without (-) 100 ng/ml of EGF for 3 min before being lysed. Equivalent amounts of protein were used to make whole cell lysates. SHC was immunoprecipitated from these cell lysates using a SHC-specific polyclonal antibody (or nonimmune rabbit serum as a negative control, data not shown), and the immunoprecipitations were electrophoresed on 7.5% polyacrylamide gels. Two identical gels were transferred to nitrocellulose for anti-phosphotyrosine (RC20H) or anti-EGF-R Western blot analysis (A). Anti-SHC Western blot analysis was also done (data not shown) to guarantee that equivalent amounts of SHC were present in all samples. The anti-EGF receptor Western blot was done using human EGF receptor-specific monoclonal antibodies (3A and 4A) to detect whether either kinase-defective (K-) or wild-type (K+) EGF receptors can associate with SHC in an EGF-dependent manner (B).



Since SHC is known to bind to the wild-type EGF-R upon EGF stimulation (12), we investigated whether SHC could also bind to the kinase-defective receptor. Anti-EGF-R Western blot analysis of the SHC immunoprecipitates (Fig. 4B) showed that SHC did bind to both the wild-type and kinase-defective EGF-Rs in an EGF-dependent manner.

In the phosphotyrosine immunoblots of Fig. 4A, 120- and 170-kDa phosphoproteins that co-immunoprecipitate with SHC were observed. The identity of the 120-kDa protein is still unknown, but the phosphorylated 170-kDa protein is probably the tyrosine-phosphorylated EGF-R, as shown in Fig. 4B.

It is also known that EGF stimulation of wild-type EGF-Rs induces SHC binding to Grb2 at the cell membrane (14) . By performing anti-Grb2 Western blot analysis on SHC immunoprecipitates (Fig. 5) we determined that Grb2 was also able to bind to SHC following stimulation of kinase-defective EGF-Rs.


Figure 5: SHC associates with Grb2 following EGF stimulation of wild-type or kinase-defective EGF-Rs. B82L mouse parental cells or B82L cells overexpressing either the wild-type (B82L K+) or kinase-defective (B82L K-) human EGF receptor were grown to confluence. The cells were then serum-starved overnight and treated with (+) or without (-) 100 ng/ml of EGF for 3 min before being lysed. SHC was immunoprecipitated from these cell lysates using a SHC-specific polyclonal antibody, and the immunoprecipitations were electrophoresed on a 15% polyacrylamide gel. The gel was transferred to nitrocellulose and Western blot analysis was done using a Grb2-specific monoclonal antibody. Whole cell lysates were separated on the same gel to show that equivalent amounts of Grb2 were present in the cell lysates. Anti-SHC Western blot analysis was also done (data not shown) to guarantee that equivalent amounts of SHC were present in all samples. IP, immunoprecipitation.



Tyrosine Phosphorylations Which Fail to be Induced by EGF Stimulation of Kinase-defective EGF-Rs

Besides SHC, other cellular proteins which have been reported to become tyrosine-phosphorylated following stimulation of wild-type EGF receptors include GAP (10) , the tyrosine kinase JAK1 and the tran-scription factor p91STAT1 (13) . Protein tyrosine phosphorylations with M values consistent with possible phosphorylation of these proteins were observed following stimulation of both wild-type and (to a lesser extent) kinase defective EGF-Rs ( Fig. 3and Fig. 4). To determine whether kinase-defective receptors can cause these phosphorylations, the proteins were immunoprecipitated, separated by PAGE, and their tyrosine phosphorylation assessed by Western blotting with an anti-phosphotyrosine antibody.

As expected, both GAP and the p62 GAP binding protein were clearly phosphorylated upon EGF stimulation of the wild-type receptor. However, there was no induction of tyrosine phosphorylation observed on either GAP or p62 following EGF stimulation of kinase-defective EGF-Rs (Fig. 6A). These results suggest that GAP and p62 phosphorylation is not required for kinase-defective EGF-R signaling to the MAP kinase pathway.


Figure 6: GAP, GAP-associated protein p62, JAK1, and p91STAT1 are tyrosine-phosphorylated following EGF stimulation of wild-type, but not kinase-defective, EGF-Rs. B82L mouse parental cells (data not shown) or B82L cells overexpressing either the kinase-defective (B82L K-) or wild-type (B82L K+) human EGF receptor were grown to confluence. The cells were then serum-starved overnight and treated with (+) or without (-) 100 ng/ml of EGF for 3 min before being lysed. A, GAP was immunoprecipitated from these cell lysates using GAP-specific monoclonal or polyclonal antibodies and the immunoprecipitates were electrophoresed on a 7.5% polyacrylamide gel. The gel was transferred to nitrocellulose and Western blot analysis was done using either a GAP-specific polyclonal antibody or a phosphotyrosine specific horseradish peroxidase-linked antibody (RC20H). Either JAK1 (B) or JAK2 (C) or p91STAT1 (D) was immunoprecipitated (using specific polyclonal antibodies) from these cell lysates, and the immunoprecipitations were electrophoresed on 7.5% polyacrylamide gels. The gel was transferred to nitrocellulose, and Western blot analysis was done using either JAK1 or JAK2 or p91STAT1-specific polyclonal antibodies or a phosphotyrosine-specific horseradish peroxidase-linked antibody (RC20H). IP, immunoprecipitation.



Similar experiments were also done with p91STAT1 (13, 39) , since a 90-kDa protein was tyrosine-phosphorylated following EGF stimulation of kinase-defective EGF-Rs. As was found with GAP and p62, phosphorylation of p91STAT1 occurred through the wild-type receptors, but not kinase-defective EGF-Rs (Fig. 6D).

We hypothesize that there is an endogenous (murine) tyrosine kinase (other than the human EGF-R itself) which associates with the receptor following EGF stimulation. JAK family members are nonreceptor tyrosine kinases which are known to become tyrosine phosphorylated and activated by a variety of growth factor receptors, including EGF. Therefore, we sought to investigate whether the 120-kDa band seen in Fig. 3and Fig. 4 might be a JAK family member. It was observed (Fig. 6, B and C) that JAK1, but not JAK2, became tyrosine-phosphorylated upon EGF stimulation of wild-type EGF-Rs, which is consistent with previously published results (13) . However, neither JAK1 nor JAK2 became tyrosine-phosphorylated with stimulation of kinase-defective EGF-Rs. Therefore, phosphorylation and presumably activation of the JAK-STAT pathway does not appear to take place upon EGF stimulation of kinase-defective EGF-Rs. Consequently, we hypothesize that MAP kinase activation by the kinase-defective receptor occurs independently of this signaling pathway.

No Endogenous Murine EGF-Rs Are Detected in the B82L Parental Cells

It has previously been observed that B82L cells do not contain detectable endogenous EGF-R mRNA or protein, as assessed by RNA blot analysis and ligand binding studies (37) . However, Hack et al.(40) have suggested that a very low level of endogenous murine EGF-Rs was detectable by anti-EGF-R immunoprecipitation from B82L cells and phosphotyrosine Western blot analysis. These authors proposed that endogenous wild-type EGF-Rs might somehow be responsible for EGF-induced signaling by the overexpressed human kinase-defective EGF-Rs. We performed similar tests for endogenous murine EGF-Rs in B82L cells, by doing immunoprecipitations and Western analysis using rabbit polyclonal antibodies that recognize both murine and human EGF-Rs. We also included positive controls (using mouse Swiss 3T3 fibroblast cells and B82L cells overexpressing human kinase-defective EGF-Rs) and negative controls (by using a nonimmune rabbit serum for immunoprecipitations).

A protein band that corresponded specifically to the murine EGF-R appeared in the anti-mouse EGF-R(1382) immunoprecipitation of Swiss 3T3 cells (Fig. 7, A and B). However, this specific murine EGF-R protein band did not appear in the 1382 immunoprecipitation of B82L parental cell lysates, even when immunoprecipitations were done using as much as 24 mg of protein (Fig. 7B). Hack et al.(40) suggested that the murine EGF-R runs at a higher molecular weight than the human EGF-R. To the contrary, we observed that the murine EGF-R immunoprecipitated from Swiss 3T3 cells ran at a molecular weight indistinguishable from the human EGF-R immunoprecipitated from B82L cells overexpressing EGF-Rs (Fig. 7A). Data shown in Fig. 7B also demonstrate that lysates from B82L (24 mg of protein) and Swiss 3T3 (8 mg of protein) cells contained a protein slightly higher in molecular weight than the EGF-R which was immunoprecipitated using either the specific anti-EGF-R antibody(1382) or the nonspecific nonimmune rabbit serum (NIS) (Fig. 7B). Thus, we believe that the ``mouse EGF-R'' detected in B82L cells by Hack et al.(40) is likely to have been a nonspecific protein that ran at a molecular weight slightly higher than either the murine or human EGF-R. In contrast to what was suggested by Hack et al.(40) , we found no detectable endogenous murine EGF-R present in the B82L cells when blotting using either antibody 22 against the murine EGF-R (Fig. 7) or RC20H against phosphotyrosine (data not shown).


Figure 7: No endogenous murine EGF-Rs are detected in the B82L parental cells. B82L mouse parental and B82L cells overexpressing human kinase-defective EGF receptors (B82L K-) and mouse Swiss 3T3 cells were grown to confluence in large quantities. A, endogenous murine EGF receptors were immunoprecipitated from B82L and B82L K- (11 mg of protein/immunoprecipitation (IP) and Swiss 3T3 (2.5 mg of protein/immunoprecipitation) cell lysates using either a rabbit polyclonal antibody 1382 (which recognizes murine and human EGF-Rs) or a rabbit nonimmune serum as a negative control. B, endogenous murine EGF receptors were immunoprecipitated from B82L (24 mg of protein/immunoprecipitation) and Swiss 3T3 (8 mg of protein/ immunoprecipitation) cell lysates using either a rabbit polyclonal antibody 1382 (which recognizes mouse and human EGF-Rs) or a rabbit nonimmune serum as a negative control. The immunoprecipitates were separated on 7.5% polyacrylamide gels and transferred to nitrocellulose. Western blot analysis was then done using the rabbit polyclonal antibody 22 which recognizes mouse and human EGF receptors.



Enhanced Stimulation of ErbB2/c-Neu Enzymatic Activity and Binding to SHC following EGF Stimulation of Kinase-defective EGF-Rs

In addition to testing for endogenous mouse EGF-Rs (ErbB1), we also investigated whether other EGF-R/ErbB family members, particularly ErbB2 (or c-Neu), might participate in signaling by kinase-defective EGF-Rs. Therefore, we immunoprecipitated ErbB2/c-Neu from the B82L cell lines using a monoclonal antibody that recognizes murine ErbB2/c-Neu. Endogenous ErbB2/c-Neu was clearly present in all three cell lines (Fig. 8A) and became tyrosine-phosphorylated (Fig. 8B) and activated (Fig. 8C) upon EGF stimulation of either the wild-type or kinase-defective EGF-Rs. There was no EGF-inducible phosphorylation or activation of ErbB2/c-Neu in the B82L parental cell line and this is expected for two reasons: there are no detectable mouse ErbB1/EGF-Rs in the B82L cells (as seen in Fig. 7), and EGF does not detectably bind ErbB2/c-Neu or any other known ErbB family members, besides ErbB1 (41, 42, 43) . Therefore, we hypothesize that the kinase-defective EGF-R (as well as the wild-type receptor) is able to heterodimerize with and activate endogenous mouse ErbB2/c-Neu which is present in B82L cells.


Figure 8: Endogenous murine ErbB2/c-Neu is tyrosine-phosphorylated and activated following EGF stimulation of wild-type or kinase-defective EGF-Rs. B82L mouse parental cells or B82L cells overexpressing either the wild-type (B82L K+) or kinase-defective (B82L K-) human EGF receptor were grown to confluence. The cells were then serum-starved overnight and treated with (+) or without (-) 100 ng/ml of EGF for 3 min before being lysed. c-Neu was immunoprecipitated from these cell lysates using a c-Neu-specific monoclonal antibody and the immunoprecipitates were electrophoresed on a 7.5% polyacrylamide gel. The gel was transferred to nitrocellulose, and Western blot analysis was done using either the c-Neu-specific monoclonal antibody (A) or a horseradish peroxidase-linked anti-phosphotyrosine antibody, RC20H (B). In vitro autokinase assays with the c-Neu immunoprecipitations were also done (Fig. 7C). IP, immunoprecipitation.



It has been reported that SHC is a substrate for ErbB2/c-Neu and that phosphorylation of carboxyl-terminal sites located on ErbB/c-Neu are required for both the phosphorylation of SHC by ErbB/c-Neu and complex formation between the SHC and ErbB/c-Neu proteins (44) . Since the EGF-R and ErbB2/c-Neu can form heterodimers (45) , we investigated whether SHC binds to ErbB2/c-Neu following EGF treatment of wild-type or kinase-defective EGF-Rs. We found that ErbB2/c-Neu binds to SHC in an EGF-dependent manner as assessed by co-immunoprecipitation (Fig. 9). Interestingly, both the in vitro autokinase reaction (Fig. 8C) and the binding experiments (Fig. 9) indicate that the activation and binding of ErbB2/c-Neu to SHC is greatly enhanced upon EGF stimulation of kinase-defective EGF-Rs, compared with that seen with wild-type receptors. Perhaps the kinase activity of ErbB2/c-Neu can compensate for the lack of tyrosine kinase activity of the kinase-defective EGF-Rs. Therefore, activation of the MAP kinase pathway by kinase-defective EGF-Rs may occur via the activation of ErbB2/c-Neu.


Figure 9: ErbB2/c-Neu binds to SHC following EGF stimulation of wild-type or kinase-defective EGF-Rs. B82L mouse parental cells or B82L cells overexpressing either the wild-type (B82L K+) or kinase-defective (B82L K-) human EGF receptor were grown to confluence. The cells were then serum-starved overnight and treated with (+) or without (-) 100 ng/ml of EGF for 3 min before being lysed. SHC was immunoprecipitated from these cell lysates using a SHC-specific polyclonal antibody (or nonimmune rabbit serum as a negative control, data not shown), and the immunoprecipitates were electrophoresed on a 7.5% polyacrylamide gel. The gel was transferred to nitrocellulose, and Western blot analysis was done using a c-Neu-specific monoclonal antibody. Anti-SHC Western blot analysis was also done (data not shown) to guarantee that equivalent amounts of SHC were present in all samples. IP, immunoprecipitation.



DISCUSSION

Even though it is generally believed that EGF receptor signaling depends on its tyrosine kinase activity, it is clear that mutant, kinase-defective EGF-Rs are capable of robustly activating the MAP kinase pathway. Although one might argue that these results could be explained by residual kinase activity retained by these mutant receptors, substantial evidence indicates that this is not the case. First, virtually no in vitro kinase activity associated with the immunopurified, kinase-defective EGF-R could be detected, unless the immunopurification was performed under nonstringent conditions (which presumably allows co-isolation of a contaminating tyrosine kinase), in agreement with previously published results (34) . Second, two different kinase-defective EGF-receptor mutants have been shown to activate MAP kinase: the Lys mutant (in the ATP binding site), which was used in the experiments reported here, and an Asp mutant (in the catalytic loop) (34, 35, 36) . Whereas the first mutation likely renders the receptor unable to properly bind ATP, the second mutation eliminates the receptor's ability to catalyze phosphate transfer. Thus, although it is difficult to prove unequivocally that the mutant receptors are totally devoid of kinase activity, no evidence supports the suggestion that the partial signaling activity of kinase-defective receptors is due to residual receptor kinase activity.

One might analogously argue that very low levels of endogenous EGF-Rs are responsible for the signaling detected in cells expressing the kinase-defective receptor. However, our attempts to detect endogenous receptors in B82L cells using immunological methods have been entirely negative, which is consistent with previous reports of Northern blot analysis and ligand binding studies (37) . As described under ``Results,'' we believe that the single report (40) to the contrary could be artifactual. Furthermore, it is difficult to imagine how this hypothesis could be correct, since the parental B82L cells (which do not express transfected human EGF-Rs) do not respond detectably to EGF with respect to any signaling pathway tested. For example, as demonstrated by Soler et al.(46) , as few as 10 endogenous wild-type receptors per cell can dramatically stimulate phosphorylation of SHC. Moreover, if this hypothesis were correct, the endogenous receptors would have to be activated by a receptor mutant which has been reported to function as a ``dominant-negative'' with respect to endogenous EGF-R signaling (47, 48) . Finally, amplified signaling by low levels of endogenous receptors cannot easily be reconciled with the strikingly dichotomous signaling activated by the kinase-defective EGF-R, in which the SHC/MAP kinase pathway is substantially activated but other tyrosine phosphorylations (e.g. GAP/p62 or JAK/STAT) which are equally prominent in response to wild-type receptors are not detectably activated by the kinase-defective receptors. Thus, although it is not possible to prove that there are no endogenous EGF receptors in the B82L cells, or that very low levels of receptors do not contribute to the signaling, the weight of the evidence argues strongly against this possibility.

It is conceivable that the EGF receptor possesses a signaling activity which does not rely on its kinase activity. For example, EGF-induced phosphorylation and activation of phospholipase C- in rat hepatocytes is reportedly inhibited by pertussis toxin (49, 50) , implying that the EGF-R is able, directly or indirectly, to activate G in this cell type. This suggestion is further strengthened by reports that G directly associates with the EGF receptor in an EGF-dependent manner and that pertussis toxin pretreatment of hepatocytes inhibits this association (50). It has also been demonstrated that thrombin treatment of CCL39 hamster fibroblasts activates a MAP kinase pathway by stimulating a pertussis toxin-sensitive G-protein (51) . Therefore, it is possible that kinase-defective EGF-Rs might signal to MAP kinase through an EGF-inducible, pertussis toxin-sensitive G-protein, such as G. However, we have found that pertussis toxin treatment of B82L cells causes only a marginal inhibition of EGF-induced MAP kinase activation, by either wild-type or kinase-defective EGF-Rs (data not shown). Thus, pertussis toxin-sensitive G-proteins appear not to be significantly involved in signaling to MAP kinase in this system.

Furthermore, the relative activation of ERK1, ERK2, MEK1, and MEK2 is similar whether occurring by kinase defective or wild-type receptors, consistent with the idea that the normal kinase-dependent pathway is operative. By contrast, several reports indicate that different signaling pathways preferentially activate different isoforms of ERKs and MEKS. For example, thrombin preferentially activates ERK2 versus ERK1 in platelets (52) , and v-ras preferentially activates MEK1 versus MEK2 in NIH 3T3 cells (53). Therefore, no evidence exists to support the notion that kinase-defective EGF receptors signal via a novel, kinase-independent pathway.

We feel it is most probable that kinase-defective EGF-Rs signal by activating (directly or indirectly) a heterologous endogenous tyrosine kinase. We found that the kinase-defective receptors do not cause detectable phosphorylation of JAK1 or JAK2 and thus presumably do not activate these nonreceptor tyrosine kinases. However, an unidentified protein of similar M does become tyrosine-phosphorylated in cells expressing kinase-defective EGF-Rs, and thus it is possible that another kinase of this family participates in the signaling to MAP kinase.

We also have examined activation of the Src family kinases, c-Src, c-Fyn, and c-Yes in response to activation of EGF receptors. We found no consistent activation or phosphorylation of any of these kinases in cells expressing either wild-type or kinase-defective receptors (data not shown).

By contrast, we found that endogenous ErbB2/c-Neu showed substantial EGF-induced tyrosine phosphorylation and activation in cells expressing the kinase-defective EGF receptor (Fig. 8, B and C). It has been shown previously that ErbB2/c-Neu can cluster with human kinase-defective (K721A) EGF-Rs (45) and that the kinase-defective EGF-Rs become tyrosine-phosphorylated following EGF stimulation of cells that co-express ErbB2/c-Neu. It has been suggested (54) that heterodimerization of ErbB2/c-Neu with the EGF-R occurs preferentially over homodimerization of either ErbB family member in the presence of EGF. In fact, kinase-defective ErbB2/c-Neu functions as a dominant-negative inhibitor of EGF-R signaling (55) . Therefore, we hypothesize that upon EGF stimulation, the overexpressed human EGF-R heterodimerizes with and activates endogenous B82L murine ErbB2/c-Neu.

It is uncertain how kinase-inactive EGF receptors cause the EGF-induced activation and tyrosine phosphorylation of ErbB2/c-Neu. Although it is clear that activation of ErbB family receptors is accompanied by transphosphorylation of dimerized or clustered receptors, intramolecular phosphorylations have not been ruled out (5, 54, 56) . Alternatively, ErbB2/c-Neu could be phosphorylated by transphosphorylation in receptor trimers or higher order oligomers, which have been reported to occur with ErbB family receptors upon EGF stimulation (6, 45, 57) . It is also known that dimerization and clustering of receptors are reversible processes (6) . ErbB2/c-Neu may first heterodimerize with the human EGF-R and become enzymatically activated by a mechanism independent of phosphorylation; the activated ErbB2/c-Neu could then be released and dimerize with another ErbB2/c-Neu molecule, which would precipitate transphosphorylations. Distinguishing between these possibilities will require further analysis of the physical and functional interactions between ErbB family receptors.

Surprisingly, we observed that the stimulation of ErbB2/c-Neu in vitro autokinase activity and SHC binding by kinase-defective EGF-Rs was actually greater than that seen with wild-type receptors (Fig. 8C and 9). This enhanced ErbB2/c-Neu signaling activity in the cells expressing kinase-defective receptors might reflect the failure of kinase-defective receptors to induce a desensitizing feedback loop and/or the ability of tyrosine-phosphorylated wild-type EGF-Rs to compete with ErbB2/c-Neu for binding to SHC. Whatever the mechanism, the fact that the endogenous ErbB2/c-Neu apparently compensates for the lack of EGF-R kinase activity could explain the surprisingly large activation of the MAP kinase pathway in these cells.

Substantial evidence is accumulating that receptors can form functional hetero-oligomeric complexes and that the specificity of signaling can depend on the formation of these hetero-oligomers. For example, the activated EGF receptor binds to and phosphorylates ErbB3, which apparently lacks intrinsic kinase activity. This transphosphorylation is hypothesized to be largely responsible for the activation of phosphatidylinositol 3`-kinase by ErbB3 upon EGF stimulation (58, 59) . We have observed that GAP, the GAP-associated protein p62, the nonreceptor tyrosine kinase JAK1, and p91STAT1 do not become tyrosine-phosphorylated following EGF stimulation of kinase-defective EGF-Rs in B82L cells, although all these phosphorylations are substantially stimulated by the wild-type receptor. If, as we suggest, signaling by the kinase-defective receptor is mediated by ErbB2/c-Neu and perhaps other related EGF receptor family members, these ErbB receptors may diverge from the EGF receptor (ErbB1) in the signaling pathways they activate. Perhaps ErbB2/c-Neu specifically signals to SHC and the MAP kinase pathway but not to other pathways which include GAP, JAK1, or p91STAT1. The different ErbB family members and their heterodimerization with the EGF receptor could be a route to provide different specificities for EGF in different cell types.

The ability of EGF receptors to heterodimerize with and to activate related but nonidentical endogenous receptors explains two otherwise peculiar attributes of mutant EGF receptors: (i) it has been reported that EGF receptor mutants devoid of autophosphorylation sites signal effectively to a subset of EGF-inducible pathways (45, 60, 61) . These signaling events could occur through heterologous receptors, thus bypassing the need for autophosphorylation sites on the EGF-R. (ii) Whereas most reports indicate that kinase-defective EGF-Rs do not induce c-fos(32, 34) , one recent publication reports the contrary (62) . Since the c-fos promoter contains binding sites for p91STAT1 as well as for the SRF (which can be regulated by MAP kinase), it is possible that the earlier results used conditions in which c-fos expression was dependent on p91STAT1 (which is not phosphorylated in response to kinase-defective EGF-Rs), whereas the more recent work utilized cells and conditions in which the SRF was limiting for gene expression. Thus, these findings support the interesting suggestion of Selva et al.(34) that selective, partial signaling by the kinase-defective EGF receptors may be useful in investigating the role of MAP kinase in signal transduction.

Ectopic expression of receptor mutants has been a powerful tool for understanding the physical and functional interactions of receptors with cellular signaling molecules. The results reported here, showing that kinase-defective EGF receptors can induce the phosphorylation and activation of the related ErbB2/c-Neu receptor, raise the caution that interactions between mutant receptors and endogenous receptors of the same family can confound the interpretation of such signaling studies but can also provide the opportunity to study the effects of lateral communication between regulatory receptors at the surface of cells.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants CA 39076, CA 40042, GM 47332, GM 49772, and 5-T32-GM 07055. 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.

§
Supported by a fellowship from the Deutsche Forschungsgemeinschaft (RE-864/2-2).

To whom correspondence should be addressed: Dept. of Microbiology, Box 441, 2-16 Jordan Hall, University of Virginia Health Sciences Center, Charlottesville, VA 22908. Tel.: 804-924-5022; Fax: 804-982-0689; E-mail: mjw@virginia.edu.

The abbreviations used are: EGF-R, epidermal growth factor receptor; EGF, epidermal growth factor; K-, kinase-defective; K+, wild-type (kinase-active); MAP kinase, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK or Erk kinase; GAP, GTPase-activating protein; PAGE, polyacrylamide gel electrophoresis; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline.


ACKNOWLEDGEMENTS

We are grateful to G. N. Gill for providing the B82L cell lines and to S. Parsons, C. M. Silva, D. McCarley, A. Larner, and H. S. Earp for providing both antibodies and helpful technical advice/suggestions. We also thank J. T. Parsons for insightful discussions.


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