serine 1002 is a site of in vivo and in vitro phosphorylation of the

Q 1993 by The American Society for Biochemistry and Molecular Biology, Inc THE JOURNAL OF BIOUXICAL CHEMISTRY Vol. 268, No. 25, Issue of September 5, pp. 19134-19142, 1993

Printed in U.S.A.

Serine 1002 Is a Site of in Vivo and in Vitro Phosphorylation of the Epidermal Growth Factor Receptor*

(Received for publication, January 25, 1993, and in revised form, May 4, 1993)

Dhandapani Kuppuswamy, Mark Dalton, and Linda J. Pike From the Department of Biochemistry and Molecular Biophysics, Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, Missouri 63110

We have shown previously that treatment of A431 cells with epidermal growth factor (EGF) induces desensitization of the EGF receptor. We now show that this desensitization is associated with an increase in the phosphorylation of the receptor on Ser-1002. Using a synthetic peptide corresponding to the sequence surrounding Ser-1002, ~34"""~ was identified as a kinase capable of phosphorylating this serine residue. Purified Xenopus ~34"""~ was found to phosphorylate the synthetic peptide on the serine residue corresponding to Ser-1002. This kinase also phosphorylated purified EGF receptor in vitro on Ser-1002. Phosphorylation of the EGF receptor by ~ 3 4 ~ " " ~ was associated with a decrease in its tyrosine protein kinase activity. These data indicate that the EGF receptor may be a target for phosphorylation by a cyclin-dependent kinase in vivo and imply that receptor function may be regulated in a cell cycle- dependent fashion.

The EGF' receptor is a 170-kDa transmembrane glycopro- tein that possesses intrinsic tyrosine protein kinase activity (1, 2). The EGF receptor is normally present on the cell surface as a monomer. However, binding of EGF induces dimerization of the receptor and leads to increased tyrosine protein kinase activity and transduction of the extracellular signal (3- 6). The capacity of the EGF receptor to transduce signals is negatively regulated through a variety of processes including down-regulation and desensitization. In down-regulation, treatment of cells with EGF induces the internalization and depletion of cell surface EGF receptors. This loss of receptors results in a decrease in the ability of the cells to respond to the growth factor. In desensitization, treatment of cells with EGF reduces the efficiency with which individual receptors transduce a signal.

Several mechanisms for EGF receptor desensitization have been reported. Phosphorylation of the EGF receptor by protein kinase C has been shown to decrease receptor affinity for EGF as well as tyrosine protein kinase activity (7-10) and to block receptor internalization and down-regulation (11). Thr-654 was identified as the primary site of phosphorylation in vivo following activation of protein kinase C by phorbol esters (12, 13). Site-directed mutagenesis studies have confirmed the in- volvement of this site in many of the alterations in EGF recep-

~~ ~

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked

indicate this fact. "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to

The abbreviations used are: EGF, epidermal growth factor; MAP,

formance liquid chromatography; TPA, 12-O-tetradecanoylphorbol-13- microtubule-associated protein; CSF, cytostatic factor; HPLC, high per-

acetate; DMEM, Dulbecco's modified Eagle's medium; TPCK, L-l-to- sylamido-2-phenylethyl chloromethyl ketone.

tor function observed in cells treated with phorbol esters (14, 15).

Treatment of cells with EGF, phorbol esters, or thapsigargin also leads to enhanced phosphorylation of the EGF receptor at Thr-669 (16-18). This phosphorylation appears to be catalyzed by MAP kinase (19,20), a serine-threonine protein kinase that is activated by both protein kinase C-dependent and -indepen- dent pathways. Mutational analysis of Thr-669 suggests that this residue is involved in determining substrate specificity of the EGF receptor and may also be important in ligand-induced receptor internalization (21).

More recently, phosphorylation of the EGF receptor at Ser- 1046 and Ser-1047 has been observed (22). Phosphorylation of the EGF receptor at these residues is associated with a decrease in receptor tyrosine kinase activity. These sites can be phosphorylated in vitro by calmodulin kinase 11, suggesting a potential link between EGF receptor activity and levels of intracellular calcium ions (23). Mutation of these serine residues blocks desensitization and potentiates EGF receptor tyrosine kinase activity and mitogenic signaling (23, 241, suggesting that phosphorylation of these sites in vivo is involved in the inhibition of receptor function.

We have shown previously that in A431 cells the EGF receptor undergoes a different type of desensitization following treatment with EGF. In this system, desensitization of the receptor leads to a decrease in the ability of the cells to turn over phosphatidylinositol in response to EGF (25) and to internalize receptor-bound 1251-EGF (26). The desensitization occurs rap- idly at 37 "C but does not take place at 4 "C. It is induced only by treatment of the cells with EGF and does not involve protein kinase C (26). This type of EGF receptor desensitization is distinct from others previously reported in that it is associated with a decrease in the ability of the desensitized receptors to undergo EGF-induced dimer formation (27). Since dimerization of receptor monomers is thought to be important in the path- way of signal transduction, the reduced ability of the desensitized receptors to undergo dimerization could explain their decreased capacity for signal transduction.

As previous forms of EGF receptor desensitization were associated with phosphorylation of the receptor, we undertook a study of the phosphorylation pattern of the desensitized receptors in A431 cells. We report here that EGF receptors isolated from desensitized cells contain elevated levels of phosphate on Ser-1002. Using a synthetic peptide based on the sequence of the receptor surrounding this site, we show that A431 cells contain a cytosolic kinase capable of phosphorylating this residue and that the activity of this kinase is stimulated by EGF. The kinase has been identified as ~ 3 4 " ~ ~ ' or a closely related enzyme based on its size and ability to be immunoprecipitated by a n t i - ~ 3 4 " ~ " ~ antibodies. Authentic Xenopus ~ 3 4 " ~ " is shown to phosphorylate the EGF receptor at Ser-1002 leading to a decrease in EGF receptor kinase activity. These data implicate cyclin-dependent kinases in the regulation of the EGF receptor and suggest that EGF receptor function may be linked to the cell cycle.

19134

EGF Receptor Phosphorylation at Ser-1002 19135

EXPERIMENTAL PROCEDURES

Reagent~-[~~P]Orthophosphate and [Y-~~PIATP were obtained from Du Pont-New England Nuclear. N a T was from Amersham Corp. The Sequelon radiosequencing kit was from MilligedBiosearch (Millipore). Protein A-cyclin A fusion protein and Xenopus laevis CSF extracts used to obtain active ~ 3 4 ~ ~ ~ were the generous gifts of Dr. James L. Maller (University of Colorado). Antibodies to the C terminus of ~ 3 4 ' ~ ' ~ were obtained from Upstate Biotechnologies, Inc. Peptides with the sequences Arg-Phe-Phe-Ser-Ser-Pro-Ser-Thr-Ser-Arg-Thr-Pro-Leu (ER999 peptide) and Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tp-Ala-Ala-Arg-Gly (Arg- Arg-Src) were synthesized on an Applied Biosystems peptide synthesizer and purified by ion exchange and reverse phase high pressure liquid chromatography. EGF was purified by the method of Savage and Cohen (28). TPCK-treated trypsin and V8 protease were from Worthington Bio- chemical Corp. All other reagents were obtained from Sigma.

Cell Culture-Human A431 epidermoid carcinoma cells were propa- gated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 3% fetal calf serum, 7% newborn serum, and 1% glutamine. The cells were grown on 150-mm tissue culture dishes in a humidified at- mosphere of 95% air and 5% COP at 37 "C.

In Vivo Labeling and Zkyptic Digestion of EGF Receptors-Cultures of A431 cells grown to approximately 50% confluence were rinsed with phosphate-free DMEM and then cultured for 24 h in phosphate-free DMEM containing 0.1% fetal bovine serum and 0.1 mCi/ml 32POf. Labeled cells were then stimulated with 50 n~ EGF for the times indicated by adding the growth factor directly to the culture medium. After stimulation, the plates were placed on ice and the medium was aspi- rated. The cultures were rinsed three times with ice-cold Hanks' bal- anced salt solution and the monolayers scraped into 4 ml of 50 m~ HEPES, pH 7.4,lO m~ sodium pyrophosphate, 100 mM sodium fluoride, 4 m~ EDTA, 0.2 m~ sodium orthovanadate, 2 m~ phenylmethylsulfonyl fluoride, 2 mg/ml aprotinin, 20 m~ p-nitrophenyl phosphate, 500 m~ NaCl, and 10% glycerol. The cells were centrifuged and the pellet solubilized by stirring for 1 h at 4 "C in 0.5 ml of the above buffer (containing 1% Triton X-lOO)/plate. The samples were then centrifuged for 30 min at 100,000 x g and the supernatant applied to wheat germ lectin-Sepharose. Following a 2-h incubation, the resin was washed and the receptor was released from the lectin by boiling in Laemmli SDS sample buffer. Samples were analyzed by SDS-polyacrylamide gel electrophoresis.

The 170-kDa band corresponding to the EGF receptor was identified by autoradiography and excised. The gel slice was incubated at 37 "C in 1 ml of 25 m~ ammonium carbonate containing 25 pg of TPCK-treated trypsin. After 5 h, a second aliquot of 12.5 pg of trypsin was added and the incubation continued overnight. The samples were centrifuged to pellet the gel pieces, and the supernatant was filtered through a 0.45-pm filter to remove impurities. Samples were then lyophilized to dryness.

For two-dimensional phosphopeptide mapping, the dried digest was resuspended in 25 pl of water and spotted on a Kodak cellulose plate. A bromphenol blue marker was spotted adjacent to the sample as an electrophoresis marker. The plate was lightly sprayed with 1% ammonium carbonate and electrophoresed on a Pharmacia flatbed electrophoresis apparatus at 350 V until the bromphenol blue marker moved 10 cm. The plate was air dried and subjected to thin layer chromatography in 1-butano1:pyridine:water:glacial acetic acid (5:3.33:4:1). The plate was air-dried and autoradiographed. When necessary, phosphopeptide spots were scraped from the plate, extracted from the cellulose with water, and subjected to radiosequencing according to the method of Sullivan and Wong (29) using the Sequelon radiosequencing kit.

For phosphopeptide isolation by HPLC, the lyophilized tryptic frag- ments of the EGF receptor were resuspended in 10 m~ potassium phosphate and 3% acetonitrile, pH 6.0. The sample was loaded onto a Vydac C 18 reverse-phase column equilibrated with the same buffer and run at a flow rate of 0.5 ml/min. The peptides were eluted with a 3-90% acetonitrile gradient. Elution of peptides was monitored by absorbance at 214 nm. One-milliliter fractions were collected and monitored for 32P by Cerenkov counting. The phosphopeptide peaks were pooled and dried under vacuum. When required, additional purification was achieved by resuspending the dried sample in 2 ml of 0.1% trifluoroacetic acid in water and chromatographing the material on the same column eluted with a 0-90% acetonitrile gradient with a flow rate of 1 ml/min. One- minute fractions were collected and analyzed for 32P by Cerenkov counting. Phosphopeptides obtained following this step were dried under vacuum and sequenced on an Applied Biosystems gas phase sequenator.

Preparation of Cytosolic and Membrane Fractions from A431 Cells "A431 cells were grown to subconfluence in 150-mm culture plates and

switched to serum-free DMEM containing 0.1% bovine serum albumin and incubated at 37 "C for 3 h. Cells were then stimulated with EGF for the indicated period of time by addition of the growth factor directly into the culture medium. Following EGF treatment, control and EGF- treated cultures were rinsed with 10 ml of ice-cold phosphate-buffered saline and once with lysis buffer, which contained 50 m~ p-glycerophosphate, pH 7.4, 100 p~ sodium orthovanadate, 1 nm dithiothreitol, 10 pg/ml leupeptin, 10 pgiml aprotinin, 2 pg/ml pepstatin A, and 1 m~ benzamidine. Cells were scraped into 2 ml of lysis buffer and homoge- nized with 50 strokes in a Dounce homogenizer. The lysate was centrifuged at 30,000 x g for 30 min and the clear supernatants stored frozen at -70 "C.

To prepare a membrane fraction, the starting homogenate was centrifuged at 3000 x g for 10 min to remove unbroken cells and cell debris. The supernatant was then centrifuged at 30,000 x g for 30 min. The pellet resuspended in 1 ml of 50 nm P-glycerophosphate, pH 7.4, and stored in liquid nitrogen. ER999 Peptide Kinase Assays-Assays were done in a total volume of

18 pl and contained (in final concentrations): 50 m~ P-glycerophosphate, 1.0 m~ dithiothreitol, 100 p sodium orthovanadate, 10 m~ MgC12, 100 p~ ATP, [Y-~~PIATP (3-6 x lo6 cpdassay), 2 m~ ER999 peptide, 0.1% bovine serum albumin, and an aliquot of enzyme. Tubes were incubated for 15 min at 30 "C, and the reaction was terminated by the addition of 30 p1 of 4% trichloroacetic acid. After centrifugation to pellet-precipitated proteins, 35 pl of the reaction mixture was spotted onto Whatman P81 phosphocellulose paper and washed three times for 2 min in 75 m~ phosphoric acid. The papers were dried and counted for 32P in an L-1217 Rackbeta counter. To correct for assay background, the value obtained in an identical assay done in the absence of ER999 peptide was subtracted from each sample value.

Purification of ER999 Peptide Kinase Activity-Cytosolic extract obtained from five plates of A431 cells was applied to 1.5 ml of heparin- agarose equilibrated in 50 m~ P-glycerophosphate, pH 7.4, 100 p~ sodium orthovanadate, and l mM dithiothreitol (buffer A), and the suspension was mixed for 30 min at 4 "C. The slurry was poured into a column, washed with 15 ml of buffer A and eluted with a 15-ml linear gradient of 0-0.4 M NaCl in buffer A. Fractions (0.5 ml) were collected and assayed for ER999 peptide kinase activity. Peak fractions were pooled, dialyzed against buffer A, and concentrated by dialysis against glycerol.

The concentrated heparin-agarose pool (1 ml) was chromatographed on a Sephacryl S-300 column (1.5 cm x 60 cm) equilibrated in buffer A containing 0.1 M NaCl. Fractions (1 ml) were collected and assayed for ER999 peptide kinase activity.

Immunoprecipitations-A 50-pl aliquot of the heparin-agarose pool was diluted with 150 pl of IP buffer (10 mM Tris-HC1, pH 7.4, 150 m~ NaC1, 0.1% Nonidet P-40). A 5-1.11 aliquot of ant i -~34 '~ '~ antiserum was added, and the mixture was incubated on ice for 1 h. The immune complexes were precipitated by the addition of 20 pl of a 1:l suspension of protein A-agarose beads. Following centrifugation, the supernatant was recovered for assay of ER999 peptide kinase activity. The immunoprecipitate was washed twice with IP buffer and resuspended in 50 pl of buffer A for similar assay.

Phosphorylation of the EGF Receptor by Xenopus p34c&2-The EGF receptor was purified fromA431 cell membranes by sequential chromatography on wheat germ lectin-Sepharose and heparin-agarose. The resulting preparation is greater than 90% pure as judged by silver stain of the preparation. The EGF receptor was phosphorylated with p34edc2 from Xenopus oocyte CSF extracts obtained in the following manner. A 50-pl aliquot ofXenopus CSF extract was incubated for 50 min at 25 "C with 300 pg of bacterially expressed bovine protein A-cyclin A fusion protein (30). The extract was diluted %fold with 20 m~ Tris, pH 7.5,O.l m~ NaCI, 20 mMp-nitrophenyl phosphate, 2 m~ sodium orthovanadate, 50 nm sodium fluoride, 25 pg/ml leupeptin, 20 pg/ml pepstatin, 10 pg/ml chymostatin and precleared with protein A-Sepharose beads. ~ 3 4 ~ ~ ' ~ - cyclin A complexes were precipitated by incubating the extract with 25 pl of rabbit IgG-Sepharose for 1 h at 4 OC. The beads were washed two times with RIPA buffer and used in phosphorylation reactions.

For phosphorylation of the EGF receptor by p34edc2, incubations contained 250-500 ng of purified EGF receptor in 10 m~ HEPES, 30 m~ &glycerophosphate, 10 m~ MgC12, 1 nm dithiothreitol, 100 p~ sodium orthovanadate, 0.1% Triton X-100, 0.1% bovine serum albumin, and 100 p~ [y-"PIATP in the presence of ~ 3 4 ~ ~ ' ~ bound to IgG-Sepharose. In place of the p34edc2, control incubations contained IgG-agarose beads that had been incubated withxenopus oocyte extracts lacking the cyclin A-protein A fusion protein. Reaction mixtures were incubated for the times and temperatures indicated in the figure legends and were

19136 EGF Receptor Phosphorylation at Ser-1002 stopped by the addition of Laemmli sample buffer. Samples were analyzed by SDS-polyacrylamide gel electrophoresis followed by autoradiography.

Phosphoamino acid analyses of the phosphorylated EGF receptor were carried out as described previously (16). Tryptic phosphopeptides of in vitro phosphorylated EGF receptors were generated and analyzed as described above for in vivo labeled receptors.

RESULTS

Identification of the Site of Phosphorylation of the EGF Re- ceptor"A431 cells become desensitized to EGF following a 30- min stimulation of the cells with this growth factor (25,26). To examine the phosphorylation pattern of desensitized EGF receptors, A431 cells were labeled overnight with 32PO:- and then treated with or without 50 nM EGF for 30 min at 37 "C. EGF receptors were then isolated and digested with trypsin and the peptides analyzed by two-dimensional thin layer chromatography (Fig. 1). Because our previous work had suggested that protein kinase C was not involved in the process of EGF receptor desensitization, tryptic phosphopeptide maps of 12-0-tetradecanoylphorbol-13-acetate (TPAbtreated cells were also generated for comparison. The EGF receptor was phosphorylated under all conditions, but the phosphorylation of a number of peptides was increased following treatment of the cells with EGF or TPA. Treatment with EGF led to an increase in the phosphorylation of spots labeled 1,2, 3, and 4+5 in Fig. 1. In some maps, spot 4+5 could be seen to be two closely spaced peptides; hence, this spot has been designated 4+5 to indicate the presence of two peptides. Treatment with TPA increased the phosphorylation of two spots labeled 3 and 6 in Fig. 1. Because desensitization of the A431 EGF receptor was induced by EGF but not by TPA, we concentrated on identifying peptides for which phosphorylation was enhanced following treatment with EGF but not with TPA. Based on this criterion, peptides 1, 2, and 4+5 were selected for further analysis.

Phosphoamino acid analyses (not shown) indicated that peptides 1 and 5 contained only phosphotyrosine suggesting that these represented sites of EGF receptor autophosphorylation. Radiosequence analyses indicated that peptide 1 contained Tyr-1173 and peptide 5 contained Tyr-1148. Peptide 4 was a minor spot that contained phosphoserine and radiosequence analysis suggested that it corresponded to the tryptic peptide containing Ser-967. Peptides 3 and 6 contained predominantly phosphothreonine. Since the phosphorylation of these peptides was enhanced in TPA-treated cells, they are not specific to the desensitized state. These peptides most likely represent peptides containing Thr-669 and Thr-654, which are known to be

Control

0

TPA

EGF

Origin

FIG. 1. Phosphopeptide maps of in vivo S*P-labeled EGF receptors. A431 cells were labeled overnight with 32POi- and subsequently desensitized by incubation with 50 nM EGF for 30 min at 37 "C. For comparison, one culture was treated with 100 nM TPA for 30 min a t 37 "C. EGF receptors were isolated by chromatography on wheat germ lectin-Sepharose followed by SDS-polyacrylamide gel electrophoresis. EGF receptor bands were excised from the gel and digested with trypsin. The resulting phosphopeptides were analyzed by two-dimensional thin layer chromatography followed by autoradiography.

phosphorylated in response to TPA. Peptide 2 was found to contain principally phosphoserine and appeared to be present only in receptor derived from desensitized cells. Because peptide 2 represented a major site of serine phosphorylation unique to the desensitized receptor, we focused on identifying the sequence of this peptide.

Phosphopeptide 2 was eluted from the thin layer plate and the isolated material was subjected to radiosequence analysis (Fig. 2, upper panel ). No 32P was released from the peptide in the first 12 cycles of Edman degradation. Therefore, the tryptic phosphopeptide was digested with V8 protease and the material was again subjected to radiosequence analysis. As shown in the lower panel of Fig. 2, 32P was released from this peptide a t cycle 11 implying that the phosphorylated serine residue was present 11 amino acids C-terminal to a glutamate residue. Within the EGF receptor only the tryptic peptide 976- ALMDEEDMDDWDADEYLIPQQGFFSSPSTSR-1007 has a sequence consistent with these characteristics of phosphopeptide 2. The data suggest that Ser-1002 is phosphorylated.

Further evidence for the identity of this peptide was obtained by direct protein sequencing methods. A43 1 cells were labeled with 32PO:- as before, and EGF receptors were isolated from control and desensitized cells. This radiolabeled material was added to similar preparations of EGF receptors derived from large quantities of unlabeled A431 cells treated with or without EGF. The receptor preparations were then digested with trypsin and the peptides analyzed by HPLC. As shown in Fig. 3, incorporation of 32P was increased in a t least six peaks in the EGF-desensitized as compared to the control receptors. Several peaks were further purified and submitted for amino acid sequence analysis. Direct sequencing of peak IV yielded the sequence Ala-Leu-Met-Asp-Glu-Glu-Asp-Met-Asp-Asp-Val-Val, which corresponds to the first 12 residues of the tryptic peptide encompassing residues 976-1007 of the EGF receptor. Phos- phoamino acid analysis indicated that this peptide contained

400 Undigested

300 - n

2 "E 200-

l o 0 L 0 1 2 3 4 5 6 7 8 9101112

Cycle No.

4 Digested with V8

300 { n

200-

100 -

0 1 2 3 4 5 6 7 8 9 1 0

Cycle No. 11 12

beled EGF receptors. A431 cells were labeled in vivo with 32P and FIG. 2. Radiosequence analysis of peptide 2 from in vivo la-

desensitized with EGF as described in Fig. 1. EGF receptors were isolated and tryptic peptides produced as described under "Experimental Procedures." Peptide 2 was isolated from the thin layer plate and subjected to radiosequence analysis. Upper panel, released from tryptic peptide 2. Lower panel, 32P released from tryptic peptide 2 following its digestion with V8 protease.


A BOO

e + M F 0 Control

0 10 20 30 40 50 60 70 80 90 100 110 Fraction No.

FIG. 3. HPLC analysis and sequencing of tryptic phosphopeptides derived from control and desensitized EGF receptors. A431 cells were labeled overnight with 32Pi and then desensitized by treatment with 50 IIM EGF for 30 min at 37 "C. EGF receptors were

tal Procedures." The receptor digests were analyzed by chromatography isolated and tryptic peptides produced as described under "Experimen-

on a Vydac C18 HPLC column. 32P eluting in each fraction was quantitated by Cerenkov counting.

phosphoserine. Together, the radiosequence analysis and the direct sequence data indicate that phosphorylation of the tryptic peptide containing residues 976-1007 is increased in desensitized EGF receptors and that the phosphorylation most likely occurs on Ser-1002.

Identification of a Kinase Capable of Phosphorylating the ER999 Peptide-A peptide was synthesized with the sequence Arg-Phe-Phe-Ser-Ser-Pro-Ser-Thr-Ser-Arg-Thr-Pro-Leu, corresponding to residues 999-1010 of the EGF receptor with an additional arginine residue at the N terminus. This peptide, designated ER999, was used as a substrate to identify kinases that might be capable of phosphorylating the EGF receptor at Ser-1002. ER999 peptide phosphorylating activity was found predominantly in A431 cell cytosol with only a low level of activity in A431 cell membranes (not shown). To determine whether the activity of the enzyme(s) catalyzing the phosphorylation of this peptide was affected by EGF, A431 cells were treated with 50 m EGF for times ranging from 0 to 30 min and cytosol was prepared from the treated cells. The data in Fig. 4 (upper panel) demonstrate that the activity of the kinase (or kinases) capable of phosphorylating the ER999 peptide could be stimulated 6-fold by treatment of the cells with EGF. Stim- ulation was seen as early as 2 min after the addition of EGF and was maximal by 10 min. Peptide phosphorylating activity declined thereafter but remained above basal levels for at least 40 min. As shown in the lower panel of Fig. 4, the concentration of EGF required for half-maximal activation of ER999 peptide kinase activity was approximately 5 m.

The kinase activity from control and EGF-stimulated cells was partially purified by chromatography on heparin-agarose and Sephacryl S-300. Approximately half of the ER999 peptide kinase activity present in crude cytosolic extracts could be bound to heparin-agarose. The activity that did not bind to the heparin column was shown to phosphorylate myelin basic protein but not histone H1 and to react with anti-MAP kinase antibodies, suggesting that this represents a MAP kinase. This kinase activity in the flow-through was stimulated approximately 3-fold by EGF. Given the fact that MAP kinase will phosphorylate serine or threonine residues immediately followed by a proline residue (31, 32) and that EGF is known to stimulate MAP kinase activity (33, 341, the observation that a portion of the ER999 peptide kinase activity was due to MAP kinase was not unexpected.

The ER999 peptide kinase activity that was retained on heparin-agarose was eluted from the column in a single peak at a

s 400007

v

'0 io 20 30 40 50

Time (min)

= rooootlt ' -; ' ' ' ' I ' -0 -7 -0

log [EGF] (M)

FIG. 4. Phosphorylation of ERgss by cytosols of A431 cells. Up- per panel, A431 cells were treated with 50 IIM EGF for the indicated time, then lysed and cytosols prepared as described under "Experimen- tal Procedures."Lowerpanel, A431 cells were treated with the indicated concentrations of EGF for 10 min, then lysed and cytosols prepared. Cytosols were assayed for their ability to phosphorylate the ER999 peptide. Results represent the mean 2 S.D. of duplicate determinations.

concentration of 300 mM NaCl (Fig. 5, upper panel 1. The pooled fractions from the heparin-agarose column were concentrated and chromatographed on a Sephacryl S-300 column. As shown in the lower panel of Fig. 5, ER999 peptide kinase activity from both control and EGF-stimulated extracts eluted as a single peak at a position that corresponded to a molecular weight of approximately 90,000. Increased peptide kinase activity was observed in the elutes purified from the cytosols of EGF-treated cells, suggesting that this ER999 peptide kinase activity was also stimulated by EGF. Peptide kinase activity in the pooled heparin-agarose fractions could also be purified by chromatography on a Mono-& column. However, enzyme purified by this method was unstable.

The size of the ER999 peptide kinase, as well as its ability to phosphorylate a peptide containing a Ser-Pro sequence, suggested that this activity might represent a cyclin-dependent kinase. To determine whether the activity phosphorylating the ER999 peptide was a cdc2-like kinase, antibodies to the C-terminal region of ~ 3 4 ' ~ ' ~ were used to immunoprecipitate ER999 peptide kinase activity from heparin-agarose-purified pools of enzyme derived from control and EGF-stimulated A431 cells. As shown in Fig. 6, approximately 85% of the peptide kinase activity could be immunoprecipitated using this antibody. These data suggest that the majority of the ER999 peptide kinase activity in this fraction is due to the ~ 3 4 ' ~ " ~ or a closely related kinase. Thus, both MAP kinase and a cdc2-like kinase appear to be present in A431 cell cytosols and able to phosphorylate the ER999 peptide.

Phosphorylation of ER999 and the EGF Receptor by Xenopus p34cdc2-MAP kinase has previously been shown to phosphorylate the EGF receptor at Thr-669 (19,20), a site distinct from

19138 EGF Receptor Phosphorylation at Ser-1002

80000 6oooon 40000 c 20000 1 /p

0 0 10 20 30

Fraction No.

5000 I t 0 Control I

0 0 20 40 60 80

Fraction No.

FIG. 5. Purification of ER999 peptide kinase activity from A431 cells. Upper panel, cytosols from A431 cells treated without or with 50 n~ EGF for 10 min were adsorbed to heparin-agarose and eluted with a gradient of NaCl. Fractions were assayed for ER999 peptide kinase

were concentrated and chromatographed on a Sephacryl S-300 column. activity. Lower panel, pooled fractions from the heparin-agarose column

Fractions were assayed for ER999 peptide kinase activity. Arrows show the position of standards. V, void volume; F, femtin; A, aldolase; B, bovine serum albumin; C, cytochrome c.

40000

0

Control ~

W EGF

Starting Immuno- Materlal precipitates

with ant i -~34~~"* antibodies. A431 cells were treated without or with FIG. 6. Immunoprecipitation of EM99 peptide kinase activity

EGF and ER999 peptide kinase activity purified by chromatography over heparin-agarose as described in the legend to Fig. 5. The peak of peptide kinase activity from each preparation was immunoprecipitated with antibodies directed against the C terminus of ~ 3 4 " ~ ' ~ as described under "Experimental Procedures." These antibodies are specific for p3Pdc2 and do not recognize cdk2. The immunoprecipitates were assayed for ER999 peptide kinase activity. Results represent the mean * S.D. of duplicate determinations.

the Ser-1002 we found phosphorylated in desensitized receptors. We therefore chose to examine the possibility that ~34"~"' phosphorylated the EGF receptor at this site in vitro. For these experiments we utilized a kinase preparation immunoprecipitated from Xenopus oocyte CSF extracts (see "Experimental Procedures"). These preparations contain primarily ~ 3 4 " ~ " ~ but may also contain low levels of other cyclin-dependent kinases.

For simplicity, the material is referred to as ~ 3 4 " ~ " ~ . Initially, the ability of Xenopus ~ 3 4 " ~ " ~ to phosphorylate the

ER999 peptide was examined. The p34cdc2 kinase avidly phosphorylated the ER999 peptide substrate. Phosphoamino acid analysis of the product indicated the presence of approximately 80-90% phosphoserine with the remainder as phosphothreonine (not shown). When subjected to radiosequence analysis, greater than 90% of the 32P was released at cycle 5 (Fig. 7). This position corresponds to Ser-1002 of the EGF receptor. Less than 10% of the counts were released in cycle 11, which corresponds to Thr-1008. Both of these residues immediately precede a proline residue, consistent with the known substrate specificity of ~34"~"'. No other residues appear to be phosphorylated even though the ER999 peptide contains numerous serine and threonine residues.

The ability of ~ 3 4 " ~ " ~ to phosphorylate the EGF receptor itself was next examined. Fig. 8 shows the results of an exper- iment in which purified ~ 3 4 " ~ " was incubated with [Y-~~PIATP and EGF receptors purified from A431 cells. A modest (-50%) increase in receptor phosphorylation was observed (Fig. 8A). However, this increase in phosphorylation was due entirely to an increase in the phosphoserinelphosphothreonine content of the receptor (Fig. 8B). EGF receptors phosphorylated in the absence of ~ 3 4 " ~ " ~ contained only phosphotyrosine, whereas receptor phosphorylated in the presence of ~34"~" ' contained significant amounts of phosphoserine and phosphothreonine in addition to phosphotyrosine. Neither receptor autophosphorylation (lanes 1 and 3) nor phosphorylation by ~ 3 4 " ~ ' ~ (lanes 2 and 4 ) appeared to be significantly affected by the addition of EGF. This apparent loss of responsiveness to EGF appears to be a property of highly purified receptor preparations and has been observed previously by others (1, 35).

To determine the site at which the EGF receptor was phosphorylated by ~ 3 4 " ~ " ~ in vitro, purified EGF receptor was phosphorylated in the absence or presence of ~ 3 4 " ~ " ~ and analyzed by SDS-polyacrylamide gel electrophoresis. The phosphorylated receptor band was excised from the gel and digested with trypsin, and the phosphopeptides were analyzed by two- dimensional thin layer chromatography (Fig. 9). Four major phosphopeptides were present in EGF receptor phosphorylated in vitro in the absence of ~ 3 4 " ~ ~ ~ . Phosphoamino acid analysis of these peptides (Fig. 10) indicated that all four contained almost exclusively phosphotyrosine, consistent with these be- ing sites of autophosphorylation. When the EGF receptor was phosphorylated in the presence of ~ 3 4 " ~ " ~ the same major phosphopeptides were present (Fig. 9). However, phosphoamino acid analysis demonstrated that peptide 2 now contained phos-

10000 '2000T

2 o o o L A 0 rl--"- 1 2 3 4 5 6 7 8 9101112l i

- 3

Cycle No. I s g P h b P h b S w - S w Q r o S w . ~ ~ . ~ ~ ~ ~ ~ ~

p34Odc8. ER999 peptide (2 m) was phosphorylated with Xenopus FIG. 7. Radiosequence analysis of ER999 phosphorylated by

p34cde2 kinase for 15 min. The phosphorylated peptide was isolated on p81 phosphocellulose paper and eluted from the paper with 50 m ammonium carbonate, pH 10. The purified peptide was then subjected to radiosequence analysis.

A.

EGF-R - .) B.

PS - $ PT I

py-O 0

EGF Receptor Phosphorylation at Ser-1002

Control Conlrol EGF E:F

p34CdC2 p34CdC2

CDm PS 0 249 13 220 cpm PT 0 169 8 133 cpm PY 548 488 583 541

FIG. 8. Phosphorylation of the EGF receptor in vitro by ~ 3 4 ' ~ ' ~ kinase. Purified EGF receptors were phosphorylated in vitro in the absence (lanes 1 and 3 ) or presence (lanes 2 and 4 ) of ~ 3 4 ' ~ ' ~ and in the absence (lanes 1 and 2 ) or presence (lanes 3 and 4 ) of EGF. Panel A, EGF receptors were analyzed by SDS-polyacrylamide gel electrophoresis followed by autoradiography. Panel B, the bands from panel A were excised and subjected to phosphoamino acid analysis. The spots repre- senting phosphoserine, phosphothreonine, and phosphotyrosine were excised and counted for 32P. The results are reported below each lane.

Control

. + p34CdC2

I

2.

FIG. 9. Phosphopeptide maps of EGF receptors phosphorylated in vitro with ~ 3 4 ' ~ ~ ~ . Purified EGF receptors were phosphorylated in vitro in the absence or presence of ~ 3 4 ' ~ ' ~ . The EGF receptors were analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. The EGF receptor band was excised from the gel and digested with trypsin. The resulting phosphopeptides were separated by two-dimensional thin layer chromatography. The autoradiograms of these maps are shown.

phoserine in addition to phosphotyrosine (Fig. 10). Ser-1002 lies within the tryptic peptide (residues 976-1007)

that contains Tyr-992, a site of in vitro autophosphorylation of

Control

PS - PT - PY -

+ p34CdC2

PS - PT - PY -

19139

2 3 4 Peptide No.

1 2 3 4 Peptide No.

FIG. 10. Phosphoamino acid analyses of peptides 1 4 f rom the phosphopeptide maps. Upper panel, analysis of peptides 1-4 from EGF receptors phosphorylated in the absence of ~ 3 4 ' ~ ' ~ . Lower panel, analysis of peptides 1-4 from EGF receptors phosphorylated in the presence of ~ 3 4 ' ~ ' ~ .

the EGF receptor. To determine whether peptide 2 corresponded to this tryptic peptide, spot 2 derived from control or p34cdc2-phosphorylated receptors was recovered from the thin layer plate and subjected to radiosequence analysis. If spot 2 represented the tryptic peptide containing residues 976-1007, then no release of 32P would be anticipated within the first 12 cycles as there are no phosphorylatable residues in this region. However, if the peptide were cleaved with V8 protease, the resulting C-terminal fragment would contain all possible phosphorylation sites including Tyr-992 a t position 1 and Ser-1002 at position 11. The results of the radiosequence analysis for peptide 2 derived from control and p34cdc2-phosphorylated EGF receptors are presented in Fig. 11. If the peptide was not digested with V8 before radiosequence analysis, no counts were recovered in the first 12 cycles from either sample (not shown), indicating that the site of phosphorylation was more C-terminal. However, when the tryptic peptides were further digested with V8 protease, 32P was released during the radiosequencing. For peptide 2 derived from control receptors (upper panel ), 32P was released in the first cycle with no further release of counts out to cycle 12. By contrast, when the receptor had been phosphorylated with p34cdc2, radiosequencing of peptide 2 showed not only a release of counts at the first cycle, but an additional peak of 32P released at cycle 11. These data are entirely consistent with the assignment of peptide 2 as the peptide containing residues 976-1007 of the EGF receptor. The radiosequence analysis suggests that Tyr-992 is phosphorylated in both peptides and Ser-1002 is also phosphorylated in receptors treated with ~ 3 4 ~ ~ ~ ' .

Because of the decreasing recovery associated with the repetitive yields of radiosequencing, it is difficult to estimate the

19140 EGF Receptor Phosphorylation at Ser-1002

20000 Control

500

1 2 3 4 5 6 7 8 9101112 Cycle No.

1 2 3 4 6 6 7 8 0101112 Cycle No.

FIG. 11. Radiosequence analysis of peptide 2 from in vitro phosphorylated EGF receptors. EGF receptors were phosphorylated in vitro in the absence or presence of p34edc2 and analyzed by tryptic phosphopeptide mapping. Peptide 2 (labeled in Fig. 9) was excised, digested with V8 protease, and subjected to radiosequence analysis. Upper panel, EGF receptors phosphorylated in the absence of ~ 3 4 " ~ ' ~ . Lower panel, EGF receptors phosphorylated in the presence of p34"dcz.

A

Control p34-

B

100

50

stoichiometry of the phosphorylation from this data. Compari- son of the counts recovered for Tyr-992 in the uncleaved versus cleaved peptides (i.e. cycle 17 uersus cycle 1) suggests that the repetitive yield for the procedure is about 83%. Assuming this repetitive yield, it can be calculated that the extent of phosphorylation at Ser-1002 is about 25% of that at Tyr-992. This estimate compares favorably with the relative levels of phosphoserine and phosphotyrosine found in peptide 2 following phosphoamino acid analysis (Fig. 10).

To determine whether phosphorylation of the EGF receptor by ~ 3 4 " ~ " ' was associated with any functional change in the EGF receptor, the protein tyrosine kinase activity of the receptor was measured using the Arg-Arg-Src peptide as a substrate. As shown in the upper panel of Fig. 12, phosphorylation of the EGF receptor by ~ 3 4 " ~ " was associated with a -50% decrease in EGF-stimulated phosphorylation of the tyrosine-containing Arg-Arg-Src peptide. Relatively little change in basal activity was observed under these conditions. The extent of the inhibition of EGF receptor tyrosine kinase activity was dependent on the length of preincubation of the receptor with ~34"~" ' (Fig. 12, lower panel). These data indicate that phosphorylation of the EGF receptor by ~ 3 4 ' ~ ~ ' results in an inhibition of its tyrosine protein kinase activity.

DISCUSSION We have shown previously that prolonged exposure of A431

cells to EGF leads to receptor desensitization as measured by EGF-stimulated phosphatidylinositol turnover (25) and internalization of lZ6I-EGF (26). By analogy with other mechanisms for the regulation of EGF receptor function, phosphorylation was considered as a mechanism for achieving this form of desensitization. Previous work had indicated that this desensitization of the EGF receptor did not involve protein kinase C . Therefore, initial experiments set out to identify peptides for which the phosphorylation was increased in desensitized cells but not in TPA-treated cells. Comparison of tryptic phospho-

Time with ~ 3 4 ' ~ ' ' Win)

FIG. 12. Tyrosine protein kinase activity of the phosphorylated EGF receptor. Upper panel, purified EGF receptors were prephospho- rylated for 10 min at 30 "C with ~ 3 4 " ~ " ~ as described under "Experi- mental Procedures" except unlabeled ATP was utilized. The supernatant was then transferred to tubes containing [y-32PlATP and 2 I" Arg-Arg-Src peptide and incubated at 30 "C for an additional 10 min. The extent ofArg-Arg-Src peptide phosphorylation by the EGF receptor was then quantitated using the method described for ER999 peptide phosphorylation. Results represent the mean f S.D. of duplicate determinations. Lower panel, purified EGF receptors were prephosphory- lated at 22 "C for the times indicated with ~ 3 4 ' ~ ~ ~ as described above. The supernatants were then transferred to tubes containing [y-32PlATP and 2 I" Arg-Arg-Src peptide and incubated at 22 "C for an additional 15 min. The extent of Arg-Arg-Src peptide phosphorylation by the EGF receptor was then quantitated as above. Results represent the mean * S.D. of duplicate determinations. The slightly reduced temperature was used to slow the rate of receptor phosphorylation.

peptide maps of in vivo 32POi--labeled EGF receptors isolated from control, desensitized, and TPA-treated cells permitted the identification of a major phosphopeptide with these characteristics. Subsequent sequencing and radiosequence analysis suggested that this peptide corresponded to the tryptic peptide encompassing residues 976-1007 of the EGF receptor and that phosphorylation occurred at Ser-1002. This peptide also contains Tyr-992, a site of receptor autophosphorylation in vitro (36). Phosphoamino acid analyses and radiosequence analyses confirmed that little, if any, phosphorylation occurred on this tyrosine residue in vivo. Thus, Ser-1002 represents a major site of phosphorylation of the EGF receptor isolated from desensitized cells.

Utilization of a peptide (ER999) containing the sequence surrounding Ser-1002 permitted the identification of kinase activities present in the cytosol of A431 cells capable of phosphorylating this peptide. Several pieces of evidence suggest that one kinase capable of phosphorylating the ER999 peptide is p34cd'2 or a closely related enzyme. First, the ER999 peptide contains


a Ser-Pro motif that is recognized by the ~ 3 4 " ~ " ~ kinase. Sec- ond, the kinase activity was partially purified and found to exhibit a molecular weight of 90,000 by gel filtration chromatography, a size consistent with the properties of active ~ 3 4 " ~ " ~ . Third, the majority of the ER999 peptide kinase activity in the partially purified preparation could be immunoprecipitated with antibodies to the C terminus of p34cdc2. Supporting this interpretation is the observation that the ER999 peptide was a good in vitro substrate for phosphorylation by the ~ 3 4 " ~ " ~ preparation. The phosphorylation occurred almost exclusively at the site corresponding to Ser-1002 of the EGF receptor despite the fact that the peptide contains numerous other serine and threonine residues.

Hall et al. (37) have previously reported that EGF stimulates the activity of a proline-directed protein kinase in A431 cells. These workers suggested that the stimulated enzyme represents ~ 3 4 " ~ ' ~ complexed with cyclin A. We observed an apparent increase in the cdc2-like kinase activity following treatment of cells with EGF; hence, our data are consistent with the hypothesis that EGF activates a cyclin-dependent kinase in A431 cells. However, additional work is required to better re- solve this kinase from other activities and define the nature of the enzyme.

The ~ 3 4 " ~ " ~ kinase phosphorylated the EGF receptor in vitro. Analysis of the tryptic peptides derived from the phosphorylated receptor indicated that peptide 2 (Figs. 9 and 10) isolated from control reactions lacking ~ 3 4 " ~ ~ ~ contained only phosphotyrosine, whereas the peptide isolated from receptors incubated with ~ 3 4 " ~ " ~ contained both phosphotyrosine and phosphoserine. Radiosequence analysis of a V8 digest of this tryptic peptide was consistent with the identification of Ser- 1002 as the site of in vitro phosphorylation of the receptor by ~ 3 4 ' ~ " ~ . The apparent comigration of the singly tyrosine-phosphorylated peptide 2 with a peptide containing both phosphotyrosine and phosphoserine may be explained if phosphorylation of Tyr-992 and Ser-1002 are mutually exclusive. Indeed, Ser-1002 appears to be a major site of receptor phosphorylation in vivo where phosphorylation of Tyr-992 is negligible (Fig. 1). By contrast, the extent of Ser-1002 phosphorylation in vitro appears to be substantially less while Tyr-992 phosphorylation is high (Figs. 9 and 10). Because Tyr-992 is relatively close to Ser-1002, its phosphorylation could negatively affect the phosphorylation of Ser-1002. Prior in vivo phosphorylation of Ser- 1002 or differences in the conformation of detergent-solubilized versus membrane-bound EGF receptors might also contribute to inefficient in vitro phosphorylation of Ser-1002. Because of these uncertainties, it is difficult to accurately quantitate the stoichiometry of phosphorylation of Ser-1002 in vitro.

Our studies of EGF receptors phosphorylated by ~ 3 4 ' ~ " ~ in vitro suggest that the phosphorylation is associated with a time-dependent, -50% inhibition of receptor tyrosine kinase activity assayed using a synthetic peptide substrate. This suggests that EGF receptor function can be altered by phosphorylation at Ser-1002 in vitro. Ser-1002 may therefore represent a novel site for the phosphorylation and regulation of the EGF receptor in vivo.

Ser-1002 lies in a region of the EGF receptor just outside the kinase domain (approximately residues 685-950) (38) and immediately downstream of an acid-rich sequence thought to form an amphipathic helix (39). This region of the EGF receptor has been shown to be important for receptor function. Truncation of the receptor to residue 973 resulted in the production of an internalization deficient receptor (39, 40). Additional experiments suggested that the 48 amino acids from residues 973- 1022 were critical for internalization and EGF-stimulated in- creases in cytosolic calcium (41). Phosphorylation of the EGF receptor within this functionally important region might well

influence receptor activity. Comparison of the sequences of several growth factor recep-

tors shows that the string of acidic residues followed by the Ser-Pro motif found at residues 1002-1003 of the EGF receptor is conserved in all EGF receptor family members including neu/erbB-2 (42), HER-3/erbB-3 (431, and let-23 (441, the EGF receptor homolog in C. elegans. In addition, the platelet-derived growth factor receptor (45) and fibroblast growth factor receptor (46) also contain Ser-Pro motifs in the region C-terminal to the kinase domain, although they lack the acidic region up- stream from this sequence. Thus the potential exists for a number of growth factor receptors to be phosphorylated by a proline-directed kinase in this region.

The finding that the EGF receptor is a target for phosphorylation by a cyclin-dependent kinase in vitro suggests that the receptor may be an in vivo substrate for these enzymes, although additional experiments will be required to support this hypothesis. Because the activity of the cyclin-dependent kinases varies during the cell cycle, responsiveness of cells to EGF could be regulated in a cell cycle-dependent fashion. Since EGF is involved in the regulation of cell growth, linking receptor function to the cell cycle could ensure proper coordination of extracellular signals and intracellular events. Several reports in the literature are consistent with the hypothesis that receptors are regulated in a cell cycle-dependent fashion. Membrane trafficking, including the internalization of coated pits, is inhibited during mitosis (see Ref. 47 for review). Using broken cell preparations, Pypaert et al. (48) demonstrated that the invagination of coated pits, and, hence, receptor endocytosis, could be inhibited by mitotic cytosol but not interphase cytosol. The inhibition could be partially reproduced by the addition of ~ 3 4 ' ~ " ~ . Thus, the efficiency of EGF receptor internalization may vary with the cell cycle. Cell-cycle dependent effects on receptor function may not be limited to alterations in cell surface receptors. Hsu et al. (49) have recently reported that the ability of the glucocorticoid receptor to activate the murine mammary tumor virus promoter was decreased in mouse L cells in G2. Interestingly, these workers observed changes in the phosphorylation of the glucocorticoid receptor isolated from Gz synchronized cells as compared to receptors isolated from asynchronous cultures. They suggested that cell cycle regulation of kinases and phosphatases could affect glucocorticoid receptor function.

The data presented herein demonstrate that the EGF receptor is phosphorylated in vivo on Ser-1002 following long term incubation with EGF. This same site can be phosphorylated in vitro by ~ 3 4 " ~ ' ~ , and this phosphorylation is associated with an inhibition of EGF receptor tyrosine kinase activity. These find- ings implicate the cyclin-dependent kinases in the regulation of the EGF receptor and raise the interesting possibility that the function of this growth factor receptor may be regulated in a cell-cycle dependent fashion.

REFERENCES 1. Cohen, S., Carpenter, G., and King, L., Jr. (1980) J. Biol. Chem. 266, 4834-

3. Yarden. Y., and Schlessinger, J. (1987) Bhhemistry 28, 1434-1442 2. Ushiro, H., and Cohen, S. (1980) J. Biol. Chem. 266,8363-8365

4. Yarden, Y., and Schlessinger, J. (1987) Biochemistry 28, 1443-1451 5. Boni-Schnetzler, M., and F'ilch, P. F. (1987) Proc. Natl. Acad. Sci. U. S. A. 84,

7832-7836 6. Cochet, C., Kashles, O., Chambaz, E. M., Borello, I., King, C. R., and

7. Cochet, C., Gill, G. N., Meisenhelder, J., Cooper, J. A,, and Hunter, T. (1984) J. Schlessinger, J. (1988) J. Biol. Chem. 283, 32903295

8. Friedman, B., Frackelton, A. R., Ross, A. H., COMO~S, J. M., Fqjiiki, H., Sug Biol. Chem. 269,2553-2558

imura, T., and Rosner, M. R. (1984) Proc. Natl. A d . Sci. U. S. A. 81, 3034-3038

9. Downward, J., Waterfield, M. D., and Parker, P. J. (1985) J. Bid. Chem. 280,

10. Lin, C. R., Chen, W. S., Lazar, C. S., Carpenter, C. D., Gill. G. N., Evans, R. M.,

4842

14538-14546

and Rosenfeld, M. G. (1986) Cell 44,839-848

19142 EGF Receptor Phosphorylation at Ser-1002 11.

12. 13.

14. 15.

17. 16.

18.

19.

20.

21.

22.

23.

24.

25.

26. 27.

29. 28.

30.

31.

Lund, K. A,, Lazar, C. S . , Chen, W. S., Walsh, B. J., Welsh, J. B., Herbst, J. J., Walton, G. M., Rosenfeld, M. G., Gill, G. N., and Wiley, H. S . (1990) J. Biol. Chem. 285,20517-20523

Hunter, T., Ling, N., and Cooper, J. A. (1984) Nature 311,48&483 Davis, R. J., and Czech, M. P. (1985) Proc. Natl. Acad. Sci. U. S . A. 82, 1974-

Davis, R. J. (1988) J. Biol. Chem. 263, 9462-9469 Decker, S . J., Ellis, C., Pawson, T., and Velu, T. (1990) J. Biol. Chem. 265,

Takishima, K., Friedman, B., Fujiki, H., and Rosner, M. R. (1988) Biochem. Heisermann, G. J., and Gill, G. N. (1988) J. Biol. Chem. 263, 13152-13158

Countaway, J. L., Northwood, I. C., and Davis, R. J. (1989) J. Biol. Chem. 264,

Takishima, IC, Griswold-Prenner, I., Ingebritsen, T., and Rosner, M. R. (1991)

Northwood, I. C., Gonzalez, F. A., Wartmann, M., Raden, D. L., and Davis, R.

Heisermann, G. J., Wiley, H. S., Walsh, B. J., Ingraham, H. A,, Fiol, C. J., and

Countaway, J. L., McQuilkin, P., Girones, N., and Davis, R. J. (1990) J. Biol.

Countaway, J. L., Nairn, A. C., and Davis, R. J. (1992) J. Biol. Chem. 267,

Theroux, S . J., Latour, D. A,, Stanley, K., Raden, D. L., and Davis, R. J. (1992)

Cunningham, T. W., Kuppuswamy, D., and Pike, L. J. (1989) J. Biol. Chem.

Kuppuswamy, D., and Pike, L. J. (1989) J. Biol. Chem. 264,33573363 Kuppuswamy, D., and Pike, L. J. (1991) Cell. Sigmlling 3, 107-117 Savage, C. R., and Cohen, S . (1972) J. Biol. Chem. 247, 7609-7611 Sullivan S. , and Wong, T. W. (1991)Anal. Biochem. 197,6548 Bandara, L. R., Adamczewski, J. P., Hunt, T., and LaThangue, N. B. (1991)

Sanghera, J. S., Aebersold, R., Momson, H. D., Bures, E. J., and Pelech, S. F.

1978

7009-7015

Biophys. Res. Commun. 157,740-746

10828-10835

Proc. Natl. Acad. Sci. U. S. A. 88, 2520-2524

J. (1991) J. Biol. Chem. 266, 1526615276

Gill, G. N. (1990) J. Biol. Chem. 265, 12820-12827

Chem. 265,34074416

1129-1140

J. Biol. Chem. 267,16620-16626

264, 15351-15356

Nature 352,249-251

32.

33.

34. 35.

36.

37.

38. 39.

40.

41.

42. 43.

44.

45.

46.

47. 48.

49.

Alvarez, E., Northwood, I. C., Gonzalez, F. A., Latour, D. A,, Seth, A,, Abate, C.,

Ahn, N. G., Seger, R., Bratlien, R. L., Diltz, C. D., Tonka, N. K., and Krebs, E.

Cobb, M. H., Boulton, T. G., and Robbing, D. J. (1991) Cell Regul. 2,965-978 Cohen, S., Fava, R. A,, and Sawyer, S . T. (1982) Proc. Natl. Acad. Sci. U. S. A.

(1990) FEBS Lett. 273,223-226

Curran, T., and Davis, R. J. (1991) Cell 266,15277-15285

G. (1991) J. Biol. Chem. 286,4220-4227

79,62374241 Walton, G. M., Chen, W. S . , Rosenfeld, M. G., and Gill, G. N. (1990) J. Biol.

Hall, F. L., Braun, R. K., Mihara, K., Fung, Y.-K Berndt, N., Carbonaro-Hall, Chem. 266,1750-1754

Hanks, S. K., Quinn, A. M., and Hunter, T. (1988) Science 421,42-52 D. A., and Vulliet, P. R. (1991) J. Biol. Chem. 266, 17430-17440

Chen, W. S . , Lazar, C. S., Lund, K A,, Welsh, J. B., Chang, C.-P., Walton, G. M.,

33A3 Der, C. J., Wiley, H. S. , Gill, G. N., and Rosenfeld, M. G. (1989) Cell 59,

Wells, A,, Welsh, J. B., Lazar, C. S. , Wiley, H. S., Gill, G. N., and Rosenfeld, M.

Chang, C.-P., Kao, J. P. Y., Lazar, C. S., Walsh, B. J., Wells, A,, Wiley, H. S., Gill, G. (1990) Science 247,962-964

Bargmann, C. I., Hung, M.-C., and Weinberg, R.A. (1986)Nature 319,223-230 G. N., and Rosenfeld, M. G. (1991) J. Biol. Chem. 266,23467-23470

Plowman, G. D., Whitney, G. S. , Neubauer, M. G., Green, J. M., McDonald, V. L., Todaro, G. J., and Shoyab, M. (1990) Proc. Natl. Acad. Sei. U. S. A. 87, 4905-4909

Amian, R. V., Koga, M., Mendel, J. E., Ohshima,Y., and Sternberg, P. W. (1990)

Yarden, Y., Escobedo, J. A., Kuang, W.J., Yang-Feng, T. L., Daniel, T. O., Nature 348,693-699

Tremble, P. M., Chen, E. Y., Ando, M. E., Harkins, R. N., Francke, U., Fried,

Lee, P. L., Johnson, D. E., Cousens, L. S., Fried, V. A,, and Williams, L. T. (1989) V. A,, Ullrich, A,, and Williams, L. T. (1986) Nature 323, 226-232

Science 246,5740 Warren, G. (1989) Nature 342,857-858 Pypaert, M., Mundy, D., Souter, E., Labbe, J.C., and Warren, G. (1991) J. Cell

Hsu, S.-C., Qi. M., and DeFranco, D. B. (1992) EMBO J. 11,3457-3468 Biol. 114, 1159-1166

serine 1002 is a site of in vivo and in vitro phosphorylation of the

Documents