Vol. 186, No. 2, 1992

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

July 31, 1992

Pages

765-774

A HIGHLY CONSERVED TYROSINE RESIDUE AT CODON 845 WITHIN THE KJNASE DOMAIN IS NOT REQUIRED FOR THE TRANSFORMING ACTIVITY OF HUMAN EPIDERMAL GROWTH FACTOR RECEPTOR Noriko Gotoh, *-4Arinobu Tojo,l12Masayuki Hino, Yoshio Yazakidand Masabumi Shibuyal* De~nlzen&offGeneticsand2Nemato/ogy/Oncolo~ TheInstiWe ofMedicaf Science, UnivemityofTokyo, 4-6/ShJ>okaneda/; Minato-ku, Tokyo 108, Japan .'SecondDepament of/ntemalMedicJhe, SchoolofMedicJhe, Osaka City Unive&ty, /-57Asabimachd Abeno-ku, Osaka549 Japan 4Thz~dDep~entofIntemaiMedicine, FaculfyofMedic~~e, Univezxity of TokJJo, 7--P/Hongo,Bunkyo-ku, TokyoOO3, Japan Received

June

4,

1992

Epidermal growth factor receptor (EGF-R) is a widely expressedligand-dependent tyrosine kinase. The tyrosine residue at 845 in EGF-R correspondsto Y416 of v/c-src kinase, which is highly conservedand functionally important in many tyrosine kinases.To clarify the functional role of Y845, we constructed a mutant human EGF-R in which this tyrosine was replacedwith phenylalanine and transfectedit to NIH3T3 cells. EGF-R F845 inducedEGF-dependentcellular transformationand revealedtyrosine-autophosphorylationof a 170kDaprotein, and initiated DNA synthesissimilar to the wild-type EGF-R. We conclude herethat Y845 is dispensablein the above mentionedfunctions of EGF-R tyrosine kinase. 0 1992Academic Press,Inc.

Epidermal growth factor receptor (EGF-R) is a ligand-dependenttyrosine kinase which can deliver a mitogenic signal towards the nucleus through interaction with intracellular substrate proteins (1,2). In addition to substrate phosphorylation, EGF-R phosphorylatesits own tyrosine (Y) residuesupon EGF binding. This autophosphorylation stepis thought to be essentialfor regulating the tyrosine kinaseactivity of EGF-R (3,4,5) as well as for associationwith substrateproteins such as phospholipaseC-y (PLC-y) (6,7). It was reported that all the major sites of autophosphorylation are located near the carboxyl terminus of the receptor (8,9,10). These include the tyrosine residuesat 1068, 1148, and 1173 of the mature EGF-R protein. On the other hand, there is no evidence for I> vivo phosphorylation of the tyrosine residue 845 (Y845) in the catalytic domain, which correspondsto Y416 of the v/c-src kinase (1). This tyrosine residue is highly conserved ( 1,27) and often autophosphorylated; plays a crucial role for both enzymatic and biological activity, suchas in c-src (11,12), v-fps (13), insulin receptor (14), colony stimulating factor *To whom correspondenceshouldbe addressed. 0006-291X/92

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1-receptor (CSF-I R) ( 15,16) and platelet derived growth factor receptor (PDGF-R)

(17, 22).

To examine whether or not the role of Y845 is significant in the EGF-R-mediated signal transduction, we constructed a mutant EGF-R cDNA encoding the phenylalanine (F) residue at 845 (F845) and examined its ability to induce protein-tyrosine phosphorylation and to stimulate DNA synthesis upon EGF binding in the transfected NIH3T3 cells.

MATERIALS

AND

METHODS

Plasmids. The myeloproliferative sarcoma virus-based retroviral vector pTJNeo containing the neomycin-resistance gene, the internal cytomegalovirus (CMV) immediate/early promoter and the pUC19-derived origin of replication was used. A 3.8 kb SacI-BamHI (nuclotides 171-3970) fragment containing the complete human EGF-R coding sequences was inserted downstream of the CMV promoter of pTJNeo. We refer to this final construct as pTJNeo-EGF-R. Substitution of tyrosine with phenylalanine at codon 845 was done by introducing a point mutation using polymerase chain reaction (PCR) ( 18), after constructing pTJNeo-EGFR. Briefly, the cDNA sequence extending from nucleotide 2686 to 295 1 was amplified in partial reactions from two sets of primers, A to B and C to D. Primers A and D were complementary to the marginal sequences to be amplified and flanked by restriction sites for ApaLI and BglII, respectively; primers B (antisense strand, 5’CCTTCTGCATGGAATTCTTTCTC-3’) and C (sesnse strand, 5’GAGAAAGAATTCCATGCAGAAGG-3’) represented the sequences surrounding Y845 but directed phenylalanine at this position (underlined). PCR reaction was performed for 25 cycles. The products of the two reactions were then mixed and coampiified for additional 25 cycles using primers A and D. The amplified fragment was digested with ApaLI and BglII, following substitution with the corresponding sequences of the EGF-R cDNA in pTJNeoEGFR. Transfections and transforming assay. NM3T3 cells were grown in DMEM (GIBCO) with 5% heat-inactivated new born calf serum (NBCS). Subconfluent cells in 6 centimeter dishes (LUX) were transfected with 5kg of the plasmid DNA per dish by the polybrene-dimethyl sulfoxide method (19). Two days later, the cells were subjected to neomycin-resistant selection in the presence of O.Zmg/ml of G418 (GIBCO) with or without long/ml EGF. After two weeks, the number of G4 1B-resistant colonies and transformed foci were counted. Seveml colonies were picked up and screened for [‘251]-EGF binding. [1251]-EGF binding experiments. Cells were plated at a density of 5x104cells per well in 24 well dishes, and allowed to grow overnight. Prior to an assay, the cells were starved for 2.5 hr, then incubated with O.OS@i of [*251]-EGF (~750 Ci/mmol; Amersham) in DMEM containing 20mM HEPES (pH7.5) and 0. I %BSA. After incubation for 60 min at 4oC, the cells were washed two times with ice-cold PBS(-) and lysed in 0.2N NaOH for 30min at 37” C. The radioactivity in the lysate was measured in a y-counter. Nonspecific binding measured in the presence of a IOO-fold excess of unlabeled EGF was subtracted from total binding. [3H]-thymidine incorporation. Cells were seeded at a density of 2xl04cells per well in 24-well dishes, and then starved for 2 days in 0.5% NBCS. The cells were then incubated with various concentrations of EGF for additional 24 hr and pulsed with I@i of t3Hl-thymidine (250Ci/mmol;Arnersham) for the last 4 hr. The labeled cells were lysed with 0.2N NaOH . The radioactivity incorporated into trichloroacetic acid-insoluble precipitates was determined in a liquid scintillation counter. Immunoblot analysis. NM3T3 cells expressing either the wild-type or the F845 EGF-R as well as parental cells plated in 6 centimeter dishes were starved in DMEM with 0.5% NBCS overnight and then treated with or without lOOng/ml EGF. After 5 min incubation at 37oC, the cells were rapidly frozen in liquid nitrogen and solubilized in buffer containing 50mM HEPES (pH7.4), 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 1mM EGTA, 50mM sodium fluoride, 1OmM sodium pyrophosphate, 2mM sodium orthovanadate, 500 trypsin inhibitor unit/ml aprotinin, and 1mM phenylmethylsulfonyl fluoride. After centrifugation, the supematant was collected and assayed for protein concentration. Samples containing 25pg protein /lane were subjected to SDS-polyacrylamide gel electrophoresis and then transferred to Immobilon (Millipore). The membrane was blotted

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with either polyclonal anti-phosphotyrosine antibodies or anti-EGF-R antibodies, followed by labeling with [ 1251]-protein A (30mWmg;Amersham) and subjected to autoradiography.

RESULTS Y845 (wild type) of EGF-R was mutated to phenylalanine as described above (Fig. 1A F845). Fig.lA shows the structure of the constructs which were used in this study. The oligonucleotide-directed point mutation at codon 845 generated an EcoRl enzyme cutting site, which facilitated convenient selection of mutant clones. Fig. 1B shows the nucleotide sequences of the mutant EGF-R and the EcoRI digested patterns of the wild-type and mutant EGF-R cDNA. Transfection of NIH3T3 cells with both the wild type and mutant EGF-R, resulted in several tens of G418 resistant colonies per dish in two weeks. As previously reported (20,2 l), the wild type EGF-R has the ability to induce EGF-dependent

transforming

foci

(Table 1). Cells transfected with only pTJNeo (mock) do not show any tmnsformed foci, either in the presence or absence of EGF (long/ml). In the case of the wild type and mutant EGF-R, the ratio of the number of transformed foci divided by the number of G4 18 resistant colonies amounted to less than 1% in the absence of EGF. This percentage was significantly

1

Y845

N-l Ligand

F845

I

1

f

/I

I w-1-c Binding

Domain

TM

Kinase Domain

C-terminal region

,I

m Construction of the human Y845 mutant EGF-R.(A) The structure of the coding region of EGF-R, wild type Y845 and F845; wild type human EGF-receptor drawn with extracellular domain (Ligand binding domain), transmembrane domain (TM) and kinase domain. Tyrosine residues for autophosphorylation (992, 1068, 1148, 1173) are indicated as (P). F845 carries a point mutation in which a single tyrosine (Y) at 845 was exchanged into phenylalanine (F). (B) DNA sequence of F845 and EcoRI-digested patterns of wild type Y845 and F845. 770

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TabIe

1. Transforming

AND

activity

BIOPHYSICAL

of Y845

Transformed cDNA

EGF

Y845

Exp.1

and F845

foci/C418-resistant Exp.3

COMMUNICATIONS

EGF-R

colonies

Total

%

-

O/l 6

v53

af77

l/146

0.6

i-

z/40

U61.5

Y69

V170.5

4.1

l/27

O/48

a/68

l/l43

0.7

Y12.5

12.5/142.5

8.8

1345

Mock

Exp.2

RESEARCH

+

605

-

O/56

o/101

Q/94

O/251

0

+

Of38

O/l 12

w109

0,059

0

3.5/35

NIH3T3 cells were transfected with 5Fg of the plasmid DNA per dish by the polybrenedimethyl sulfoxide method. The transfected cells were grown in the presence of 0.2mgIml of G418 with or without lOng/ml EGF. After two weeks the number of G418 resistant colonies and transformed foci were counted, respectively. Three independent experiments were done. The results indicated the mean of the two transfections in Exp.2 and 3.

increased

by EGF treatment

in the case of the wild

type (4.1%)

and the mutant

receptor(8.8%). Next, several transformed foci were picked up and propagated for further analysis. As previously reported (20), parental NIH3T3 cells did not show any detectable EGF binding sites (Fig.2A). Hence NIH3T3 cells expressing the wild-type and mutant EGF-R contained significant amounts of EGF binding sites-shown by [ ‘25I]-EGF specific binding (Fig.2A) and the 17OkDa EGF-R

protein-identified

by immunoblotting

(Fig.2B).

Binding studies

revealed that the mutant EGF-R had about 3-fold more binding sites than the wild-type (Fig.2A). Though EGF rapidly stimulate tyrosine phosphorylation of both the wild type and the mutant (Fig. 2C), the level of intensity is higher in the mutant correlating with the 3-fold higher level of expression of the mutant when compared to that of the wild type receptor. We compared the capacity of these cells to incorporate [3H]-thymidine in response to various concentration of EGF (Fig.3). Dose response curves were similar in the wild-type and F845 EGF-R. Maximal level of stimulation was about 1.5-times higher in the mutant EGF-R than in the wild-type of receptors.

EGF-R, correlating with the difference in the expression level

DISCUSSION In EGF-R tyrosine kinase, all the major autophosphorylation

sites are located at the

C-terminal end of the receptor molecule (8,9,10). The possible role of autophosphorylation of these sites reported SO far are still controvertial. Replacement of each tyrosine residue with phenylalanine showed no critical effect and exhibit rather low Km for exogenous substrate phosphorylation (4,5,28). In unphosphorylated form, these sites appear to serve as 771

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A

--+

--+

-+ -

-

205kDa

205kDa

- EGF-R -

116.5

-

80

0

80

Y845 F&l5 Parent

Ii&L Expression and tyrosine phosphorylatin of the wild type Y845 and F845 EGF-R. (A) [‘25I]-EGF binding study. Wild type or F845 EGF-R plasmidtransfected ceils were seeded and incubated overnight. Then the cells were cultivated in serum-free medium for 2.5hr, and then incubated with [~251]-EGF at 4oC for lh. The radioactivity bounds to cells was counted, subtracted with nonspecific binding, and divided by total radioactivity. The results are the meat&D of tripricates. (B) Western blotting with polyclonal anti-EGF-R antibodies. NIH3T3 cells expressing the wild type and F845 EGF-R were lysed, 25Fg of each sample protein was separated by SDS-polyacrylamide gel electrophoresis. The sample was transferred to Immobilon (Milipore) and mixed with polyclonal anti-EGF-R antibodies, then reacted with [124]-protein A. (C) Tyrosine phosphorylation of EGF-R in living cells. Quiescent cells were treated with or without lOOng/ml EGF. After Smin incubation at 37OC, cells were lysed and 25pg of each sample protein was analyzed by Western blotting using polyclonal anti- phosphotyrosine antibodies.

J&& Stimulation of [sH]-thymidine incorporation by EGF. Quiescent cells were incubated with various concentration of EGF for 24hr and pulsed with [sH]-thymidine for last 4hr (see Materials and Methods ). The results of Y845 and F845 are the meantSD of tripricates.The result of Parent is the mean of duplicate. Y axis indicates stimulation index. 772

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competitive/alternate substrates(4,5,28). In phosphorylatedform, thesesitesare required to exhibit maximal biological activity (23,24,25,26). It is still possiblehowever, that critical autophosphorylation is occurring at sevral other sitesin the EGF-R, but that phosphorylation of thesesitesare rapidly being clearedby tyrosyl-phosphatasesmaking detection very difficult (9). Y845 of the EGF-R was thought to be one such site. But in the present study, we clearly demonstratethat the F845 mutant EGF-R

retains its ability to mediate EGF-dependent morphological transformation and

mitogenic responsein NIH3T3 cells. Therefore, Y845 appearesto play no crucial role in EGF-R. Moreover, the mutant EGF-R appearsto have rather potent transforming activity when comparedto the wild-type EGF-R, since the transforming activity was about 2-times higher. This result provides an interesting contrast to that of other tyrosine kinases. Replacementof the Y845 counterpart with phenylalanineresulted in the reduction of both tyrosine kinase activity and biological responsesin the case of Y4 16 of c-src (11, 12), Y 1162/‘Y1163of the human insulin receptor (14) and Y807 of the murine CSF-IR (16). In the caseof Y809 of the human CSF-1R (15) and Y825 of the PDGF-R (17,22), similar mutation resultedin the impairmentof mitogenic responses without affecting kinaseactivity. In the latter cases,the substitutedtyrosine residuesmight be involved in the interaction with substrateproteins. The presentdata showsthe heterogeneousroles of the highly-conserved tyrosine residuesamongvarious tyrosine kinases.

ACKNOWLEDGMENTS We thank Dr. M. Hamaguchi for providing anti-phosphotyrosineantibodies, Dr. T. Akiyama for providing anti-EGF-R antibodies, Mrs. Lata Seetharam for reviewing our manuscript andMrs. S. Yamaguchi for excellent technicalassistance. This work was supportedby Grant-in-Aid for Special Project Researchon CancerBioscience 04253204 from the Ministry of Education, Science and Culture of Japan, a resarchgrant from the PrincessTakamatsuCancerResearchFund, and a researchgrant from the Foundation for Promotion of CancerResearchin Japan.

REFERENCES

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

Yarden, Y., and Ullrich, A. (1988) Ann. Rev. Biochem. 57,443-477 Ullrich, A., and Schlessinger,J. (1990) Cell. 61, 203-212. Bertics, P. J., and Gill, G. N. (1985) J. Biol. Chem. 260, 14642-14647. Bertics, P. J., Chen, W. S., Hubler, L., Lazar, C. S., Rosenfeld, M. G., and Gill, G. N. (1988) J. Biof. Chem. 263, 3610-3617. Honegger, A., Dull, T. J., Szapary, D., Komoriya, A., Kris, R., Ullrich, A., and Schlessinger,J. (1988) EMBO J. 7, 3053-3060. Margolis, B., Li, N., Koch, A., Mohammadi, M., Hurwitz, D. R., Ziberstein, A., Ullrich, A., Pawson,T., and Schlessinger,J. (1990) EMBO J. 9,4375-4380. Rotin, D., Margolos, B., Mohammadi, M., Daly, R.J., Daum, G., Li, N., Fischer, E.H., Burgess,W.H., Ullrich, A., and Schlessinger,J. (1992) EMBO J. 11, 559-567. Downward, J., Parker, P., and Waterfield, M.D. ( 1984)Nature. 3 11, 483-485. Margolis, B.L., Lax, I., Kris, R., Dombalagian, M., Honegger,A.M., Howk, R., Givol, D., Ullrich, A., and Schlessinger,J. (1989) J. Biol. Chem. 264. 10667-10671. 773

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10. Walton, G.M., Chen, W.S., Rosenfeld, M.G., and Gill, G.N. (1990) J. Biol. Chem. 265, 1750-1754. 11. Piwnica-Worms, H., Saunders,K,B, Roberts, T.M., Smith, A.E., and Cheng, S.H. (1987) Cell. 49, 75-82. Kmiecik, T.E., and Shalloway, D. (1987) Cell. 49, 65-73. ii: Weinmaster, G., Zoller, M-J., Smith, M., Hinze, E., and Pawson,T. (1984) Cell. 37, 559-568. 14. Ellis, L., Clauser,E., Morgan, D.O., Edery, M., Roth, R.A., and Rutter, W.J. (1986) Cell. 45, 721-732. 15. Roussel,M.F., Shurtleff, S.A., Downing, J.R., and Sherr, C.J. (1990) Proc. Natl. Acad. Sci. USA. 87, 6738-6742. Geer, P., and Hunter, T. (1991) Mol. Cell. Biol. 11, 4698-4709. :;: Fantl, W.J., Escobedo,J.A., and Williams, L.T. (1989) Mol. Cell. Biol. 9,4473-4478. 18. Ho, S.N., Hunt, H.D., Horton, R.M., Pullen, J.K., and Pease,L.R. (1989) Gene. 77, 51-59. Kawai, S., and Nishizawa, M. (1984) Mol. Cell. Biol. 4, 1172-l 174. E: Fiore, P.P.D., Pierce, J.H., Fleming, T.P., Hazan, R., Ullrich, A., King, C. R., Schlessinger,J., and Aaronson, S.A. (1987) Cell. 51, 1063-1070. 21. Velu, T.J., Beguinot, L., Vass, WC., Willingham, M.C., Merlino, G.T., Pastan,I., and Lowy, D.R. (1987) Science. 238, 1408 1410. 22. Morrison, D.K., Kaplan, D.R., Rhee, S.G., and Williams, L.T. (1990) Mol. Cell. Biol. 10, 2359-2366. 23. Velu, T.J., Vass, W.C., Lowy, D.R., and Beguinot, L. (1989) Mol. Cell. Biol. 9, 1772-1778. 24. Chen, W.S., Lazar, C.S., Lund, K.A., Weish, J.B., Chang, C.P., Walton, G.M., Der, C.J., Wiley, H.S., Gill, G.N., and Rosenfeld,M.G. (1989) Cell. 59, 33-43. 25. Helin, K., Velu, T., Martin, P., Vass, W.C., Allevato, G., Lowy, D.R., and Beguinot, L. (1991) Oncogene, 6, 825-832. 26. So&in, A., Helin, K., Warers, C.M., Carpenter, G., and Beguinot, L. (1992) J. Biol. Chem. 267, 8672-8678. 27. Hanks, S.K., Quinn, A.M., and Hunter, T. (1988) Science. 241, 42-52. 28. Honegger, A., Dull, T.J., Bellot, F., Obberghen, E. V., Szapary, D., Schmidt, A., Ullrich, A., and Schlessinger,J. (1988) EMBO J. 7, 3045-3052.

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A highly conserved tyrosine residue at codon 845 within the kinase domain is not required for the transforming activity of human epidermal growth factor receptor.

Epidermal growth factor receptor (EGF-R) is a widely expressed ligand-dependent tyrosine kinase. The tyrosine residue at 845 in EGF-R corresponds to Y...
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