Vol. 180, No. 3, 1991 November 14, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages ]490-]497
Highly conserved eight amino acid sequence in SH2 is important for recognition of phosphotyrosine site Michihiro Hidaka, Yoshimi Homma and Tadaomi Takenawa* Department of Biosignal Research, Tokyo Metropolitan Institute of Gerontology, 35-2, Sakae-cho, Itabashi-ku, Tokyo 173 Japan Received October ii, 1991
SUMMARY:Src homology region 2(SH2) has been demonstrated to recognize phosphotyrosine site. To clarify the precise mechanism of the recognition, we developed in vitro binding assay system using EGF receptor and SH2/SH3 region of phospholipase C(PLC) y1. Phosphorylated EGF receptor bound to immobilized SH2/SH3 of PLC y1 in Sepharose beads, while nonphosphorylated EGF receptor did not bind. In SH2 domain of PLC y1, there are several highly conserved amino acid sequences that are common in a variety of SH2-containing proteins. Especially the eight amino acid sequence, G(S/T)FLVR(E/D)S is highly conserved in these proteins. We synthesized several peptides related to these sequences and examined the effect of peptides on the binding of EGF receptor to SH2 of PLC y1. P1, GSFLVRES was the most effective inhibitor to suppress the binding. P2, GSFLVAES in which one amino acid, arginine of P1 is substituted by alanine is still effective. But a peptide, P3, SFLVRE in which two amino acids are deleted from P1 did not inhibit markedly. Moreover, P1 peptide immobilized in Sepharose beads also bound phosphorylated EGF receptor. These data suggest that highly conserved amino acid sequence GSFLVRES is the minimum essential unit to recognize tyrosine phosphorylated site. ~ 1991 A c a d e m i c P . . . . . I n c .
Src homology region, SH2 and SH3 have been found in a variety of proteins including PLC y , ras GAP, PI 3-kinase 85 K subunit and Crk besides Src family tyrosine kinases(reviewed in ref. 1). Concerning the role of SH2 domain, direct evidence was first obtained when oncogene v - c r k was found to encode a protein primarily consisting of viral gag protein fused with SH2 and SH3 domain(2). In p47gag -crk ¢¢
To whom correspondence should be addressed. 0006-291X/91
$1.50
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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transformed cells, an endogenous tyrosine kinase activity was enhanced, even though p47gag -crk itself was not a tyrosine kinase(3). It has also been shown that p47gag -crk binds to phosphotyrosine-containing proteins in these transformed cells(4). Subsequently, many investigators have demonstrated that SH2 domains in a variety of proteins also recognize peptide sequence that encompasses tyrosine phosphorylation site(5-9). The mitogenic effect of growth factors such as epidermal growth factor(EGF) or platelet derived growth factor (PDGF) absolutely requires the tyrosine kinase activity. In this tyrosine kinase-signaling system, it has been found that SH2 domain-containing proteins such as PLC ~,, rasGAP and PI 3-kinase bind the phosphorylation site of receptor tyrosine kinase in response to PDGF or EGF, thereby mediating the formation of protein complexes. The formation of these complexes appears to activate SH2-containing proteins, which induces new signals. Thus, it has been considered that SH2 domain plays crucial role in the signaling system of growth factors. In this paper, to clarify the essential amino acid sequence in SH2 for recognition of phosphotyrosine, we synthesized several peptides related to the conserved sequences that are common in a variety of SH2containing proteins and examined the effect of peptides on the binding of EGF receptor to SH2 of PLC ~(1. The eight amino acid sequence, GSFLVRES was found to be most effective inhibitor, suggesting that this sequence plays an important role in the recognition of phosphotyrosine site.
Experimental Procedures
Materials--Anti-phosphotyrosine antibody(PY 20) was purchased from ICN Immunobiologicals. Anti-EGF receptor antibody(Ab-1) was from Oncogene Science Inc. SH2/SH3 domain of PLC yl(amino acid 517901) was prepared as described before(10). Affigel 10 was from BioRad Labo. CNBr-activated Sepharose 4B was from Pharmacia Fine Chemicals. Synthesis and Purification ofPeptides-- Peptides used in the binding inhibition experiments were prepared by 430 A peptide synthesizer(Applied Biosystems) using t-butoxycarbonyl-protected amino acids. The peptides were purified by high performance liquid chromatography on a C18 reverse phase column in 0.1% trifluoroacetic acid by using a linear gradient of acetonitril over 60 min. 1491
Vol. 180, No. 3, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Preparation of SH2/SH3-Sepharose--SH2/SH3 domain of phospholipase Cy1 was expressed and produced in E. coli(10). The SH2/SH3 proteins were solubilized with the buffer consisting of 0.5 % sodium dodecyl suffate(SDS), 1 0 % glycerol and 50 mM Hepes/NaOH(pH, 6.8). The proteins were covalently bound to activated CNBr-Sepharose (Pharmacia). This beads contained 0.5 nmol SH2/SH3 in 15 ~tl. Preparation of P1 peptide-Agarose --P1 peptide was coupled to Affigel 10 as follows: P1 peptide (5 rag) was mixed with 2 ml of Affigel 10 beads in 10 mM Hepes/NaOH buffer(pH, 7.4) for 2 h at room temperature. The reaction was stopped with the addition of 0.1 ml of 1 M ethanolamine and then washed with 1 M NaC1. The Pl-beads were stored in 4 °C untill use. Binding assay of EGF receptor to SH2/SH3-Sepharose--For EGF receptor, shed membranes from A431 cells were used. The shed membranes were prepared according to the method previously described(11 ). The membranes were incubated with 20 l.tM ATP or 20 ktM [y-32p]ATP in the phosphorylation buffer in a total volume of 50 I.tl( 20 mM Hepes/NaOH (pH. 7.5) containing 25 mM MgC12, 4 mM MnC12 and 0.1 mM sodium vanadate) for 5 min at 30 °C. The reaction was stopped by the addition of ice-cold 50 mM Hepes/NaOH(pH, 7.4), 150 mM NaC1, 10 % glycerol, 1% Triton X-100 and 200 ~M sodium vanadate. The mixture was sonicated for 10 s and then centrifuged at 20,000 X g for 10 min. The supematants were mixed with 50 ~tl of SH2/SH3-Sepharose or Pl-agarose, and then shaken for 2 h at 4 °C. The beads were washed 5 times with RIPA buffer (20 mM Hepes/NaOH(pH, 7.4), 150 mM NaC1, 10 % glycerol, 1% Triton X100, 0.1% SDS and 1% sodium deoxycholate ). The radioactivity retained in the beads was counted. SDS gel electrophoresis and Western blotting--The proteins retained in SH2/SH3-Sepharose were resolved by SDS-polyacrylamide gel electrophoresis. The gels were electroblotted on nylon membranes(Clear Membrane P, ATTO, Tokyo), and the blots were blocked with 5 % skim milk in phosphate-buffered saline and incubated with EGF receptor antibody. The membrane was stained with alkaline phosphate conjugated anti-mouse IgG antibody. Results and Discussion
In SH2 domain, there are several highly conserved amino acid sequences that are common in many SH2-containing proteins.(Fig. 1A). Especially, the eight amino acid sequence G(S/T)FLVR(E/D)S is common to c-Src, c-Abl, PLCy1, PLCT2, v-Crk and PI 3-kinase 85 K subunit and WFHGK is identical in PLCT1 and GAP. Considering that these G(S/T)FLVR(E/D)S is very conservative among SH2- containing protein, this part seems to be important to recognize phosphotyrosine site. It is very interesting to clarify the precise mechanism of 1492
V o l . 180, No. 3, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A PLC 71 N PLC 71 C PLC ~2 N PLC "(2 C GAP N GAP C PI3KN PI3KC v-SRC v-CRK
la
TEYO,ETGAPOOSF , VRSETV__00
ASLT VES Y YDR L S
AQ TS E
S
KQ EE
T LS
RE GD
DIS
HM M RVPDG~IFLMR EP K Q E Y C A E m GA KID q i ~ F L VR E S EIT F P IA~EED~RM~IM . , P R D LAs~L MR K~L~sN E P
EIAIY NLL. LIM T V G Q A C VNEK RDTA S
R S
LNPEN QGQRH
SFLVR FLVR PR
N NDIYTLS I N sS~F~JL~sS
SDNTP GD STKMHG
FLVRESETTG FLVR .. Sl
P
KA G
PLC y1 SH2 N SH2 C SH3
X
H
H
H
/ P4:WFHGK
Y
H P5:YPINEEALE
P1 :GSFLVRES P2:GSFLVAES P3:SFLVAE
Fig.1. Conservative sequences in SH2 domain. A. Alignment of the SH2 domain of various proteins is done. Highly conserved residues are boxed and homologous amino acids are shaded. The suffix N indicates the more N-terminal SH2 domain of PLC 7, GAP or PI 3 kinase 85 K subunit, whereas C indicates the more C-terminal domain. B. Location of synthetic peptides in PLC 71 amino acid sequence. recognition of phosphotyrosine site by SH2 domain. Therefore, we synthesized several peptides related to the sequence and examined the effect of peptides on the binding of EGF receptor to SH2 of PLCT1. We synthesized 5 peptides(FiglB) according to the amino acid sequence of PLC 71. P1 was a peptide, GSFLVRES which is highly conserved in SH2. P2 is a peptide, GSFLVAES in which one amino acid, arginine of P1 is substituted by alanine. P3 is a peptide, SFLVRE in which two amino acids are deleted from P1. P4 is a peptide, WYHGK which is another conserved sequence in SH2. P5 is a peptide, of which sequence is not included in SH2 region. The association of PLC 71 and EGF receptor has already demonstrated by stimulating A431 cells with EGF and immunoprecipitating the cell lysates with EGF receptor antibody(l, 12,13). Therefore, We developed in vitro binding assay system to detect the association between SH2 of PLC 71 and phosphorylated EGF receptor. The protein containing SH2 regions of PLC 71( amino acid 517-901 ) was produced in E. coli expression system. The protein was coupled to Sepharose beads (Pharmacia CNBr-Sepharose). The immobilized SH2/SH3 was incubated with EGF receptor from A431 cells and the proteins bound 1493
Y
Vol. 180, No. 3, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
c~ EGF Receptor Ab
1
Autoradiography
2
3
_Fig. 2. Binding of EGF receptor to the SH2/SH3 of PLC T1. The shed membranes from A431 cells were phosphorylated(lane 1) or not phosphorylated(lane 2) in vitro and then solubilized. Binding of EGF receptor to SH2/SH3-Sepharose was examined as described in Experimental Procedures. Western blot analysis was done using antiEGF receptor antibody(lane 1 and 2). Autoradiography of the 32p_ labeled proteins retained in SH2/SH3-Sepharose was shown in lane 3. to beads were analyzed(Fig. 2). Immunoblot analysis showed that only phosphorylated EGF receptor could bind to the SH2/SH3-Sepharose without any significant binding of unphosphorylated EGF receptor. Moreover, in this system, it was found that phosphorylated EGF receptor was an only protein detected by autoradiography, suggesting that most of 32p bound to the beads are due to the 32p-EGF receptor. Using this binding method, We examined the inhibitory effect of synthesized peptides on the binding of EGF receptor to SH2/SH3 region of PLCy1. As indicated in Table 1, P1 was the most effective inhibitor for the binding. P2 was a moderate inhibitor and P4 was less effective than P1 or P2. P3 and P5 did not affect the binding markedly even when 1000-fold higher concentrations of peptide were used compared to those of immobilized SH2/SH3. These results suggest that highly conserved eight amino acid sequence is an important site for recognition of phosphorylated tyrosine. Concerning P1, P2 and P4, dose-dependent inhibition curve was determined(Fig 3). Similarly, P1 was the strongest inhibitor. It inhibited the binding by 50 % with 10-fold higher concentrations than immobilized SH2. P2 and P4 were also effective to suppress the binding. P2 was stronger than P4, suggesting that GSFLVRES in SH2 is important for recognition of phosphotyrosine 1494
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Table 1. Inhibitory effect of various peptides on binding of EGF-receptor to SH2/SH3 regions Bound EGF-receptor (% of Control) Concentration (n-fold)
P1
P2
P3
P4
P5
0
100
100
100
100
100
10
54
55
90
86
95
100
27
42
84
62
88
1000
23
33
72
40
70
EGF-receptors32 in shed membranes were autophosphorylated with" 20 ~tM [T- P]ATP(1 I.tCi) in 50 ~1 of phosphorylation buffer for 5 min at 37"C and then 50 ~tl of lysis buffer was added. The mixture was centrifuged at 10000 X g for 10 min. The supematant was added to 50 ~1 of SH2/SH3-beads in the presence or absence of peptides, shaken for 2 h, and then washed with RIPA buffer. The radioactivity retained in the beads was counted. Concentration is indicated as n-fold to that of immobilized SH2/SH3.
and the substitution of arginine in P1 by alanine does not afford a critical damage to the recognition. Even a penta peptide, WYHGK remains a activity though its potency is fairly low. Next, we examined
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.
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.
.
,
•
.
40
,
60
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.
,
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.
,
100
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.
120
Peptide (Concentration, n-fold)
Fig. 3. D o s e - d e p e n d e n c e curve of the inhibition of peptides on the b i n d i n g of E G F receptor. Inhibitory effect of P1, P2 and P4 on the b i n d i n g of E G F receptor to SH2/SH3 was e x a m i n e d as described in Table 1. Concentration is indicated as n-fold to that of i m m o b i l i z e d SH2/SH3 of PLC T1. 1495
Vol. 180, No. 3, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
200 -
ii!ii!iiiiii',;~i ' iili~lilli~iliiiiii
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:::::::::::::::::::
I
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2 Fig. 4. Eight amino acid peptide, GSFLVRES can bind to phosphorylated EGF receptor. Pl-agarose(50 Ixl) was incubated with [32p]phosphorylated EGF receptor in the presence (lane 2) or absence(lane 1) of 500 laM P 1, and then washed 5 times with phosphate-buffered saline containing 0.1% Tween 20. The proteins retained in Pl-agarose was analyzed with SDS-polyacrylamide gel electrophoresis and autoradiography.
whether the peptide P1 could bind EGF receptor or not. P1 peptide was covalently coupled to Affigel 10(Biorad). The beads were incubated with solubilized membranes of A431 cells after membranes were labeled with [7-32p]ATP. As shown in Fig. 4. EGF receptor bound to P1 peptide-beads though its binding affinity appears to be much weak. When the beads were washed vigorously with the buffer( 20 mM Hepes/NaOH, pH, 7.4 containing 0.1% SDS, 1% Triton X-100, 1% sodium deoxyclolate, 150 mM NaC1 and 10 % glycerol), the binding was diminished. All these data show that eight amino acid sequence, GSFLVRES is minimum essential sequence for recognition of phosphotyrosine site. References 1. Koch, C. A., Anderson, D., Moran, M. F., Ellis, C. and Pawson, M. F. (1991) Science 252, 668-674 2. Mayer, B. j., Hamaguchi, M. and Hanafusa, H. (1988) Nature 332, 272-275 3. Mayer, B. J. and Hanafusa, H. (1990) Proc. Natl. Acad. Sci. USA 87, 2638-2642 4. Matsuda, M., Mayer, B. J., Fukui, Y. and Hanafusa, H. (1990) Science 248, 1537-1539 1496
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
5. Anderson, D., Koch, C. A., Grey, L., Ellis, C., Morran, H. F. and Pawson, T. (1990) Science 250, 979-985 6. Morran, M. F., Koch, C. A., Anderson, D., Ellis, C., England, L., Martin, C. S. and Pawson, T. (1990) Proc. Natl. Acad. Sci. USA 87, 8622-8626 7. Margolis, B., Li, N., Koch, A., Mohammadi, M., Hurwitz, D. R., Ziberstein, A., Ullich, A., Pawson, T., A, D. Sclessinger, J. (1990) EMBO J. 9, 4375-4380 8. Morrison, D. E., Kaplan, D., Rhee, S. G. and Williams, L. (1990) Mol. Cell. Biol. 10, 2359-2366 9. Cantly, E. C., Auger, K. R., Carpenter, C., Duckworth, B., Graziani, A., Kapeller, R. and Soltoff, S. (1991) Cell 64, 281-302 10. Emori, Y. Homma, Y., Sorimachi, H., Kawasaki, H., Nakanishi, O., Suzuki, K. and Takenawa, T. (1989) J. Biol. Chem. 264, 21885-21890 11. Choen, S., Ushiro, H., Stoscheck, L. and Chinkers, M. (1982) J. Biol. Chem. 10, 1523-1531 12. Wahl, M., Daniel, T. O. Carpenter, G. (1988) Science 241, 968-970 13. Margolis, B., Bellot, F., Honegger, A. M., Ullich, A., Schlessinger, J. and Ziberstein, A. (1990) Mol. Cell. Biol. 10, 435-441
1497
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Highly conserved eight amino acid sequence in SH2 is important for recognition of phosphotyrosine site Michihiro Hidaka, Yoshimi Homma and Tadaomi Takenawa* Department of Biosignal Research, Tokyo Metropolitan Institute of Gerontology, 35-2, Sakae-cho, Itabashi-ku, Tokyo 173 Japan Received October ii, 1991
SUMMARY:Src homology region 2(SH2) has been demonstrated to recognize phosphotyrosine site. To clarify the precise mechanism of the recognition, we developed in vitro binding assay system using EGF receptor and SH2/SH3 region of phospholipase C(PLC) y1. Phosphorylated EGF receptor bound to immobilized SH2/SH3 of PLC y1 in Sepharose beads, while nonphosphorylated EGF receptor did not bind. In SH2 domain of PLC y1, there are several highly conserved amino acid sequences that are common in a variety of SH2-containing proteins. Especially the eight amino acid sequence, G(S/T)FLVR(E/D)S is highly conserved in these proteins. We synthesized several peptides related to these sequences and examined the effect of peptides on the binding of EGF receptor to SH2 of PLC y1. P1, GSFLVRES was the most effective inhibitor to suppress the binding. P2, GSFLVAES in which one amino acid, arginine of P1 is substituted by alanine is still effective. But a peptide, P3, SFLVRE in which two amino acids are deleted from P1 did not inhibit markedly. Moreover, P1 peptide immobilized in Sepharose beads also bound phosphorylated EGF receptor. These data suggest that highly conserved amino acid sequence GSFLVRES is the minimum essential unit to recognize tyrosine phosphorylated site. ~ 1991 A c a d e m i c P . . . . . I n c .
Src homology region, SH2 and SH3 have been found in a variety of proteins including PLC y , ras GAP, PI 3-kinase 85 K subunit and Crk besides Src family tyrosine kinases(reviewed in ref. 1). Concerning the role of SH2 domain, direct evidence was first obtained when oncogene v - c r k was found to encode a protein primarily consisting of viral gag protein fused with SH2 and SH3 domain(2). In p47gag -crk ¢¢
To whom correspondence should be addressed. 0006-291X/91
$1.50
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
1490
Vol. 180, No. 3, 1991
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transformed cells, an endogenous tyrosine kinase activity was enhanced, even though p47gag -crk itself was not a tyrosine kinase(3). It has also been shown that p47gag -crk binds to phosphotyrosine-containing proteins in these transformed cells(4). Subsequently, many investigators have demonstrated that SH2 domains in a variety of proteins also recognize peptide sequence that encompasses tyrosine phosphorylation site(5-9). The mitogenic effect of growth factors such as epidermal growth factor(EGF) or platelet derived growth factor (PDGF) absolutely requires the tyrosine kinase activity. In this tyrosine kinase-signaling system, it has been found that SH2 domain-containing proteins such as PLC ~,, rasGAP and PI 3-kinase bind the phosphorylation site of receptor tyrosine kinase in response to PDGF or EGF, thereby mediating the formation of protein complexes. The formation of these complexes appears to activate SH2-containing proteins, which induces new signals. Thus, it has been considered that SH2 domain plays crucial role in the signaling system of growth factors. In this paper, to clarify the essential amino acid sequence in SH2 for recognition of phosphotyrosine, we synthesized several peptides related to the conserved sequences that are common in a variety of SH2containing proteins and examined the effect of peptides on the binding of EGF receptor to SH2 of PLC ~(1. The eight amino acid sequence, GSFLVRES was found to be most effective inhibitor, suggesting that this sequence plays an important role in the recognition of phosphotyrosine site.
Experimental Procedures
Materials--Anti-phosphotyrosine antibody(PY 20) was purchased from ICN Immunobiologicals. Anti-EGF receptor antibody(Ab-1) was from Oncogene Science Inc. SH2/SH3 domain of PLC yl(amino acid 517901) was prepared as described before(10). Affigel 10 was from BioRad Labo. CNBr-activated Sepharose 4B was from Pharmacia Fine Chemicals. Synthesis and Purification ofPeptides-- Peptides used in the binding inhibition experiments were prepared by 430 A peptide synthesizer(Applied Biosystems) using t-butoxycarbonyl-protected amino acids. The peptides were purified by high performance liquid chromatography on a C18 reverse phase column in 0.1% trifluoroacetic acid by using a linear gradient of acetonitril over 60 min. 1491
Vol. 180, No. 3, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Preparation of SH2/SH3-Sepharose--SH2/SH3 domain of phospholipase Cy1 was expressed and produced in E. coli(10). The SH2/SH3 proteins were solubilized with the buffer consisting of 0.5 % sodium dodecyl suffate(SDS), 1 0 % glycerol and 50 mM Hepes/NaOH(pH, 6.8). The proteins were covalently bound to activated CNBr-Sepharose (Pharmacia). This beads contained 0.5 nmol SH2/SH3 in 15 ~tl. Preparation of P1 peptide-Agarose --P1 peptide was coupled to Affigel 10 as follows: P1 peptide (5 rag) was mixed with 2 ml of Affigel 10 beads in 10 mM Hepes/NaOH buffer(pH, 7.4) for 2 h at room temperature. The reaction was stopped with the addition of 0.1 ml of 1 M ethanolamine and then washed with 1 M NaC1. The Pl-beads were stored in 4 °C untill use. Binding assay of EGF receptor to SH2/SH3-Sepharose--For EGF receptor, shed membranes from A431 cells were used. The shed membranes were prepared according to the method previously described(11 ). The membranes were incubated with 20 l.tM ATP or 20 ktM [y-32p]ATP in the phosphorylation buffer in a total volume of 50 I.tl( 20 mM Hepes/NaOH (pH. 7.5) containing 25 mM MgC12, 4 mM MnC12 and 0.1 mM sodium vanadate) for 5 min at 30 °C. The reaction was stopped by the addition of ice-cold 50 mM Hepes/NaOH(pH, 7.4), 150 mM NaC1, 10 % glycerol, 1% Triton X-100 and 200 ~M sodium vanadate. The mixture was sonicated for 10 s and then centrifuged at 20,000 X g for 10 min. The supematants were mixed with 50 ~tl of SH2/SH3-Sepharose or Pl-agarose, and then shaken for 2 h at 4 °C. The beads were washed 5 times with RIPA buffer (20 mM Hepes/NaOH(pH, 7.4), 150 mM NaC1, 10 % glycerol, 1% Triton X100, 0.1% SDS and 1% sodium deoxycholate ). The radioactivity retained in the beads was counted. SDS gel electrophoresis and Western blotting--The proteins retained in SH2/SH3-Sepharose were resolved by SDS-polyacrylamide gel electrophoresis. The gels were electroblotted on nylon membranes(Clear Membrane P, ATTO, Tokyo), and the blots were blocked with 5 % skim milk in phosphate-buffered saline and incubated with EGF receptor antibody. The membrane was stained with alkaline phosphate conjugated anti-mouse IgG antibody. Results and Discussion
In SH2 domain, there are several highly conserved amino acid sequences that are common in many SH2-containing proteins.(Fig. 1A). Especially, the eight amino acid sequence G(S/T)FLVR(E/D)S is common to c-Src, c-Abl, PLCy1, PLCT2, v-Crk and PI 3-kinase 85 K subunit and WFHGK is identical in PLCT1 and GAP. Considering that these G(S/T)FLVR(E/D)S is very conservative among SH2- containing protein, this part seems to be important to recognize phosphotyrosine site. It is very interesting to clarify the precise mechanism of 1492
V o l . 180, No. 3, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A PLC 71 N PLC 71 C PLC ~2 N PLC "(2 C GAP N GAP C PI3KN PI3KC v-SRC v-CRK
la
TEYO,ETGAPOOSF , VRSETV__00
ASLT VES Y YDR L S
AQ TS E
S
KQ EE
T LS
RE GD
DIS
HM M RVPDG~IFLMR EP K Q E Y C A E m GA KID q i ~ F L VR E S EIT F P IA~EED~RM~IM . , P R D LAs~L MR K~L~sN E P
EIAIY NLL. LIM T V G Q A C VNEK RDTA S
R S
LNPEN QGQRH
SFLVR FLVR PR
N NDIYTLS I N sS~F~JL~sS
SDNTP GD STKMHG
FLVRESETTG FLVR .. Sl
P
KA G
PLC y1 SH2 N SH2 C SH3
X
H
H
H
/ P4:WFHGK
Y
H P5:YPINEEALE
P1 :GSFLVRES P2:GSFLVAES P3:SFLVAE
Fig.1. Conservative sequences in SH2 domain. A. Alignment of the SH2 domain of various proteins is done. Highly conserved residues are boxed and homologous amino acids are shaded. The suffix N indicates the more N-terminal SH2 domain of PLC 7, GAP or PI 3 kinase 85 K subunit, whereas C indicates the more C-terminal domain. B. Location of synthetic peptides in PLC 71 amino acid sequence. recognition of phosphotyrosine site by SH2 domain. Therefore, we synthesized several peptides related to the sequence and examined the effect of peptides on the binding of EGF receptor to SH2 of PLCT1. We synthesized 5 peptides(FiglB) according to the amino acid sequence of PLC 71. P1 was a peptide, GSFLVRES which is highly conserved in SH2. P2 is a peptide, GSFLVAES in which one amino acid, arginine of P1 is substituted by alanine. P3 is a peptide, SFLVRE in which two amino acids are deleted from P1. P4 is a peptide, WYHGK which is another conserved sequence in SH2. P5 is a peptide, of which sequence is not included in SH2 region. The association of PLC 71 and EGF receptor has already demonstrated by stimulating A431 cells with EGF and immunoprecipitating the cell lysates with EGF receptor antibody(l, 12,13). Therefore, We developed in vitro binding assay system to detect the association between SH2 of PLC 71 and phosphorylated EGF receptor. The protein containing SH2 regions of PLC 71( amino acid 517-901 ) was produced in E. coli expression system. The protein was coupled to Sepharose beads (Pharmacia CNBr-Sepharose). The immobilized SH2/SH3 was incubated with EGF receptor from A431 cells and the proteins bound 1493
Y
Vol. 180, No. 3, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
c~ EGF Receptor Ab
1
Autoradiography
2
3
_Fig. 2. Binding of EGF receptor to the SH2/SH3 of PLC T1. The shed membranes from A431 cells were phosphorylated(lane 1) or not phosphorylated(lane 2) in vitro and then solubilized. Binding of EGF receptor to SH2/SH3-Sepharose was examined as described in Experimental Procedures. Western blot analysis was done using antiEGF receptor antibody(lane 1 and 2). Autoradiography of the 32p_ labeled proteins retained in SH2/SH3-Sepharose was shown in lane 3. to beads were analyzed(Fig. 2). Immunoblot analysis showed that only phosphorylated EGF receptor could bind to the SH2/SH3-Sepharose without any significant binding of unphosphorylated EGF receptor. Moreover, in this system, it was found that phosphorylated EGF receptor was an only protein detected by autoradiography, suggesting that most of 32p bound to the beads are due to the 32p-EGF receptor. Using this binding method, We examined the inhibitory effect of synthesized peptides on the binding of EGF receptor to SH2/SH3 region of PLCy1. As indicated in Table 1, P1 was the most effective inhibitor for the binding. P2 was a moderate inhibitor and P4 was less effective than P1 or P2. P3 and P5 did not affect the binding markedly even when 1000-fold higher concentrations of peptide were used compared to those of immobilized SH2/SH3. These results suggest that highly conserved eight amino acid sequence is an important site for recognition of phosphorylated tyrosine. Concerning P1, P2 and P4, dose-dependent inhibition curve was determined(Fig 3). Similarly, P1 was the strongest inhibitor. It inhibited the binding by 50 % with 10-fold higher concentrations than immobilized SH2. P2 and P4 were also effective to suppress the binding. P2 was stronger than P4, suggesting that GSFLVRES in SH2 is important for recognition of phosphotyrosine 1494
Vol. 180, No. 3, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table 1. Inhibitory effect of various peptides on binding of EGF-receptor to SH2/SH3 regions Bound EGF-receptor (% of Control) Concentration (n-fold)
P1
P2
P3
P4
P5
0
100
100
100
100
100
10
54
55
90
86
95
100
27
42
84
62
88
1000
23
33
72
40
70
EGF-receptors32 in shed membranes were autophosphorylated with" 20 ~tM [T- P]ATP(1 I.tCi) in 50 ~1 of phosphorylation buffer for 5 min at 37"C and then 50 ~tl of lysis buffer was added. The mixture was centrifuged at 10000 X g for 10 min. The supematant was added to 50 ~1 of SH2/SH3-beads in the presence or absence of peptides, shaken for 2 h, and then washed with RIPA buffer. The radioactivity retained in the beads was counted. Concentration is indicated as n-fold to that of immobilized SH2/SH3.
and the substitution of arginine in P1 by alanine does not afford a critical damage to the recognition. Even a penta peptide, WYHGK remains a activity though its potency is fairly low. Next, we examined
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Fig. 3. D o s e - d e p e n d e n c e curve of the inhibition of peptides on the b i n d i n g of E G F receptor. Inhibitory effect of P1, P2 and P4 on the b i n d i n g of E G F receptor to SH2/SH3 was e x a m i n e d as described in Table 1. Concentration is indicated as n-fold to that of i m m o b i l i z e d SH2/SH3 of PLC T1. 1495
Vol. 180, No. 3, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
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2 Fig. 4. Eight amino acid peptide, GSFLVRES can bind to phosphorylated EGF receptor. Pl-agarose(50 Ixl) was incubated with [32p]phosphorylated EGF receptor in the presence (lane 2) or absence(lane 1) of 500 laM P 1, and then washed 5 times with phosphate-buffered saline containing 0.1% Tween 20. The proteins retained in Pl-agarose was analyzed with SDS-polyacrylamide gel electrophoresis and autoradiography.
whether the peptide P1 could bind EGF receptor or not. P1 peptide was covalently coupled to Affigel 10(Biorad). The beads were incubated with solubilized membranes of A431 cells after membranes were labeled with [7-32p]ATP. As shown in Fig. 4. EGF receptor bound to P1 peptide-beads though its binding affinity appears to be much weak. When the beads were washed vigorously with the buffer( 20 mM Hepes/NaOH, pH, 7.4 containing 0.1% SDS, 1% Triton X-100, 1% sodium deoxyclolate, 150 mM NaC1 and 10 % glycerol), the binding was diminished. All these data show that eight amino acid sequence, GSFLVRES is minimum essential sequence for recognition of phosphotyrosine site. References 1. Koch, C. A., Anderson, D., Moran, M. F., Ellis, C. and Pawson, M. F. (1991) Science 252, 668-674 2. Mayer, B. j., Hamaguchi, M. and Hanafusa, H. (1988) Nature 332, 272-275 3. Mayer, B. J. and Hanafusa, H. (1990) Proc. Natl. Acad. Sci. USA 87, 2638-2642 4. Matsuda, M., Mayer, B. J., Fukui, Y. and Hanafusa, H. (1990) Science 248, 1537-1539 1496
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
5. Anderson, D., Koch, C. A., Grey, L., Ellis, C., Morran, H. F. and Pawson, T. (1990) Science 250, 979-985 6. Morran, M. F., Koch, C. A., Anderson, D., Ellis, C., England, L., Martin, C. S. and Pawson, T. (1990) Proc. Natl. Acad. Sci. USA 87, 8622-8626 7. Margolis, B., Li, N., Koch, A., Mohammadi, M., Hurwitz, D. R., Ziberstein, A., Ullich, A., Pawson, T., A, D. Sclessinger, J. (1990) EMBO J. 9, 4375-4380 8. Morrison, D. E., Kaplan, D., Rhee, S. G. and Williams, L. (1990) Mol. Cell. Biol. 10, 2359-2366 9. Cantly, E. C., Auger, K. R., Carpenter, C., Duckworth, B., Graziani, A., Kapeller, R. and Soltoff, S. (1991) Cell 64, 281-302 10. Emori, Y. Homma, Y., Sorimachi, H., Kawasaki, H., Nakanishi, O., Suzuki, K. and Takenawa, T. (1989) J. Biol. Chem. 264, 21885-21890 11. Choen, S., Ushiro, H., Stoscheck, L. and Chinkers, M. (1982) J. Biol. Chem. 10, 1523-1531 12. Wahl, M., Daniel, T. O. Carpenter, G. (1988) Science 241, 968-970 13. Margolis, B., Bellot, F., Honegger, A. M., Ullich, A., Schlessinger, J. and Ziberstein, A. (1990) Mol. Cell. Biol. 10, 435-441
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