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Phosphoamino acid specificity of the EGF-receptor kinase system in normal mitogenically responsive human cells
Cell Biology
International
Reports,
533
Vol. 7, No. 7, July 1983
PHOSPHOAMINOACID SPECIFICITY OF THE EGF-RECEPTOR KINASE SYSTEM IN NORMAL MITOGENICALLY RESPONSIVE HUMAN CELLS Y. YARDEN & J. SCHLESSINGER Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot 76100,
Israel.
The binding of epidermal growth factor (EGF) to its membrane receptor activates a cyclic nucleotide independent protein kinase which appears to be tyrosine specific, The EGF-sensitive-kinase autophosan integral part of the receptor molecule (1,2). phorylates the receptor molecule in membranes and in intact A-431 cells (Cohen et al. Most studies concerning the EGF 1980; Buhrow et al. 1982; Hunter and Cooper, 1981). large induced phosphorylation were done on A-431 cells which possess an unusually number of EGF-receptors but do not respond mitogenically to the growth factor. In this report we explore the EGF-induced phosphorylation in normal human foreskin This cells proliferate in response to EGF fibroblasts (HFF) (Yarden et al., 1983). and thus provide a good model for exploring the relationship between tyrosine phosphorylation and DNA synthesis. The phosphoamino acid content of EGF-receptor in HFF and A-431 cells. Crude membrane from either A-431 cells or from HFF were solubilized and immunoprecipitated with either monoclonal or polyclonal antibodies directed against EGF-recepSchreiber et al., 1981; Schreiber et al., 1983). After extensive washes, tor Phosphofor 10 minutes at 4’C. [y-p s 2]ATP and Mn+2 were added to the precipitate amino acid analysis of the phosphorylated receptor extracted from the polyacrylamide gel (see Fig. 1) shows that tyrosine is the only phosphorylated residue in EGF-receptor from A-431 cells, while both -serine and -tyrosine residues are phosphorylated residues in EGF-receptor from HFF. EGF-induced phosphbamino acids in a /-\ P- SER membranes from HFF and A-431 cells. t.J ) We have further analyzed the phosphoamino content of EGF-receptor in the presence or P-TYR absence of EGF. Table I summarizes this P-THR /: In A-431 cells the only residue analysis. which is phosphorylated in the absence or (i presence of EGF is tyrosine (%98%). v However, in EGF-receptor from HFF 85% of the phosphate is associated with serine residues and only 5% is bound to tyrosine Upon the addition of EGF the residues. major increase occurs on tyrosine residues (25% phosphoserine and 75% phosphotyrosine). Hence the EGF-receptor-kinase complex has b different phosphoamino acid content in HFF However, upon binding of and A-431 cells. EGF to its receptor only the tyrosinespecific kinase activity is stimulated in both cell lines. P-TYR DISCUSSION EGF induces the phosphorylation of various endogenous membrane proteins in membrane preparations and in intact cells. The addition of EGF to membranes prepared from either HFF or A-431 cells leads to the enhancement of phosphotyrosine content of EGF receptors of these systems. However, in the absence of EGF, and in the immunoprecipitated EGF receptor from HFF the phosphorylation occurs also on serine residues. Hence, the basal phosphorylation of EGF-receptor from HFF, even in the receptor immunopurified with monoclonal antibodies, occurs on serine residues.
0309-1651/83/070533-02/$03.00/O
I, Fig. 1. Analysis of the phosphoaminoacidresidues of EGF receptor in immunoprecipitates, EGF receptor was phosphorylated in immunoprecipitates from either A-431 cells (a)-or from HFF (b). The phosphoamino acids in the receptor excised from SDS-PAGE were analysed according to published procedures.
0
1983
Academic
Press
Inc.
(London)
Ltd.
Cell Biology
534 Table
I.
international
Phosphamino
Acid Analysis P-Thr (%I
System
Reports,
Vol. 7, No. 7, July
1983
of EGF-Receptor. P-Ser (%I
P-Tyr (%I
Total (cpm)
Human fibroblast a.
Membranes Membranes + EGF
b.
Immunoprec.
receptor
Immunoprec.:receptor Human carcinoma a.
+ EGF
10
85
5
60
1
24
76
250
6
28
66
438
3
24
73
450
(A431)
Membranes
2
2
96
60
Membranes + EGF
0
0
100
150
3
0.2
97
6100
1
0.1
99
7080
b.
Immunoprec.
receptor
Immunoprec.:
receptor
+ EGF
Quantitative analysis of the phosphoamino acid content of EGF-receptor. The phosphoamino acids were extracted from the thin layer chromatography plates of the various samples and their radioactive content was determined. Our results show that EGF activates a tyrosine specific kinase in normal mitogenitally responsive human cells. Nevertheless, the total phosphoamino content (basal plus stimulated) of EGF receptor in the mitogenically responsive cells occurs on tyrosine and serine residues, while in A-431 phosphorylation occurs almost exclusively on tyrosine residues. Interestingly, in both intact HFF and A-431 cells the main phosphorylated aminoacids of EGF-receptor appear to be serine residues (Yarden et al. 1983). The differential phosphoamino content of EGF-receptor in HFF and A-431 cells could lead to dissimilar phosphorylation patterns of cellular substrates in these two cells which may play a role in the mitogenic signal mediated by EGF. Acknowledgments This work was supported by grants from the NIH (CA-25820), from the U.S. Binational Science Foundation and from Stiftung Volkswagenwerk.
Israel
REFERENCES J.V. (1982) J. Biol. Chem. 257, 4019-4022. Buhrow, S.A., Cohen, S. and Staros, Cohen, S., Carpenter, E. and King, L.J. (1980) J. Biol. Chem. 255, 4834-4842. Hunter, T. and Cooper, J.A. (1981) Cell 24, 741-752. J. (1981) Proc. Schreiber, A.B., Lax, I., Yarden, Y., Eshhar, Z. and Schlessinger Natl. Acad. Sci. U.S.A. 2, 7535-7539. Schreiber, A.B., Libermann, T.A., Lax, I., Yarden, Y. and Schlessinger, J. (1983) J. Biol. Chem. 258, 846-853. A.B., Lax, I., and Schlessinger, J. (1983) Yarden, Y., Libermann, T.A., Schreiber, J. Biol. Chem. (submitted).
International
Reports,
533
Vol. 7, No. 7, July 1983
PHOSPHOAMINOACID SPECIFICITY OF THE EGF-RECEPTOR KINASE SYSTEM IN NORMAL MITOGENICALLY RESPONSIVE HUMAN CELLS Y. YARDEN & J. SCHLESSINGER Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot 76100,
Israel.
The binding of epidermal growth factor (EGF) to its membrane receptor activates a cyclic nucleotide independent protein kinase which appears to be tyrosine specific, The EGF-sensitive-kinase autophosan integral part of the receptor molecule (1,2). phorylates the receptor molecule in membranes and in intact A-431 cells (Cohen et al. Most studies concerning the EGF 1980; Buhrow et al. 1982; Hunter and Cooper, 1981). large induced phosphorylation were done on A-431 cells which possess an unusually number of EGF-receptors but do not respond mitogenically to the growth factor. In this report we explore the EGF-induced phosphorylation in normal human foreskin This cells proliferate in response to EGF fibroblasts (HFF) (Yarden et al., 1983). and thus provide a good model for exploring the relationship between tyrosine phosphorylation and DNA synthesis. The phosphoamino acid content of EGF-receptor in HFF and A-431 cells. Crude membrane from either A-431 cells or from HFF were solubilized and immunoprecipitated with either monoclonal or polyclonal antibodies directed against EGF-recepSchreiber et al., 1981; Schreiber et al., 1983). After extensive washes, tor Phosphofor 10 minutes at 4’C. [y-p s 2]ATP and Mn+2 were added to the precipitate amino acid analysis of the phosphorylated receptor extracted from the polyacrylamide gel (see Fig. 1) shows that tyrosine is the only phosphorylated residue in EGF-receptor from A-431 cells, while both -serine and -tyrosine residues are phosphorylated residues in EGF-receptor from HFF. EGF-induced phosphbamino acids in a /-\ P- SER membranes from HFF and A-431 cells. t.J ) We have further analyzed the phosphoamino content of EGF-receptor in the presence or P-TYR absence of EGF. Table I summarizes this P-THR /: In A-431 cells the only residue analysis. which is phosphorylated in the absence or (i presence of EGF is tyrosine (%98%). v However, in EGF-receptor from HFF 85% of the phosphate is associated with serine residues and only 5% is bound to tyrosine Upon the addition of EGF the residues. major increase occurs on tyrosine residues (25% phosphoserine and 75% phosphotyrosine). Hence the EGF-receptor-kinase complex has b different phosphoamino acid content in HFF However, upon binding of and A-431 cells. EGF to its receptor only the tyrosinespecific kinase activity is stimulated in both cell lines. P-TYR DISCUSSION EGF induces the phosphorylation of various endogenous membrane proteins in membrane preparations and in intact cells. The addition of EGF to membranes prepared from either HFF or A-431 cells leads to the enhancement of phosphotyrosine content of EGF receptors of these systems. However, in the absence of EGF, and in the immunoprecipitated EGF receptor from HFF the phosphorylation occurs also on serine residues. Hence, the basal phosphorylation of EGF-receptor from HFF, even in the receptor immunopurified with monoclonal antibodies, occurs on serine residues.
0309-1651/83/070533-02/$03.00/O
I, Fig. 1. Analysis of the phosphoaminoacidresidues of EGF receptor in immunoprecipitates, EGF receptor was phosphorylated in immunoprecipitates from either A-431 cells (a)-or from HFF (b). The phosphoamino acids in the receptor excised from SDS-PAGE were analysed according to published procedures.
0
1983
Academic
Press
Inc.
(London)
Ltd.
Cell Biology
534 Table
I.
international
Phosphamino
Acid Analysis P-Thr (%I
System
Reports,
Vol. 7, No. 7, July
1983
of EGF-Receptor. P-Ser (%I
P-Tyr (%I
Total (cpm)
Human fibroblast a.
Membranes Membranes + EGF
b.
Immunoprec.
receptor
Immunoprec.:receptor Human carcinoma a.
+ EGF
10
85
5
60
1
24
76
250
6
28
66
438
3
24
73
450
(A431)
Membranes
2
2
96
60
Membranes + EGF
0
0
100
150
3
0.2
97
6100
1
0.1
99
7080
b.
Immunoprec.
receptor
Immunoprec.:
receptor
+ EGF
Quantitative analysis of the phosphoamino acid content of EGF-receptor. The phosphoamino acids were extracted from the thin layer chromatography plates of the various samples and their radioactive content was determined. Our results show that EGF activates a tyrosine specific kinase in normal mitogenitally responsive human cells. Nevertheless, the total phosphoamino content (basal plus stimulated) of EGF receptor in the mitogenically responsive cells occurs on tyrosine and serine residues, while in A-431 phosphorylation occurs almost exclusively on tyrosine residues. Interestingly, in both intact HFF and A-431 cells the main phosphorylated aminoacids of EGF-receptor appear to be serine residues (Yarden et al. 1983). The differential phosphoamino content of EGF-receptor in HFF and A-431 cells could lead to dissimilar phosphorylation patterns of cellular substrates in these two cells which may play a role in the mitogenic signal mediated by EGF. Acknowledgments This work was supported by grants from the NIH (CA-25820), from the U.S. Binational Science Foundation and from Stiftung Volkswagenwerk.
Israel
REFERENCES J.V. (1982) J. Biol. Chem. 257, 4019-4022. Buhrow, S.A., Cohen, S. and Staros, Cohen, S., Carpenter, E. and King, L.J. (1980) J. Biol. Chem. 255, 4834-4842. Hunter, T. and Cooper, J.A. (1981) Cell 24, 741-752. J. (1981) Proc. Schreiber, A.B., Lax, I., Yarden, Y., Eshhar, Z. and Schlessinger Natl. Acad. Sci. U.S.A. 2, 7535-7539. Schreiber, A.B., Libermann, T.A., Lax, I., Yarden, Y. and Schlessinger, J. (1983) J. Biol. Chem. 258, 846-853. A.B., Lax, I., and Schlessinger, J. (1983) Yarden, Y., Libermann, T.A., Schreiber, J. Biol. Chem. (submitted).