Expression of epidermal growth factor receptor sequences as E. coli fusion proteins: applications in the study of tyrosine kinase function.
To investigate the functions of key domains of the epidermal growth factor receptor (EGFR), various EGFR-derived peptide sequences were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins. The purified fusion proteins (GST-TK0-8) were tested as substrates for the tyrosine kinase activities of the EGFR and c-src. Both the GST-TK4 fusion protein, which contains the major C-terminal tyrosine autophosphorylation sites of the EGFR, and GST-TK7, which contains the connecting sequence between the EGFR kinase domain and the C-terminal autophosphorylation domain, were strongly phosphorylated by the EGFR and c-src. Hence the candidate tyrosine phosphorylation sites present in the connecting sequences of the EGFR, as well as the known autophosphorylation sites of the EGFR, can be phosphorylated by the two tyrosine kinases. The protein GST-TK7 was phosphorylated by c-src with a KM of 5-10 microM, which indicated a potential interaction between the connecting segment of the EGFR and the c-src kinase. The GST fusion proteins were also used to map the sites recognized by two anti-EGFR monoclonal antibodies and a polyclonal serum raised against an EGFR tyrosine kinase domain fragment. The recognition site of one monoclonal antibody was determined to be in a short sequence surrounding tyr1068, a primary site of autophosphorylation in the C-terminal domain of the receptor. The anti-peptide polyclonal serum recognized only sequences in the GST-TK7 fusion protein, and hence binds to the connecting sequence between the kinase core and the C-terminal domain. These antibodies will therefore be useful reagents for studying the function of two key structural elements of the EGFR tyrosine kinase. The GST-TK fusion proteins should have many other applications in the study of EGFR catalysis and mitogenic signalling.
Koland
J G
JG
Department of Pharmacology, NYS College of Veterinary Medicine, Cornell University, Ithaca 14853.
O'Brien
K M
KM
Cerione
R A
RA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D066246:ErbB Receptors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000235:genetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010957:Plasmids; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping; D012550:Schistosoma mansoni / Q000201:enzymology / Q000235:genetics
D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases