EXPRESSION OF EPIDERMAL GROWTH-FACTOR RECEPTOR SEQUENCES AS ESCHERICHIA-COLI FUSION PROTEINS - APPLICATIONS IN THE STUDY OF TYROSINE KINASE FUNCTION
EXPRESSION OF EPIDERMAL GROWTH-FACTOR RECEPTOR SEQUENCES AS ESCHERICHIA-COLI FUSION PROTEINS - APPLICATIONS IN THE STUDY OF TYROSINE KINASE FUNCTION
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DOI：
10.1016/0006-291x(90)91915-f
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发表时间：
1990-01-15
影响因子：
3.1
通讯作者：
CERIONE, RA
中科院分区：
文献类型：
--
作者：
KOLAND, JG;OBRIEN, KM;CERIONE, RA
为了研究表皮生长因子受体（EGFR）关键结构域的功能，在大肠杆菌中表达了多种EGFR衍生肽序列作为谷胱甘肽S-转移酶（GST）融合蛋白。将纯化的融合蛋白（GST-TK 0 -8）作为EGFR和c-src的酪氨酸激酶活性的底物进行测试。GST-TK 4融合蛋白（包含EGFR的主要C-末端酪氨酸自磷酸化位点）和GST-TK 7（包含EGFR激酶结构域和C-末端自磷酸化结构域之间的连接序列）均被EGFR和c-src强烈磷酸化。因此，EGFR的连接序列中存在的候选酪氨酸磷酸化位点以及EGFR的已知自磷酸化位点可以被两种酪氨酸激酶磷酸化。蛋白质GST-TK 7被c-src磷酸化，KM为5-10 μ M，这表明EGFR的连接片段与c-src激酶之间存在潜在的相互作用。GST融合蛋白也被用来映射的网站识别的两个抗EGFR单克隆抗体和多克隆血清提出了对EGFR酪氨酸激酶结构域片段。一种单克隆抗体的识别位点被确定为在围绕tyr 1068的短序列中，tyr 1068是受体C-末端结构域中自磷酸化的主要位点。抗肽多克隆血清仅识别GST-TK 7融合蛋白中的序列，因此结合激酶核心和C-末端结构域之间的连接序列。因此，这些抗体将是用于研究EGFR酪氨酸激酶的两个关键结构元件的功能的有用试剂。GST-TK融合蛋白在EGFR催化和促有丝分裂信号传导的研究中应该具有许多其他应用。
To investigate the functions of key domains of the epidermal growth factor receptor (EGFR), various EGFR-derived peptide sequences were expressed in Escherchia 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 .mu.M, 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.