Efficient expression in insect cells of a soluble, active human insulin receptor protein-tyrosine kinase domain by use of a baculovirus vector.
The human insulin receptor (IR) is a transmembrane glycoprotein, whose cytoplasmic domain contains an insulin-activated protein-tyrosine kinase (EC 2.7.1.112). By the use of an appropriately engineered baculovirus expression vector, a soluble cytoplasmic derivative of this domain was expressed in the insect cell line Spodoptera frugiperda (Sf9). At 24 to 48 h after Sf9 cells were infected with recombinant virus, a protein of the size expected for this domain (approximately 48 kilodaltons) constituted a major band when total cell lysates of metabolically labeled cells were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. This protein (designated AchIRPTK) was immunoprecipitated by three monoclonal antibodies, each of which recognizes a distinct antigenic site of the IR cytoplasmic domain and requires the native structure of the protein for recognition and one of which binds at or near the physiologically relevant site(s) of IR autophosphorylation. In vivo, AchIRPTK was phosphorylated on both tyrosine and serine residues. When affinity purified, the kinase was active in vitro; it autophosphorylated exclusively on tyrosine residues, and phosphorylated the exogenous substrates histone H2b and poly(Glu-Tyr). The expression of an active IR protein-tyrosine kinase molecule in this heterologous cell system provides an efficient experimental method for producing this domain in quantity for enzymatic and structural studies.
Ellis
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Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas 75235-9050.
Levitan
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A
Cobb
M H
MH
Ramos
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P
D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D005786:Gene Expression Regulation; D007304:Insect Viruses / Q000235:genetics; D007915:Lepidoptera / Q000235:genetics*; D008970:Molecular Weight; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000235:genetics*; D011972:Receptor, Insulin; D012694:Serine / Q000378:metabolism; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism
D000911:Antibodies, Monoclonal; D014443:Tyrosine; D012694:Serine; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin