Synthesis, purification, and characterization of the cytoplasmic domain of the human insulin receptor using a baculovirus expression system.
The cytoplasmic domain of the beta subunit of the human insulin receptor has been overexpressed in insect cells using the baculovirus expression system. A recombinant baculovirus (BIR-2) was constructed by inserting the human insulin proreceptor cDNA fragment that encodes the cytoplasmic domain of the receptor into the genome of Autographa californica nuclear polyhedrosis virus adjacent to the strong polyhedrin promoter. Synthesis of the protein (baculovirus insulin receptor kinase (BIRK), Mr 48,000) in BIR-2-infected Spodoptera frugiperda (Sf9) cells was detected 24 h after infection and maximal accumulation (2-3% of the cytosolic protein) was achieved 48-72 h post-infection. The expressed protein is active as a soluble protein tyrosine kinase, both in Sf9 cells and in vitro. Rapid purification to near homogeneity was accomplished by sequential chromatography on Fast-Q-Sepharose and phenyl-Superose with an overall yield of 35% and a specific activity with histone as substrate of 20 nmol/min/mg protein. Autophosphorylation activated the intrinsic kinase activity of BIRK and decreased its mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using a combination of tryptic digestion and immunoprecipitation with specific antipeptide antisera, it was ascertained that 30-40% of the 32P incorporated into BIRK by autophosphorylation is in the carboxyl-terminal domain (that includes tyrosyl residues 1316 and 1322 of the human proreceptor). Of the remaining radioactivity, 75% is in the amino-terminal domain (that includes tyrosyl residues 953, 960, 972, 999, and 1075) and 25% is in the conserved autophosphorylation domain (including tyrosyl residues 1146, 1150, and 1151). Limited digestion of BIRK with trypsin yielded a fragment, Mr 38,000, that lacks the carboxyl-terminal domain. This fragment exhibits protein tyrosine kinase activity that is stimulated by autophosphorylation. The properties of the soluble, monomeric BIRK are similar to those of the intact, activated, oligomeric insulin receptor kinase with respect to specificity, immunoreactivity, divalent cation requirements, and specific activity. These observations coupled with the ease of producing 0.4 mg of purified enzyme from 100 ml of suspension culture suggest that BIRK will be useful for biochemical and biophysical analysis of the insulin receptor protein tyrosine kinase.
D002845:Chromatography; D003593:Cytoplasm / Q000378:metabolism; D004274:DNA, Recombinant / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007163:Immunosorbent Techniques; D007304:Insect Viruses / Q000235:genetics* / Q000378:metabolism; D046911:Macromolecular Substances; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D010446:Peptide Fragments; D010766:Phosphorylation; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014357:Trypsin; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D004274:DNA, Recombinant; D046911:Macromolecular Substances; D010446:Peptide Fragments; D019000:Phosphotyrosine; D014443:Tyrosine; D008345:Manganese; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D014357:Trypsin