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Herbimycin A is an ansamycin antibiotic isolated as an agent that reverses morphological transformation induced by v-src. Although herbimycin A is widely used as a tool for inhibiting multiple tyrosine protein kinases and tyrosine kinase-activated signal transduction, its mechanism of action is not well defined and includes a decrease in both tyrosine kinase protein levels and activity (Uehara, Y., Murakami, Y., Sugimoto, Y., and Mizuno, S. (1989) Cancer Res. 49, 780-785). We now show that herbimycin A induces a profound decrease in the total cellular activity of transmembrane tyrosine kinase receptors, such as insulin-like growth factor, insulin, and epidermal growth factor receptors. A substantial proportion of the in vivo inhibition could be explained by an increase in the rate of degradation. The enhanced degradation of insulin-like growth factor-insulin receptor was prevented by inhibitors of the 20S proteasome, whereas neither lysosomotropic agents nor general serine- and cysteine-protease inhibitors were active in preventing receptor degradation induced by herbimycin A. Moreover, in a temperature-sensitive mutant cell line defective in the E1-catalyzed activation of ubiquitin, herbimycin A treatment at the restrictive temperature did not result in the degradation of insulin receptor. These results suggest that herbimycin A represents a novel class of drug that targets the degradation of tyrosine kinases by the 20S proteasome. The ubiquitin dependence of this process indicates that this degradation of tyrosine kinases might involve the 20S proteasome as the proteolytic core of the ubiquitin-dependent 26S protease.

We have transfected mouse L cells with a recombinant membrane-anchored insulin receptor kinase (called MARK), under the transcriptional control of a glucocorticoid-responsive element. The transfected construct includes only 15 extracellular residues, with the transmembrane and intracellular kinase domains of the human insulin receptor cDNA (amino acid residues -27 to 12 and 915 to 1343 (see Ullrich, A., Bell, J. R., Chen, E. Y., Herrera, R., Petruzzelli, L. M., Dull, T. J., Gray, A., Coussens, L., Liao, Y. C., Tsubokawa, M., Mason, A., Seeburg, P. H., Grunfeld, C., Rosen, O. M., and Ramachandran, J. (1985) Nature 313, 756-761), predicted Mr = 56,000 with signal sequence, 53,000 without). Transfected cells which are exposed to dexamethasone express two proteins of approximate Mr = 54,000 which (a) react with anti-peptide antisera raised to human insulin receptor sequences, (b) localize to the membrane fraction, and (c) possess ligand (insulin)-independent tyrosine kinase activity. In extracts of steroid-treated MARK cells, a phosphotyrosine-containing protein of Mr = 185,000 is detected, which corresponds in size to a known endogenous substrate for the insulin receptor. Control studies were performed with the nontransfected parent line, and with L cells transfected with an inactive human insulin receptor protein tyrosine kinase. Dexamethasone induced no change in the proteins detected by an anti-phosphotyrosine antibody in the two control lines. In studies of deoxyglucose uptake, dexamethasone stimulated increase in deoxyglucose uptake 2-fold in the MARK cells compared to the same cells studied in the absence of dexamethasone. Dexamethasone had no effect in the control cell lines. These studies demonstrate that a membrane-anchored insulin receptor kinase, devoid of virtually the entire extracellular domain of the insulin receptor, is sufficient to induce enhanced deoxyglucose uptake.

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.