CHARACTERIZATION OF THE GENE PRODUCTS OF THE ETS LOCUS

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The human ETS1 proto-oncogene proteins have been isolated from the T-cell line, CEM, by immunoaffinity chromatography and their identity confirmed by N-terminal amino acid sequencing. The p5l and p42 ETS1 isoforms react with monoclonal antibodies directed against a bacterially-expressed ETS1 protein and to an oligopeptide directed to the carboxyl-terminal 13 amino acids of the human ETS1 protein. The p42 human ETS1 does not react with an antibody directed to exon VII of the human ETSI, indicating that it is the product of alternatively-spliced mRNA lacking exon VII. The p48 and the p39 isoforms of the human ETSI are shown to be derived from the p5l and p42 isoforms of the human ETS1 by the covalent modification of -SH groups by the protease inhibitor (N(alpha)-p-tosyl-L-lysine chloromethyl ketone (TLCK). The renatured human ETS1 was shown to have DNA sequence-specific binding to the PEA-3 (AGGAAGT) motif; this complex can be observed by electrophoretic mobility shift assays (EMSA). The purified ETSI retards a complex which is exactly the same size complex as is retarded from nuclear extracts prepared from the T-cell leukemia cell line, CEM. Reduced ETSI is required to form the ETS1. PEA-3 complex, but modification of the ETSI -SH groups by N-ethylmaleiamide or by TLCK does not inhibit formation of the ETS1.PEA-3 complex. The ETSI.PEA-3 complex formed with TLCK-modified ETSI has a slower mobility than does the complex formed with unmodified ETSI. Six monoclonal antibodies were prepared from mice immunized with a bacterially-expressed human ETS2 protein. These antibodies specifically recognize the two human ETS2-encoded proteins, p56 and p54, but failed to react with chicken, mouse, rat, bovine, or monkey proteins, suggesting that the antibodies recognize epitopes specific to the human ETS2 protein. The biochemical analysis of the ETS2 protein will be facilitated by the development of these monoclonal antibodies, which may be useful as both domain-specific probes and tools for specifically detecting the human ETS2 protein in heterologous expression systems.