Human ETS1 oncoprotein. Purification, isoforms, -SH modification, and DNA sequence-specific binding.
The human ETS1 proto-oncogene proteins have been isolated from the T-cell leukemia line, CEM, by immunoaffinity chromatography and their identity confirmed by NH2-terminal amino acid sequencing. Incubation of CEM cells with N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) indicates that ETS proteins can be modified in their cellular context and that pretreatment of the cells with N-ethylmaleimide (NEM) protects ETS1 proteins from TLCK modification. These data show that ETS1 proteins can exist in at least two different states, -SH-available and -SH-protected. Renatured human ETS1 has DNA sequence-specific binding to the PEA3 (CAGGAAGT) motif. The ETS1.PEA3 complex can be observed by electrophoretic mobility shift assays (EMSA). Purified ETS1 retards a band which is exactly the same size as a complex that is retarded from nuclear extracts prepared from CEM cells. Reduced ETS1 is required to form the ETS1.PEA3 complex, however; modification of the ETS1 -SH groups by either NEM or by TLCk does not inhibit formation of the complex. The ETS1.PEA3 complex formed with TLCK-modified ETS1 has a slower mobility than the complex formed with unmodified ETS1. Zone sedimentation analysis of purified ETS1 indicates that it is the monomer of ETS1 which binds to the PEA3 oligonucleotide.
Fisher
R J
RJ
Laboratory of Cellular Biochemistry, Program Resources, Inc./DynCorp, Frederick Cancer Research and Development Center, Maryland.
Koizumi
S
S
Kondoh
A
A
Mariano
J M
JM
Mavrothalassitis
G
G
Bhat
N K
NK
Papas
T S
TS
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D002501:Centrifugation, Zonal; D002846:Chromatography, Affinity; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide / Q000494:pharmacology; D006801:Humans; D015458:Leukemia, T-Cell; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism*; D009842:Oligopeptides / Q000138:chemical synthesis / Q000276:immunology; D000090063:Proto-Oncogene Mas; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D050783:Proto-Oncogene Proteins c-ets; D014107:Tosyllysine Chloromethyl Ketone / Q000494:pharmacology; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; C494041:ETS1 protein, human; C000715470:MAS1 protein, human; D009838:Oligodeoxyribonucleotides; D009842:Oligopeptides; D000090063:Proto-Oncogene Mas; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D014157:Transcription Factors; D014107:Tosyllysine Chloromethyl Ketone; D005033:Ethylmaleimide