Biochemical characterization of human GCF transcription factor in tumor cells.
GCF is a transcriptional regulator that was found to repress transcription of the epidermal growth factor (EGF) receptor and several other genes and is encoded by a 3-kb mRNA (R. Kageyama and I. Pastan, Cell, 59: 815-825, 1989; A. C. Johnson et al., J. Biol. Chem., 267: 1689-1694, 1992). To identify and characterize the GCF gene product at the cellular level, we have developed antibodies against a bacterially expressed GCF fusion protein. GCF antibodies recognize GCF present in extracts from human cells and causes a "supershift" of a protein DNA complex containing a GCF oligonucleotide binding site. The major form of GCF has a molecular weight of approximately M(r) 97,000, identical to that of GCF transiently expressed in CV1 cells by the vaccinia virus system. In addition, other less abundant species with slightly higher and lower apparent molecular weight are specifically recognized, suggesting extensive posttranslational modification. GCF is highly expressed in EGF receptor-negative human cell lines (HUT102, U266, and CA46) and in lower amounts in several EGF receptor-expressing cells (KB, A431, TMK, and HeLa). Cell fractionation studies indicate that GCF is predominantly localized in the nucleus. GCF is a stable protein with a relatively long half-life. In addition, GCF is a phosphoprotein, and the phosphorylated form is found to be associated with the nuclear compartment in both HUT102 and KB cells. Phosphorylation occurs on serine and threonine residues and is stimulated by okadaic acid, phorbol myristate acetate, and cyclic AMP, but not vanadate.(ABSTRACT TRUNCATED AT 250 WORDS)
Beguinot
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Division of Cancer Biology, Diagnosis and Centers, National Cancer Institute, NIH, Bethesda, Maryland 20892-4255, USA.
Yamazaki
H
H
Pastan
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I
Johnson
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AC
D016475:3T3 Cells / Q000187:drug effects; D000818:Animals; D002461:Cell Line, Transformed; D002522:Chlorocebus aethiops; D000242:Cyclic AMP / Q000494:pharmacology; D018076:DNA, Complementary / Q000235:genetics; D015536:Down-Regulation / Q000187:drug effects; D066246:ErbB Receptors / Q000096:biosynthesis* / Q000235:genetics; D004988:Ethers, Cyclic / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects; D006801:Humans; D007106:Immune Sera; D051379:Mice; D008970:Molecular Weight; D019319:Okadaic Acid; D010766:Phosphorylation / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000378:metabolism; D012097:Repressor Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000187:drug effects
D018076:DNA, Complementary; D004988:Ethers, Cyclic; C072278:GCFC2 protein, human; D007106:Immune Sera; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D019319:Okadaic Acid; D000242:Cyclic AMP; D066246:ErbB Receptors; D017346:Protein Serine-Threonine Kinases; D013755:Tetradecanoylphorbol Acetate