2-Aminopurine abolishes epidermal growth factor-stimulated phosphorylation of complexed and chromatin-associated forms of a 33 kDa phosphoprotein.
We have recently reported that several purified polypeptide mitogens such as epidermal growth factor, embryonal carcinoma-derived growth factor, basic fibroblast growth factor and Bombesin induce the rapid appearance of a 33 kDa chromatin-associated phosphoprotein in the nuclei of murine fibroblasts. We show here that in both mouse and human cell lines, a second form of the 33 kDa phosphoprotein exists in a detergent-extractable complexed form which may be pelleted by ultracentrifugation. When quiescent [32P]-labelled cells are treated with EGF, both complexed and chromatin-associated forms of the labelled phosphoprotein are detectable within 10 min, the response peaking at about 1 h and being substantially over 3 h after EGF stimulation. By chymotryptic and cyanogen bromide phosphopeptide mapping studies, the two forms of the 33 kDa phosphoprotein are indistinguishable, as are the mouse and human forms of the protein. The protein kinase inhibitor 2-aminopurine, which has recently been shown to block growth factor-stimulated c-fos and c-myc induction, specifically abolishes the mitogen-stimulated appearance of both forms of the 33 kDa phosphoprotein without affecting the phosphorylation of other cellular proteins. The 33 kDa protein has been purified from Hela cells by a combination of sucrose density gradient centrifugation, preparative electrophoresis and reverse-phase HPLC during which the protein is resolved into two closely-eluting peaks which differ markedly in their specific activity. These results are discussed in relation to the possible role of these events in coupling growth factor-receptor interaction at the cell surface to the early responses of transcriptional activation in the nucleus.
Mahadevan
L C
LC
Department of Biochemistry, Oxford University, UK.
Targett
K
K
Heath
J K
JK
D015075:2-Aminopurine / Q000494:pharmacology*; D000225:Adenine / Q000031:analogs & derivatives*; D000818:Animals; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D002843:Chromatin / Q000187:drug effects / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D008934:Mitogens / Q000494:pharmacology; D008970:Molecular Weight; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D012330:RNA, Double-Stranded / Q000494:pharmacology; D038601:Ribosomal Protein S6; D012269:Ribosomal Proteins / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic
D002843:Chromatin; D008934:Mitogens; D010750:Phosphoproteins; D012330:RNA, Double-Stranded; D038601:Ribosomal Protein S6; D012269:Ribosomal Proteins; D015075:2-Aminopurine; D004815:Epidermal Growth Factor; D000225:Adenine