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2-Aminopurine abolishes epidermal growth factor-stimulated phosphorylation of complexed and chromatin-associated
L C Mahadevan1, K Targett, J K Heath
1Department of Biochemistry, Oxford University, UK.
Abstract:
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.
Insights
Growth factors rapidly induce a 33 kDa nuclear phosphoprotein in fibroblasts. This protein, existing in both chromatin-associated and complexed forms, is crucial for early transcriptional responses following growth factor stimulation.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Polypeptide mitogens like epidermal growth factor (EGF) trigger rapid cellular responses.
- A 33 kDa chromatin-associated phosphoprotein appears in the nucleus of murine fibroblasts upon mitogen stimulation.
Purpose of the Study:
- To investigate the forms and regulation of the 33 kDa phosphoprotein induced by mitogens.
- To elucidate the role of this phosphoprotein in coupling growth factor signaling to nuclear events.
Main Methods:
- Cell culture (murine fibroblasts, mouse and human cell lines, Hela cells)
- Radioactive labeling ([32P])
- EGF stimulation
- Ultracentrifugation
- Chymotryptic and cyanogen bromide phosphopeptide mapping
- Protein kinase inhibitor (2-aminopurine) treatment
- Sucrose density gradient centrifugation
- Preparative electrophoresis
- Reverse-phase HPLC
Main Results:
- A second, detergent-extractable complexed form of the 33 kDa phosphoprotein was identified in mouse and human cells.
- Both forms appeared rapidly (within 10 min) after EGF stimulation, peaking at 1 hour.
- Phosphopeptide mapping showed the two forms and mouse/human forms of the protein were indistinguishable.
- 2-aminopurine specifically abolished the mitogen-stimulated appearance of both phosphoprotein forms.
- Purification of the 33 kDa protein from Hela cells yielded two peaks with differing specific activities.
Conclusions:
- The 33 kDa phosphoprotein exists in at least two forms, both rapidly induced by mitogens.
- Its induction is specifically blocked by a protein kinase inhibitor, suggesting a role in signaling pathways.
- The findings suggest this phosphoprotein is involved in linking cell surface growth factor receptor activation to nuclear transcriptional responses.