2-Aminopurine abolishes EGF- and TPA-stimulated pp33 phosphorylation and c-fos induction without affecting the activation of protein kinase C.
Epidermal Growth Factor (EGF) and Tetradecanoyl Phorbol Acetate (TPA) initiate signalling cascades in C3H 10T1/2 fibroblasts by primarily activating distinct protein kinases, the EGF receptor tyrosine kinase and protein kinase C respectively; there is no signal crossover at the initiation of signalling. Nevertheless, we show here that both agents rapidly elicit common intracellular responses, including the phosphorylation of complexed and chromatin-associated forms of a 33 kDa phosphoprotein (pp33), that of a 15 kDa chromatin-associated phosphoprotein (pp15), as well as the transcriptional activation of a common subset of genes including the c-fos proto-oncogene. 2-aminopurine specifically abolishes complexed and chromatin-associated pp33 phosphorylation in response to EGF and TPA, as well as the induction of c-fos by both agents. The activation of protein kinase C and the levels of transcription factors that bind to the serum response element (SRE), TPA response element (TRE) or NFkB sites in stimulated cells are relatively unaffected by 2-aminopurine. This, to our knowledge, is the first demonstration that it is possible, by using 2-aminopurine which selectively blocks TPA-stimulated pp33 phosphorylation, to block c-fos induction in TPA-treated cells although protein kinase C remains fully active. Further, we show here that although EGF- and TPA-stimulated induction of c-fos is abolished by 2-aminopurine, the appearance of TRE-binding activity in nuclear extracts of stimulated cells is unaffected, suggesting that EGF- and TPA-stimulated induction of TRE-binding activity utilises existing proteins and is not dependent on fresh c-FOS synthesis. These results imply that 2-aminopurine-sensitive complexed and chromatin-associated pp33 phosphorylation may be crucial to c-fos induction in response to EGF and TPA.
Mahadevan
L C
LC
Department of Biochemistry, Oxford University.
Wills
A J
AJ
Hirst
E A
EA
Rathjen
P D
PD
Heath
J K
JK
D015075:2-Aminopurine / Q000494:pharmacology*; D000225:Adenine / Q000031:analogs & derivatives*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004790:Enzyme Induction; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000096:biosynthesis*; D011505:Protein-Tyrosine Kinases / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics*; D016760:Proto-Oncogene Proteins c-fos; D011519:Proto-Oncogenes; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014157:Transcription Factors / Q000378:metabolism
D008565:Membrane Proteins; D015345:Oligonucleotide Probes; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D016760:Proto-Oncogene Proteins c-fos; D014157:Transcription Factors; D015075:2-Aminopurine; D004815:Epidermal Growth Factor; D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C; D000225:Adenine; D013755:Tetradecanoylphorbol Acetate