The 33-kDa C-terminal domain of Raf-1 protein kinase exhibits a Ras-independent serum- and phorbol ester-induced shift in gel mobility.
Experiments were carried out to determine Raf-1 protein kinase domain fragments which exhibit a characteristic electrophoretic mobility shift noted with Raf-1 protein kinase in response to serum and phorbol ester (PMA) treatment of serum-deprived NIH 3T3 cells. Epsilon-epitope tagged 84 kDa Raf-1 holoenzyme (HR-epsilon), as well as the epsilon-epsilon pitope tagged 35 kDa N-terminal (RI-epsilon), 33 kDa mid-portion (RII-epsilon), and 33 kDa C-terminal (RIII-epsilon) fragments of Raf-1 were overexpressed in NIH 3T3 cells. The overexpressed HR-epsilon exhibited a serum- and PMA-induced shift in gel mobility similar to that noted with endogenous Raf-1. The C-terminal RIII-epsilon fragment exhibited a similar shift in gel mobility while the electrophoretic mobility of the N-terminal RI-epsilon fragment remained unchanged. These results suggest that modification(s) within the 33 kDa C-terminal portion of Raf-1 which occur independently of association with Ras may be responsible for the band shift observed with serum and PMA treatment of serum-deprived NIH 3T3 cells.
Oláh
Z
Z
Laboratory of Cellular Oncology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Ferrier
A
A
Lehel
C
C
Anderson
W B
WB
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001769:Blood; D003470:Culture Media; D016895:Culture Media, Serum-Free; D017931:DNA Primers; D000939:Epitopes; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D016133:Polymerase Chain Reaction; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D016324:Sequence Tagged Sites; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014162:Transfection; D018631:ras Proteins / Q000378:metabolism*
D003470:Culture Media; D016895:Culture Media, Serum-Free; D017931:DNA Primers; D000939:Epitopes; D010446:Peptide Fragments; D010710:Phosphates; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D018631:ras Proteins; D013755:Tetradecanoylphorbol Acetate