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.