Mitotic Raf-1 is stimulated independently of Ras and is active in the cytoplasm

Raf-1 is a Ser/Thr protein kinase that is involved in regulation of proliferation, differentiation, and apoptosis, Recently, we and others showed that Raf-1 is mot only activated in mitogenic pathways leading to cell cycle entry but also during mitosis, Transient expression studies in COS cells now demonstrate that, in contrast to growth factor-dependent activation of Raf-1, mitotic activation of Raf-1 is Ras-independent, Dominant negative RasS17N does not interfere with mitotic activation of Raf-1, whereas epidermal growth factor-dependent stimulation of Raf-1 is inhibited, In addition, the Raf-1 mutant RafR89L, which cannot bind to activated Ras, is still stimulated in mitotic cells, Mitotic activation of Raf-1 seems to be partially dependent on tyrosine phosphorylation since the kinase activity of the Raf mutant RafYY340/341FF, which can no longer be activated by Src, is reduced in mitotic cells. Surprisingly, cell fractionation experiments showed that mitotic-activated Raf-1 is predominantly located in the cytoplasm in contrast to the mitogen-activated Raf-1 that is hound to the plasma membrane, In addition, mitotic activation of Raf-1 does not lead to stimulation of the mitogen-activated protein kinase kinase (MAPKK or MEK) and the extracellular signal-regulated protein kinase (ERK). These data demonstrate that in mitotic cells a Ras-independent mechanism results in a cytoplasmic active Raf-1 kinase which does not signal via the MEW ERK pathway,These data demonstrate that in mitotic cells a Ras-independent mechanism results in a cytoplasmic active Raf-1 kinase which does not signal via the MEK/ERK pathway.