MITOTIC RAF-1 IS STIMULATED INDEPENDENTLY OF RAS AND IS ACTIVE IN THECYTOPLASM

Raf-1 is a Ser/Thr protein kinase that is involved in regulation of pr oliferation, differentiation, and apoptosis, Recently, we and others s howed that Raf-1 is mot only activated in mitogenic pathways leading t o cell cycle entry but also during mitosis, Transient expression studi es in COS cells now demonstrate that, in contrast to growth factor-dep endent activation of Raf-1, mitotic activation of Raf-1 is Ras-indepen dent, Dominant negative RasS17N does not interfere with mitotic activa tion of Raf-1, whereas epidermal growth factor-dependent stimulation o f Raf-1 is inhibited, In addition, the Raf-1 mutant RafR89L, which can not bind to activated Ras, is still stimulated in mitotic cells, Mitot ic activation of Raf-1 seems to be partially dependent on tyrosine pho sphorylation since the kinase activity of the Raf mutant RafYY340/341F F, which can no longer be activated by Src, is reduced in mitotic cell s. Surprisingly, cell fractionation experiments showed that mitotic-ac tivated 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 ext racellular signal-regulated protein kinase (ERK). These data demonstra te that in mitotic cells a Ras-independent mechanism results in a cyto plasmic active Raf-1 kinase which does not signal via the MEW ERK path way,These data demonstrate that in mitotic cells a Ras-independent mec hanism results in a cytoplasmic active Raf-1 kinase which does not sig nal via the MEK/ERK pathway.