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 not 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 bound 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 MEK/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.
Ziogas
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A
Institute of Medical Virology, University of Zurich, Gloriastrasse 30, CH-8028 Zurich, Switzerland.
Lorenz
I C
IC
Moelling
K
K
Radziwill
G
G
D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002453:Cell Cycle / Q000502:physiology*; D003593:Cytoplasm / Q000201:enzymology; D004789:Enzyme Activation; D008938:Mitosis; D016297:Mutagenesis, Site-Directed; D015739:Nocodazole / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D016283:Proto-Oncogene Proteins p21(ras); D015739:Nocodazole