title: Site-specific phosphorylation of raf in cells containing oncogenic ras-p21 is likely mediated by jun-N-terminal kinase.
abstract: In a study of interactions between the raf-MEK-MAPK (ERK) and JNK-jun pathways, we found previously that JNK can induce phosphorylation of raf but not vice versa. In this study, we investigate the nature of the JNK-induced phosphorylation of raf. In in vitro experiments in which immunobead-bound raf is phosphorylated by activated JNK, we find strong phosphorylation signals at raf-Ser259 and Ser338. The Ser259 phosphorylation is surprising since it is associated with inhibition of migration of raf to the cell membrane where it can interact with ras-p21. We also find that in oocytes induced to mature with oncogenic ras-p21, which induces high levels of phosphorylated JNK and MAPK, the same pattern of phosphorylation of raf occurs. In contrast, in oocytes induced to mature with insulin, which requires activation of wild-type ras-p21, phosphorylation of raf-Ser338 but not raf-Ser259 occurs. In oncogenic ras-transformed human pancreatic cancer MIA-PaCa-2 cells, phosphorylation of both raf serines occurs. Treatment of these cells with the ras peptide, PNC-2 attached to a penetrating sequence that blocks JNK and MAPK phosphorylation and induces tumor cell necrosis, results in a marked decrease in phosphorylation of raf-Ser259, but not that of raf-Ser338. These results suggest that oncogenic ras-p21 induces phosphorylation of both raf-Ser259 and Ser338 and that raf-Ser 259 phosphorylation may be effected by activated JNK.
authors.affiliation: Department of Pathology, SUNY Downstate Medical Center, Brooklyn, New York, USA.
authors.forename: Victor
authors.initials: V
authors.lastname: Adler
authors.forename: Wilbur
authors.initials: W
authors.lastname: Bowne
authors.forename: Josef
authors.initials: J
authors.lastname: Michl
authors.forename: Kelley A
authors.initials: KA
authors.lastname: Sookraj
authors.forename: Kamran
authors.initials: K
authors.lastname: Ikram
authors.forename: Sidney
authors.initials: S
authors.lastname: Pestka
authors.forename: Lara
authors.initials: L
authors.lastname: Izotova
authors.forename: Michael
authors.initials: M
authors.lastname: Zenilman
authors.forename: Fred K
authors.initials: FK
authors.lastname: Friedman
authors.forename: Yongxia
authors.initials: Y
authors.lastname: Qu
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Pincus
mesh_terms: D000818:Animals; D000918:Antibody Specificity; D045744:Cell Line, Tumor; D002471:Cell Transformation, Neoplastic; D004789:Enzyme Activation; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008954:Models, Biological; D015689:Oncogene Protein p21(ras) / Q000378:metabolism*; D010190:Pancreatic Neoplasms / Q000378:metabolism; D010748:Phosphopeptides / Q000378:metabolism; D010766:Phosphorylation; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D014981:Xenopus
chemical_list: D010748:Phosphopeptides; D019908:Proto-Oncogene Proteins c-raf; D048031:JNK Mitogen-Activated Protein Kinases; D015689:Oncogene Protein p21(ras)