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Annals of Clinical & Laboratory Science 38:47-56 (2008)
© 2008 Association of Clinical Scientists

Site-Specific Phosphorylation of raf in Cells Containing Oncogenic ras-p21 Is Likely Mediated by jun-N-Terminal Kinase

Victor Adler1,2, Wilbur Bowne3,4, Josef Michl1,5, Kelley A. Sookraj3,4, Kamran Ikram3,4, Sidney Pestka6, Lara Izotova6, Michael Zenilman3, Fred K. Friedman7, Yongxia Qu1,2 and Matthew R. Pincus1,2
1 Department of Pathology, SUNY Downstate Medical Center, and 2 Department of Pathology & Laboratory Medicine, New York Harbor VA Medical Center, Brooklyn, NY; 3 Department of Surgery, SUNY Downstate Medical Center, and 4 Department of Surgery, New York Harbor VA Medical Center, Brooklyn, NY; 5 Departments of Microbiology, Anatomy, and Cell Biology, SUNY Downstate Medical Center, Brooklyn, NY; 6 Department of Molecular Genetics, Microbiology and Immunology, Robert Wood Johnson Medical School, Piscataway, NJ; and 7 National Institutes of Health, Bethesda, MD

Address correspondence to Matthew R. Pincus, M.D., Ph.D., New York Harbor VA Medical Center, 800 Poly Place, Brooklyn, NY 11209, USA; tel 718 630 3688; fax 718 630 2960; e-mail: matthew.pincus2{at}med.va.gov.

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 penetratin 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.

Abbreviations: MEK, mitogen (extracellular)-activated kinase, the direct activator of MAPK; MAPK, mitogen-activated protein kinase, also called ERK; TOPK, Lymphokine-Activated Killer T-Cell-Originated Protein Kinase (TOPK); DYRK1A, Dual-Specificity-Tyrosine-Phosphorylation-Regulated Kinase-1A; PNC-2, Ha-ras peptide 96-110; PNC-7, Ha-ras peptide, 35–47; leader peptide or penetratin, trans-membrane-penetrating peptide; PNC-28, peptide containing human p53 sequence 17–26 linked to penetratin sequence; JNK, jun-N-terminal kinase; PKC, protein kinase C.

Keywords: oncogenic ras-p21, raf, phosphorylation, raf-Ser259, raf-Ser338, JNK






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