IDENTIFICATION OF RAF-1 SER(621) KINASE-ACTIVITY FROM NIH 3T3 CELLS AS AMP-ACTIVATED PROTEIN-KINASE

Raf-1 is extensively phosphorylated on Ser(621) in both quiescent and mitogen-stimulated cells, To identify the responsible kinase(s), cytos olic fractions of NIH 3T3 cells were analyzed for Ser(621) peptide kin ase activity, One major peak of activity was detected and identified a s AMP-activated protein kinase (AMPK) by immunodepletion experiments, AMPK phosphorylated the catalytic domain of Raf-l, expressed in Escher ichia coli as a soluble GST fusion protein, to generate a single trypt ic [P-32]phosphopeptide containing exclusively phospho-Ser(621). AMPK also phosphorylated full-length, kinase-defective Raf-1 (K375M) to gen erate two [P-32]phosphopeptides, one co-migrating with synthetic trypt ic peptide containing phospho-Ser(621) and the other with phospho-Ser( 259). (C) Federation of European Biochemical Societies.