Identification of Raf-1 Ser621 kinase activity from NIH 3T3 cells as AMP-activated protein kinase.
Raf-1 is extensively phosphorylated on Ser621 in both quiescent and mitogen-stimulated cells. To identify the responsible kinase(s), cytosolic fractions of NIH 3T3 cells were analyzed for Ser621 peptide kinase activity. One major peak of activity was detected and identified as AMP-activated protein kinase (AMPK) by immunodepletion experiments. AMPK phosphorylated the catalytic domain of Raf-1, expressed in Escherichia coli as a soluble GST fusion protein, to generate a single tryptic [32P]phosphopeptide containing exclusively phospho-Ser621. AMPK also phosphorylated full-length, kinase-defective Raf-1 (K375M) to generate two [32P]phosphopeptides, one co-migrating with synthetic tryptic peptide containing phospho-Ser621 and the other with phospho-Ser259.
Sprenkle
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Howard Hughes Medical Institute, Department of Medicine, University of Virginia, Charlottesville 22908, USA.
Davies
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SP
Carling
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D
Hardie
D G
DG
Sturgill
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TW
D016475:3T3 Cells / Q000201:enzymology*; D055372:AMP-Activated Protein Kinases; D000595:Amino Acid Sequence; D000818:Animals; D051379:Mice; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000032:analysis; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D012694:Serine / Q000378:metabolism*
D009097:Multienzyme Complexes; D010748:Phosphopeptides; D011518:Proto-Oncogene Proteins; D012694:Serine; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D055372:AMP-Activated Protein Kinases