Article
Identification Of Raf‐1 Ser621 Kinase Activity From NIH 3T3 Cells As AMP‐Activated Protein Kinase
FEBS Letters
(1997)
Abstract
Raf-1 is extensively phosphorylated on Ser in both quiescent and mitogen-stimulated cells. To identify the responsible kinase(s), cytosolic fractions of NIH 3T3 cells were analyzed for Ser6 21 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-Ser 2 . AMPK also phosphorylated full-length, kinasedefective Raf-1 (K375M) to generate two [32P]phosphopeptides, one co-migrating with synthetic tryptic peptide containing phospho-Ser621 and the other with phospho-Ser.
Keywords
- Raf-1,
- AMP-activated protein kinase,
- cAMP-dependent protein kinase,
- 14-3-3 protein AMPK,
- MAP,
- mitogen-activated protein PKA,
- cAMP-dependent protein kinase GST,
- glutathione S-transferase,
- DMEM,
- Dulbecco's modified Eagle's medium,
- MCLR,
- microcystin-LR,
- DTT,
- dithiothreitol,
- 2-ME,
- 2-mercaptoethanol,
- HVE,
- high-voltage electrophoresis,
- FSBA,
- p-fluorosulfonylbenzoyl 5′-adenosine
Disciplines
Publication Date
February 24, 1997
DOI
10.1016/S0014-5793(97)00062-8
Citation Information
Amy B Sprenkle, Stephen P Davies, David Carling, D.Grahame Hardie, et al.. "Identification Of Raf‐1 Ser621 Kinase Activity From NIH 3T3 Cells As AMP‐Activated Protein Kinase" FEBS Letters Vol. 403 Iss. 3 (1997) p. 254 - 258 Available at: http://works.bepress.com/amy-sprenkle/5/