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Cyclic AMP-Dependent Kinase Regulates Raf-1 Kinase Mainly by Phosphorylation of Serine 259

Amardeep S. Dhillon,¹ Claire Pollock,¹ Helge Steen,^2, Peter E. Shaw,² Harald Mischak,³ and Walter Kolch^1,4*

The Beatson Institute for Cancer Research, Bearsden, Glasgow G61 1BD,¹ School of Biomedical Sciences, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH,² Institute for Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, United Kingdom,⁴ Department of Nephrology, Medizinische Hochschule Hannover, 30625 Hannover, Germany³

Received 27 July 2001/ Returned for modification 19 September 2001/ Accepted 8 February 2002

The Raf-1 kinase activates the ERK (extracellular-signal-regulated kinase) pathway. The cyclic AMP (cAMP)-dependent protein kinase (PKA) can inhibit Raf-1 by direct phosphorylation. We have mapped all cAMP-induced phosphorylation sites in Raf-1, showing that serines 43, 259, and 621 are phosphorylated by PKA in vitro and induced by cAMP in vivo. Serine 43 phosphorylation decreased the binding to Ras in serum-starved but not in mitogen-stimulated cells. However, the kinase activity of a RafS43A mutant was fully inhibited by PKA. Mutation of serine 259 increased the basal Raf-1 activity and rendered it largely resistant to inhibition by PKA. cAMP increased Raf-1 serine 259 phosphorylation in a PKA-dependent manner with kinetics that correlated with ERK deactivation. PKA also decreased Raf-1 serine 338 phosphorylation of Raf-1, previously shown to be required for Raf-1 activation. Serine 338 phosphorylation of a RafS259A mutant was unaffected by PKA. Using RafS259 mutants we also demonstrate that Raf-1 is the sole target for PKA inhibition of ERK and ERK-induced gene expression, and that Raf-1 inhibition is mediated mainly through serine 259 phosphorylation.

Present address: BioChip Technologies GmbH, GeneScan Europe AG, D-79108 Freiburg, Germany.

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