Mechanism of Inhibition of Raf-1 by Protein Kinase A

• Published in: Molecular and cellular biology Vol. 14; no. 10; pp. 6696 - 6703
• Main Authors: Häfner, Susanne, Adler, Henric S., Mischak, Harald, Janosch, Petra, Heidecker, Gisela, Wolfman, Alan, Pippig, Susanne, Lohse, Martin, Ueffing, Marius, Kolch, Walter
• Format: Journal Article
• Language: English
• Published: Taylor & Francis 01.10.1994
• ISSN: 1098-5549; 1098-5549
• DOI: 10.1128/mcb.14.10.6696-6703.1994

The cytoplasmic Raf-1 kinase is essential for mitogenic signalling by growth factors, which couple to tyrosine kinases, and by tumor-promoting phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate, which activate protein kinase C (PKC). Signalling by the Raf-1 kinase can be blocked by activation of the cyclic AMP (cAMP)-dependent protein kinase A (PKA). The molecular mechanism of this inhibition is not precisely known but has been suggested to involve attenuation of Raf-1 binding to Ras. Using purified proteins, we show that in addition to weakening the interaction of Raf-1 with Ras, PKA can inhibit Raf-1 function directly via phosphorylation of the Raf-1 kinase domain. Phosphorylation by PKA interferes with the activation of Raf-1 by either PKCα or the tyrosine kinase Lck and even can downregulate the kinase activity of Raf-1 previously activated by PKCα or amino-terminal truncation. This type of inhibition can be dissociated from the ability of Raf-1 to associate with Ras, since (i) the isolated Raf-1 kinase domain, which lacks the Ras binding domain, is still susceptible to inhibition by PKA, (ii) phosphorylation of Raf-1 by PKCα alleviates the PKA-induced reduction of Ras binding but does not prevent the downregulation of Raf-1 kinase activity by PKA and (iii) cAMP agonists antagonize transformation by v-Raf, which is Ras independent.