Abstract
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 PKC alpha or the tyrosine kinase Lck and even can downregulate
the kinase activity of Raf-1 previously activated by PKC alpha 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
PKC alpha 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.
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