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Mechanism of inhibition of Raf-1 by protein kinase A
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.
Insights
Protein kinase A (PKA) directly inhibits Raf-1 kinase activity through phosphorylation, independent of Ras binding. This provides a novel mechanism for regulating mitogenic signaling pathways.
Area of Science:
- Cellular signaling
- Molecular biology
- Biochemistry
Background:
- Raf-1 kinase is crucial for mitogenic signaling pathways activated by growth factors and phorbol esters.
- Cyclic AMP-dependent protein kinase A (PKA) is known to inhibit Raf-1 signaling, but the precise mechanism remains unclear.
- Previous hypotheses suggested PKA inhibits Raf-1 by reducing its binding to Ras.
Purpose of the Study:
- To elucidate the molecular mechanism by which PKA inhibits Raf-1 kinase.
- To investigate whether PKA's inhibition of Raf-1 involves direct phosphorylation of the kinase domain.
- To determine if PKA-mediated inhibition of Raf-1 is dependent on its interaction with Ras.
Main Methods:
- Utilized purified proteins to study interactions and enzymatic activities.
- Investigated the effect of PKA phosphorylation on Raf-1 kinase activity and its activation by other kinases (PKC alpha, Lck).
- Examined the functional consequences of PKA phosphorylation on Raf-1's interaction with Ras and its kinase domain activity.
Main Results:
- PKA directly inhibits Raf-1 kinase activity via phosphorylation of its kinase domain, independent of Ras binding.
- PKA phosphorylation interferes with Raf-1 activation by PKC alpha and Lck.
- PKA can downregulate the kinase activity of already activated Raf-1.
- An isolated Raf-1 kinase domain, lacking the Ras-binding domain, remains susceptible to PKA inhibition.
- PKC alpha phosphorylation reduces PKA's effect on Ras binding but not on kinase activity inhibition.
Conclusions:
- PKA directly phosphorylates and inhibits the Raf-1 kinase domain, offering a mechanism beyond Ras interaction modulation.
- This direct inhibition by PKA provides a critical regulatory point in mitogenic signaling pathways.
- Understanding this PKA-mediated inhibition is key for targeting signaling in diseases like cancer.