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Negative regulation of Raf-1 by phosphorylation of serine 621
H Mischak1, T Seitz, P Janosch
1GSF-Institut für Klinische Molekularbiologie und Tumorgenetik, Munich, Germany.
Abstract:
The elevation of cyclic AMP (cAMP) levels in the cell downregulates the activity of the Raf-1 kinase. It has been suggested that this effect is due to the activation of cAMP-dependent protein kinase (PKA), which can directly phosphorylate Raf-1 in vitro. In this study, we confirmed this hypothesis by coexpressing Raf-1 with the constitutively active catalytic subunit of PKA, which could fully reproduce the inhibition previously achieved by cAMP. PKA-phosphorylated Raf-1 exhibits a reduced affinity for GTP-loaded Ras as well as impaired catalytic activity. As the binding to GTP-loaded Ras induces Raf-1 activation in the cell, we examined which mechanism is required for PKA-mediated Raf-1 inhibition in vivo. A Raf-1 point mutant (RafR89L), which is unable to bind Ras, as well as the isolated Raf-1 kinase domain were still fully susceptible to inhibition by PKA, demonstrating that the phosphorylation of the Raf-1 kinase suffices for inhibition. By the use of mass spectroscopy and point mutants, PKA phosphorylation site was mapped to a single site in the Raf-1 kinase domain, serine 621. Replacement of serine 621 by alanine or cysteine or destruction of the PKA consensus motif by changing arginine 618 resulted in the loss of catalytic activity. Notably, a mutation of serine 619 to alanine did not significantly affect kinase activity or regulation by activators or PKA. Changing serine 621 to aspartic acid yielded a Raf-1 protein which, when expressed to high levels in Sf-9 insect cells, retained a very low inducible kinase activity that was resistant to PKA downregulation. The purified Raf-1 kinase domain displayed slow autophosphorylation of serine 621, which correlated with a decrease in catalytic function. The Raf-1 kinase domain activated by tyrosine phosphorylation could be downregulated by PKA. Specific removal of the phosphate residue at serine 621 reactivated the catalytic activity. These results are most consistent with a dual role of serine 621. On the one hand, serine 621 appears essential for catalytic activity; on the other hand, it serves as a phosphorylation site which confers negative regulation.
Insights
Elevated cyclic AMP (cAMP) activates protein kinase A (PKA), which inhibits Raf-1 kinase activity by phosphorylating serine 621. This phosphorylation reduces Ras binding and catalytic function, crucial for Raf-1 regulation.
Area of Science:
- Molecular Biology
- Cell Signaling
- Enzymology
Background:
- Cyclic AMP (cAMP) signaling pathway regulates cellular processes.
- Raf-1 kinase is a key component in signal transduction pathways.
- cAMP-dependent protein kinase (PKA) is implicated in regulating Raf-1 activity.
Purpose of the Study:
- To confirm the hypothesis that PKA activation by cAMP inhibits Raf-1 kinase.
- To elucidate the mechanism of PKA-mediated Raf-1 inhibition in vivo.
- To identify the specific phosphorylation site on Raf-1 responsible for PKA-mediated inhibition.
Main Methods:
- Coexpression of Raf-1 and constitutively active PKA catalytic subunit.
- Site-directed mutagenesis of Raf-1.
- Mass spectrometry to map phosphorylation sites.
- In vitro kinase assays and analysis of Ras binding affinity.
Main Results:
- PKA directly phosphorylates Raf-1, leading to inhibition of its catalytic activity and reduced affinity for Ras.
- PKA-mediated inhibition of Raf-1 occurs independently of Ras binding.
- Serine 621 was identified as the critical PKA phosphorylation site conferring negative regulation.
- Phosphorylation at serine 621 is essential for catalytic activity and serves as a negative regulatory site.
Conclusions:
- PKA-mediated phosphorylation of serine 621 is sufficient to inhibit Raf-1 kinase activity.
- Serine 621 plays a dual role, being essential for catalytic function and acting as a negative regulatory phosphorylation site.
- This study clarifies the molecular mechanism by which cAMP signaling regulates Raf-1 activity.