No 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.

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.The Raf-1 kinase is at the interface of a signaling pathway that connects cell surface receptors to nuclear transcription factors. Raf-1 is the entry point to the ERK/MAPK (extracellular-signal-regulated kinase/mitogen-activated protein kinase) pathway. It phosphorylates and activates MEK (MAPK/ERK kinase), which in turn phosphorylates and activates ERK. Raf-1 activation is initiated by binding of Raf-1 to GTP-loaded Ras, which results in the translocation of Raf-1 from the cytosol to the cell membrane, where activation takes place (reviewed in references 4, 18, 21, 22, and 26). Activation involves phosphorylation on serine 338 (17, 23) and tyrosine 341 (10, 23) as well as other yet-unknown modifications. It has been extremely difficult to identify the activating modifications, in part because presumably only a small fraction of Raf-1 becomes activated (14). Hence, despite receiving intensive attention during the last 10 years, several facets of Raf-1 regulation have evaded elucidation. This also pertains to the cross talk between the Raf-MEK-ERK and cyclic AMP (cAMP) signaling systems; and the mechanism of Raf-1 regulation by cAMP has not been completely clarified (15).In resting cells Raf-1 is phosphorylated on serines 43, 259, and 621 (27). Serines 43 and 621 have been previously described as target sites for the cAMP-regulated protein kinase, PKA, which inhibits Raf-1 (13, 25, 38). Phosphorylation of serine 43 was reported to diminish the affinity of Raf-1 for Ras (38) and thereby to interfere with Raf-1 activation (30, 38), although recent results dispute this (34). The role of serine 621 phosphorylation is more difficult to study, because mutation of this residue is incompatible with kinase function (25,27). This observation was taken as an indication that serine 621 phosphorylation is essential for Raf-1 function. Biochemical experiments confirmed that th...