Regulation of Raf-1 by Direct Feedback Phosphorylation

Abstract

The Raf-1 kinase is an important signaling molecule, functioning in the Ras pathway to transmit mitogenic, differentiative, and oncogenic signals to the downstream kinases MEK and ERK. Because of its integral role in cell signaling, Raf-1 activity must be precisely controlled. Previous studies have shown that phosphorylation is required for Raf-1 activation, and here, we identify six phosphorylation sites that contribute to the downregulation of Raf-1 after mitogen stimulation. Five of the identified sites are proline-directed targets of activated ERK, and phosphorylation of all six sites requires MEK signaling, indicating a negative feedback mechanism. Hyperphosphorylation of these six sites inhibits the Ras/Raf-1 interaction and desensitizes Raf-1 to additional stimuli. The hyperphosphorylated/desensitized Raf-1 is subsequently dephosphorylated and returned to a signaling-competent state through interactions with the protein phosphatase PP2A and the prolyl isomerase Pin1. These findings elucidate a critical Raf-1 regulatory mechanism that contributes to the sensitive, temporal modulation of Ras signaling.

Identification of Novel In Vivo Raf-1 Phosphorylation Sites Mediating Positive Feedback Raf-1 Regulation by Extracellular Signal-regulated Kinase

“…Our present findings are in agreement with these previous reports offering a positive role of MEK and ERK in Raf-1 regulation. More recently, after the initial submission of this manuscript, two groups reported Raf-1 phosphorylation on similar sites as reported here (Dougherty et al, 2005;Hekman et al, 2005). Both studies proposed a negative regulatory role for the identified phosphorylations sites in Raf-1 regulation, leading to the enticing hypothesis that ERK participates in a negative feedback loop responsible for down-regulating Raf-1 activity (Dumaz and Marais, 2005).…”

“…Our data, in contrast to the authors claims that this site is regulated downstream of ERK, show little or no change in S43 phosphorylation in the MEK inhibitor-treated cells, whereas resulting in a complete block of the ERK-induced sites (Figure 3). In addition, in our experiments, we did not observe an increase in the Raf-1 activation kinetics in MEK-inhibited cells ( Figure 6B) as observed by Dougherty et al (2005); however, we used COS-7 cells and EGF as stimulus, whereas Dougherty et al (2005) used NIH 3T3 cells and PDGF stimulation. Although remotely likely, it remains a possibility that the differences in our results regarding this specific observation are cell type/mitogen dependent.…”

“…Thus, the difference at 2 min after EGF stimulation between wild type and the phosphorylation site Raf-1 mutants observed by Hekman et al (2005)may not necessarily represent the effects of ERK-induces Raf-1 phosphorylation. At an early time point, also Dougherty et al (2005) did not observe differences in the activities of wild type and mutant Raf-1. The effect of ERK on Raf-1 activity either in vitro or in vivo has not been examined in this work.…”

“…The effect of ERK on Raf-1 activity either in vitro or in vivo has not been examined in this work. Dougherty et al (2005) reported that Raf-1 is phosphorylated in response to platelet-derived growth factor (PDGF) treatment, resulting in an upshift in Raf-1 mobility on SDS-PAGE that coincided with decreased kinase activity of Raf-1. The authors identified five ERK-mediated Raf-1 phosphorylation sites, including S29, S289, S296, S301, and S642 and showed that their mutation together with S43, a previously identified negative Raf-1 regulatory site targeted by PKA, prevents the upshift in mobility and decreases its inactivation rate.…”

Activation of Ras/Raf protects cells from melanoma differentiation-associated gene-5-induced apoptosis

“…Additionally, impairment of ras activity hinders raf activation. 6,[9][10][11] Aberrant activation of this pathway is frequently linked with tumor progression and malignancy, which can contribute to the low efficacy of chemotherapy in patients. 6 Mutations leading to constitutive activation of ras have been found in approximately 30% of all human cancers.…”