Oncogenes, growth factors and phorbol esters regulate Raf-1 through common mechanisms (original) (raw)
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Regulation of the Raf-1 kinase domain by phosphorylation and 14-3-3 association
Biochemical Journal, 2000
The Raf-1 kinase domain is kept in an inactive state by the Nterminal regulatory domain. Activation of the kinase domain occurs following release from the N-terminal repression and possible catalytic upregulation. To distinguish the regulatory mechanisms that directly influence the catalytic activity of the enzyme from those which act through the inhibitory domain, the catalytic domain of Raf-1 (CR3) was expressed in COS-7 cells. The role of phosphorylation in the direct regulation of this domain was determined by substituting non-phosphorylatable amino acids for known serine and tyrosine phosphorylation sites. The intrinsic activity of each mutant protein was determined as well as stimulation by v-Src and phorbol esters. Both v-Src and phorbol esters were potent activators of CR3, requiring the serine 338\339 (p21-activated protein kinase, Pak) and tyrosine 340\341 (Src) phosphorylation sites for full stimulation of CR3. In contrast, loss of the serine 497\499 protein kinase C phosphorylation sites had little effect on CR3 activation by either v-Src or phorbol esters. Loss of serine 621, a 14-3-3 adaptorprotein-binding site, prevented activation of CR3 by v-Src or phorbol esters and partially decreased the high basal activity of
Phosphatase and Feedback Regulation of Raf-1 Signaling
Cell Cycle, 2007
The Raf-1 kinase is an effector of Ras GTPases that lies at the apex of the three-tier Raf/MEK/ERK pathway. Raf-1 activation is a complex process that entails two major events-relief of autoinhibition imposed by the regulatory domain and kinase domain activation. Recent studies indicate that the transition of Raf-1 from an active to an inactive state bears similar complexity to the activation process. Both these events require dynamic changes in Raf-1 phosphorylation. Here, we discuss the critical role of phosphatases and feedback phosphorylation during activation and inactivation of Raf-1 signaling. IntroDuctIon The Ras/Raf/MEK/ERK pathway regulates cell fate by relaying many extracellular growth and mitogenic signals to the nucleus. Depending on the cellular context, activation of this pathway can alter fundamental processes such as proliferation, differentiation, migration and apoptosis. Not surprisingly, irregularities in the activation of this pathway are associated with several diseases, most notably with cancer. 1 The mammalian Raf family of serine/threonine kinases comprises three members, A-Raf, B-Raf and Raf-1, of which the latter is the best studied. Extracellular signals are transmitted to Raf-1 through receptor-induced activation of Ras GTPases. Active, GTP-bound Ras, binds and recruits Raf-1 from the cytosol to the plasma membrane, setting in motion a multi-step activation process involving dynamic changes in intra-and inter molecular interactions as well as phosphorylation. Raf-1 consists of a N-terminal regulatory domain and a C-terminal catalytic domain (Fig. 1). The N-terminal regulatory domain of Raf-1 encompasses a region that binds GTP-loaded Ras (CR1) and a serine-rich region (CR2) that binds 14-when serine 259 (S259) is phosphorylated. The CR region of Raf-1 consists of the kinase domain and makes up the bulk of the catalytic domain. A region known as the "N-region" lies at the N-terminal of the kinase domain and contains two important activating phosphorylation sites, serine 8 (S8) and tyrosine 41 (Y41). Phosphorylation of Y41 in response to physiological stimuli has proven difficult to detect, but mutational data suggest an important role for this residue in Raf-1 activation. The C-terminus of the catalytic domain contains a second phosphorylation-dependent 14-binding site, serine 621 (S621). Activation of Raf-1 by growth factors entails two key steps. First, the autoinhibition imposed by the regulatory domain of Raf-1 on its catalytic domain must be neutralised. Next, multiple phosphorylations in catalytic domain are required to elevate its basal activity.
Current Biology, 2000
Activation of the protein kinase Raf-1 is a complex process involving association with the GTP-bound form of Ras (Ras-GTP), membrane translocation and both serine/threonine and tyrosine phosphorylation (reviewed in [1]). We have reported previously that p21-activated kinase 3 (Pak3) upregulates Raf-1 through direct phosphorylation on Ser338 [2]. Here, we investigated the origin of the signal for Pak-mediated Raf-1 activation by examining the role of the small GTPases Cdc42, Rac and Ras, and of phosphatidylinositol (PI) 3-kinase. Pak3 acted synergistically with either Cdc42V12 or Rac1V12 to stimulate the activities of Raf-1, Raf-CX, a membranelocalized Raf-1 mutant, and Raf-1 mutants defective in Ras binding. Raf-1 mutants defective in Ras binding were also readily activated by RasV12. This indirect activation of Raf-1 by Ras was blocked by a dominant-negative mutant of Pak, implicating an alternative Ras effector pathway in Pak-mediated Raf-1 activation. Subsequently, we show that Pak-mediated Raf-1 activation is upregulated by both RasV12C40, a selective activator of PI 3-kinase, and p110-CX, a constitutively active PI 3-kinase. In addition, p85∆ ∆, a mutant of the PI 3-kinase regulatory subunit, inhibited the stimulated activity of Raf-1. Pharmacological inhibitors of PI 3-kinase also blocked both activation and Ser338 phosphorylation of Raf-1 induced by epidermal growth factor (EGF). Thus, Raf-1 activation by Ras is achieved through a combination of both physical interaction and indirect mechanisms involving the activation of a second Ras effector, PI 3-kinase, which directs Pak-mediated regulatory phosphorylation of Raf-1.
Molecular and Cellular Biology, 1997
Activation of the Raf serine/threonine protein kinases is tightly regulated by multiple phosphorylation events. Phosphorylation of either tyrosine 340 or 341 in the catalytic domain of Raf-1 has been previously shown to induce the ability of the protein kinase to phosphorylate MEK. By using a combination of mitogenic and enzymatic assays, we found that phosphorylation of the adjacent residue, serine 338, and, to a lesser extent, serine 339 is essential for the biological and enzymatic activities of Raf-1. Replacement of S338 with alanine blocked the ability of prenylated Raf-CX to transform Rat-1 fibroblasts. Similarly, the loss of S338-S339 in Raf-1 prevented protein kinase activation in COS-7 cells by either oncogenic Ras[V12] or v-Src. Consistent with phosphorylation of S338-S339, acidic amino acid substitutions of these residues partially restored transforming activity to Raf-CX, as well as kinase activation of Raf-1 by Ras[V12] or v-Src. Two-dimensional phosphopeptide mapping o...
Negative regulation of Raf-1 by phosphorylation of serine 621
Molecular and cellular biology, 1996
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...
Cyclic AMP-Dependent Kinase Regulates Raf-1 Kinase Mainly by Phosphorylation of Serine 259
Molecular and Cellular Biology, 2002
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.
Regulation of Raf-1 activation and signalling by dephosphorylation
The EMBO Journal, 2002
The Raf-1 kinase is regulated by phosphorylation, and Ser259 has been identi®ed as an inhibitory phosphorylation site. Here we show that the dephosphorylation of Ser259 is an essential part of the Raf-1 activation process, and further reveal the molecular role of Ser259. The fraction of Raf-1 that is phosphorylated on Ser259 is refractory to mitogenic stimulation. Mutating Ser259 elevates kinase activity because of enhanced binding to Ras and constitutive membrane recruitment. This facilitates the phosphorylation of an activating site, Ser338. The mutation of Ser259 also increases the functional coupling to MEK, augmenting the ef®ciency of MEK activation. Our results suggest that Ser259 regulates the coupling of Raf-1 to upstream activators as well as to its downstream substrate MEK, thus determining the pool of Raf-1 that is competent for signalling. They also suggest a new model for Raf-1 activation where the release of repression through Ser259 dephosphorylation is the pivotal step.
Molecular Biology of the Cell, 2005
The Ras-Raf-mitogen-activated protein kinase cascade is a key growth-signaling pathway, which uncontrolled activation results in transformation. Although the exact mechanisms underlying Raf-1 regulation remain incompletely understood, phosphorylation has been proposed to play a critical role in this regulation. We report here three novel epidermal growth factor-induced in vivo Raf-1 phosphorylation sites that mediate positive feedback Raf-1 regulation. Using mass spectrometry, we identified Raf-1 phosphorylation on three SP motif sites: S289/S296/S301 and confirmed their identity using twodimensional-phosphopeptide mapping and phosphospecific antibodies. These sites were phosphorylated by extracellular signal-regulated kinase (ERK)-1 in vitro, and their phosphorylation in vivo was dependent on endogenous ERK activity. Functionally, ERK-1 expression sustains Raf-1 activation in a manner dependent on Raf-1 phosphorylation on the identified sites, and S289/296/301A substitution markedly decreases the in vivo activity of Raf-1 S259A. Importantly, the ERKphosphorylated Raf-1 pool has 4 times higher specific kinase activity than total Raf-1, and its phosphopeptide composition is similar to that of the general Raf-1 population, suggesting that the preexisting, phosphorylated Raf-1, representing the activatable Raf-1 pool, is the Raf-1 subpopulation targeted by ERK. Our study describes the identification of new in vivo Raf-1 phosphorylation sites targeted by ERK and provides a novel mechanism for a positive feedback Raf-1 regulation.
Second nature: Biological functions of the Raf-1 “kinase”
FEBS Letters, 2005
More than 20 years ago, Raf was discovered as a cellular oncogene transduced by transforming retroviruses. Since then, the three Raf isoforms have been intensively studied, mainly as the kinases linking Ras to the MEK/ERK signaling module. As this pathway is activated in human cancer, the Raf kinases are considered promising therapeutic targets, and we have learned a lot about their regulation, targets, and functions. Do they still hold surprises? Recent gene targeting studies indicate that they do. This review focuses on the regulation and biology of the best-studied Raf isoform, Raf-1, in the context of its kinase-independent functions.