Regulation of the protein kinase Raf-1 by oncogenic Ras through phosphatidylinositol 3-kinase, Cdc42/Rac and Pak

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 GTPase 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 membrane-localized 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, p85Delta, 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.     

Microtubule integrity regulates Pak leading to Ras-independent activation of Raf-1. insights into mechanisms of Raf-1 activation

Growth factors activate Raf-1 by engaging a complex program, which requires Ras binding, membrane recruitment, and phosphorylation of Raf-1. The present study employs the microtubule-depolymerizing drug nocodazole as an alternative approach to explore the mechanisms of Raf activation. Incubation of cells with nocodazole leads to activation of Pak1/2, kinases downstream of small GTPases Rac/Cdc42, which have been previously indicated to phosphorylate Raf-1 Ser(338). Nocodazole-induced stimulation of Raf-1 is augmented by co-expression of small GTPases Rac/Cdc42 and Pak1/2. Dominant negative mutants of these proteins block activation of Raf-1 by nocodazole, but not by epidermal growth factor (EGF). Thus, our studies define Rac/Cdc42/Pak as a module upstream of Raf-1 during its activation by microtubule disruption. Although it is Ras-independent, nocodazole-induced activation of Raf-1 appears to involve the amino-terminal regulatory region in which the integrity of the Ras binding domain is required. Surprisingly, the Raf zinc finger mutation (C165S/C168S) causes a robust activation of Raf-1 by nocodazole, whereas it diminishes Ras-dependent activation of Raf-1. We also show that mutation of residues Ser(338) to Ala or Tyr(340)-Tyr(341) to Phe-Phe immediately amino-terminal to the catalytic domain abrogates activation of both the wild type and zinc finger mutant Raf by both EGF/4beta-12-O-tetradecanoylphorbol-13-acetate and nocodazole. Finally, an in vitro kinase assay demonstrates that the zinc finger mutant serves as a better substrate of Pak1 than the wild type Raf-1. Collectively, our results indicate that 1) the zinc finger exerts an inhibitory effect on Raf-1 activation, probably by preventing phosphorylation of (338)SSYY(341); 2) such inhibition is first overcome by an unknown factor binding in place of Ras-GTP to the amino-terminal regulatory region in response to nocodazole; and 3) EGF and nocodazole utilize different kinases to phosphorylate Ser(338), an event crucial for Raf activation.     

Stretch-induced Raf-1 activation in mesangial cells requires actin cytoskeletal integrity

Glomerular capillary hypertension is a determinant of glomerulosclerosis and is modelled in vitro by exposure of mesangial cells to cyclic mechanical strain. In response to strain, Erk is activated and mediates extracellular matrix accumulation and mesangial cell proliferation. Erk activation is dependent on an intact cytoskeleton. Since Raf-1 lies upstream of Erk in response to numerous stimuli, and since its activation is dependent on membrane recruitment, we postulated that the cytoskeleton was essential for Raf-1 membrane recruitment and Erk activation. Primary rat mesangial cells (passages 8-20) were stretched at 1 Hz and 27 kPa. Raf-1 was both phosphorylated on serine-338 (S338) and activated within 2 min of strain. The Raf-1 inhibitor, GW5074, dose-dependently blocked strain-induced Erk activation and Raf-1 phosphorylation. Although phosphatidylinositol-3-kinase (PI3-K) may mediate Raf-1 activation, PI3-K inhibition with wortmannin or LY294002 had no effect on stretch-induced Raf-1 activation. Cytoskeletal disruption with cytochalasin D and the Rho-kinase inhibitor, Y-27632, however, blocked both Raf-1 phosphorylation and activation. Furthermore, membrane localization of Raf-1 was increased by strain and prevented by cytoskeletal disruption. Thus, strain leads to rapid membrane localization, S338 phosphorylation, and activation of Raf-1. These events are independent of PI3-K, but require Rho-kinase activation and an intact actin cytoskeleton.     

14-3-3 antagonizes Ras-mediated Raf-1 recruitment to the plasma membrane to maintain signaling fidelity

We have investigated the role that S259 phosphorylation, S621 phosphorylation, and 14-3-3 binding play in regulating Raf-1 activity. We show that 14-3-3 binding, rather than Raf-1 phosphorylation, is required for the correct regulation of kinase activity. Phosphorylation of S621 is not required for activity, but 14-3-3 binding is essential. When 14-3-3 binding to conserved region 2 (CR2) was disrupted, Raf-1 basal kinase activity was elevated and it could be further activated by (V12,G37)Ras, (V23)TC21, and (V38)R-Ras. Disruption of 14-3-3 binding at CR2 did not recover binding of Raf-1 to (V12,G37)Ras but allowed more efficient recruitment of Raf-1 to the plasma membrane and stimulated its phosphorylation on S338. Finally, (V12)Ras, but not (V12,G37)Ras, displaced 14-3-3 from full-length Raf-1 and the Raf-1 bound to Ras. GTP was still phosphorylated on S259. Our data suggest that stable association of Raf-1 with the plasma membrane requires Ras-mediated displacement of 14-3-3 from CR2. Small G proteins that cannot displace 14-3-3 fail to recruit Raf-1 to the membrane efficiently and so fail to stimulate kinase activity.     

Phosphorylation site specificity of the Pak-mediated regulation of Raf-1 and cooperativity with Src

The p21-activated kinase, Pak, has recently been shown to phosphorylate Raf-1 on serine 338 (S338), a critical regulatory residue. The specificity requirements for Pak-mediated phosphorylation of S338 were examined by substitution analysis of Raf-1 peptides and conserved region 3 (CR3) proteins. Phosphorylation was found to be very sensitive to alterations in amino acid side chains proximal to S338. Loss of N-terminal arginines resulted in decreased peptide phosphorylation while loss of these residues, as well as C-terminal glutamates and bulky C-terminal hydrophobic residues, decreased phosphorylation of the CR3 protein. Phosphorylation of Raf-1 on tyrosine 341 is significant in epidermal growth factor- and Src-mediated signaling, suggesting that cooperativity may exist between Pak and Src phosphorylation of Raf-1. Purified Pak and Src were found not to be cooperative in phosphorylating peptides or purified CR3 protein. However, the phosphorylation of Raf-1 S338 by Pak was increased in the presence of Src. The complexity of this signaling module could thus account for the different levels of Raf-1 activation required for fulfillment of different biological roles within the cell.     

Calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylates Raf-1 at serine 338 and mediates Ras-stimulated Raf-1 activation

The calcium/calmodulin-dependent kinase II (CaMKII) participates with Ras to Raf-1 activation, and it is necessary for activation of the extracellular signal-regulated kinase (ERK) by different factors in epithelial and mesenchimal cells. Raf-1 activation is a complex multistep process, and its maximal activation is achieved by phosphorylation at Y341 by Src and at S338 by other kinase/s. Although early data proposed the involvement of p21-activated kinase 3 (Pak3), the kinase phosphorylating S338 remains to be definitively identified. In this study, we verified the hypothesis that CaMKII phosphorylates Raf-1 at Ser338. To do so, we determined the role of CaMKII in Raf-1 and ERK activation by oncogenic Ras and other factors. Serum, fibronectin, Src^Y527 and Ras^V12 activated CaMKII and ERK, at different extents. The inhibition of CaMKII attenuated Raf-1 and ERK activation by all these factors. CaMKII was also necessary for the phosphorylation of Raf-1 at S338 by serum, fibronectin and Ras. Conversely, inhibition of Pak3 activation by blocking phosphatidylinositol 3-kinase was ineffective. The direct phosphorylation of S338 Raf-1 by CaMKII was demonstrated in vitro by interaction of purified kinases. These results demonstrate that Ras activates CaMKII, which, in turn, phosphorylates Raf-1 at S338 and participates in ERK activation upon different stimuli.     

Ras couples phosphoinositide 3-OH kinase to the epithelial Na channel

Aldosterone induces the expression of the small G protein K-Ras. Both K-Ras and its 1st effector phosphoinositide 3-OH kinase (PI3-K) are necessary and sufficient for the activation of ENaC increasing channel open probability. The cell signaling mechanism by which K-Ras enhances ENaC activity, however, is uncertain. We demonstrate here that K-Ras significantly activates human ENaC reconstituted in Chinese hamster ovary cells ∼3-fold. Activation in response to K-Ras was sensitive to the irreversible PI3-K inhibitor wortmannin but not the competitive LY294002 inhibitor of this phospholipid kinase. Similarly, a PI3-K 1st effector-specific Ras mutant (G12:C40) enhanced ENaC activity in a wortmannin but not LY294002 sensitive manner. Constitutively active PI3-K also enhanced ENaC activity but in a wortmannin and LY294002 sensitive manner with the effects of PI3-K and K-Ras not being additive. The activation of ENaC by PI3-K was also sensitive to intracellular GDPβS. Constitutively active PI3-K that is incapable of interacting with K-Ras (K227E p110α) acted as dominant negative with respect to the regulation of ENaC even in the presence of K-Ras. K-Ras is known to directly interact with PI3-K with aldosterone promoting this interaction. Here we demonstrate that K-Ras also interacts with ENaC through an, as yet, undetermined mechanism. We conclude that K-Ras enhances ENaC activity by localizing PI3-K near the channel and stimulating of PI3-K activity.     

Phosphorylation of Raf-1 serine 338-serine 339 is an essential regulatory event for Ras-dependent activation and biological signaling.

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 of wild-type Raf-CX and Raf-CX[A338A339] confirmed the presence of a phosphoserine-containing peptide with the predicted mobility in the wild-type protein which was absent from the mutant. This peptide could be quantitatively precipitated by an antipeptide antibody specific for the 18-residue tryptic peptide containing S338-S339 and was demonstrated to contain only phosphoserine. Phosphorylation of this peptide in Raf-1 was significantly increased by coexpression with Ras[V12]. These data demonstrate that Raf-1 residues 338 to 341 constitute a unique phosphoregulatory site in which the phosphorylation of serine and tyrosine residues contributes to the regulation of Raf by Ras, Src, and Ras-independent membrane localization.     

Ras couples phosphoinositide 3-OH kinase to the epithelial Na+ channel

Aldosterone induces the expression of the small G protein K-Ras. Both K-Ras and its 1st effector phosphoinositide 3-OH kinase (PI3-K) are necessary and sufficient for the activation of ENaC increasing channel open probability. The cell signaling mechanism by which K-Ras enhances ENaC activity, however, is uncertain. We demonstrate here that K-Ras significantly activates human ENaC reconstituted in Chinese hamster ovary cells approximately 3-fold. Activation in response to K-Ras was sensitive to the irreversible PI3-K inhibitor wortmannin but not the competitive LY294002 inhibitor of this phospholipid kinase. Similarly, a PI3-K 1st effector-specific Ras mutant (G12:C40) enhanced ENaC activity in a wortmannin but not LY294002 sensitive manner. Constitutively active PI3-K also enhanced ENaC activity but in a wortmannin and LY294002 sensitive manner with the effects of PI3-K and K-Ras not being additive. The activation of ENaC by PI3-K was also sensitive to intracellular GDPbetaS. Constitutively active PI3-K that is incapable of interacting with K-Ras (K227E p110alpha) acted as dominant negative with respect to the regulation of ENaC even in the presence of K-Ras. K-Ras is known to directly interact with PI3-K with aldosterone promoting this interaction. Here we demonstrate that K-Ras also interacts with ENaC through an, as yet, undetermined mechanism. We conclude that K-Ras enhances ENaC activity by localizing PI3-K near the channel and stimulating of PI3-K activity.     

Serine and tyrosine phosphorylations cooperate in Raf-1, but not B-Raf activation

The Raf family of serine/threonine protein kinases couple growth factor receptor stimulation to mitogen activated protein kinase activation, but their own regulation is poorly understood. Using phospho-specific antisera, we show that activated Raf-1 is phosphorylated on S338 and Y341. Expression of Raf-1 with oncogenic Ras gives predominantly S338 phosphorylation, whereas activated Src gives predominantly Y341 phosphorylation. Phosphorylation at both sites is maximal only when both oncogenic Ras and activated Src are present. Raf-1 that cannot interact with Ras-GTP is not phosphorylated, showing that phosphorylation is Ras dependent, presumably occurring at the plasma membrane. Mutations which prevent phosphorylation at either site block Raf-1 activation and maximal activity is seen only when both are phosphorylated. Mutations at S339 or Y340 do not block Raf-1 activation. While B-Raf lacks a tyrosine phosphorylation site equivalent to Y341 of Raf-1, S445 of B-Raf is equivalent to S338 of Raf-1. Phosphorylation of S445 is constitutive and is not stimulated by oncogenic Ras. However, S445 phosphorylation still contributes to B-Raf activation by elevating basal and consequently Ras-stimulated activity. Thus, there are considerable differences between the activation of the Raf proteins; Ras-GTP mediates two phosphorylation events required for Raf-1 activation but does not regulate such events for B-Raf.     

The T-cell receptor regulates Akt (protein kinase B) via a pathway involving Rac1 and phosphatidylinositide 3-kinase

The serine/threonine kinase Akt (also known as protein kinase B) (Akt/PKB) is activated upon T-cell antigen receptor (TCR) engagement or upon expression of an active form of phosphatidylinositide (PI) 3-kinase in T lymphocytes. Here we report that the small GTPase Rac1 is implicated in this pathway, connecting the receptor with the lipid kinase. We show that in Jurkat cells, activated forms of Rac1 or Cdc42, but not Rho, stimulate an increase in Akt/PKB activity. TCR-induced Akt/PKB activation is inhibited either by PI 3-kinase inhibitors (LY294002 and wortmannin) or by overexpression of a dominant negative mutant of Rac1 but not Cdc42. Accordingly, triggering of the TCR rapidly stimulates a transient increase in GTP-Rac content in these cells. Similar to TCR stimulation, L61Rac-induced Akt/PKB kinase activity is also LY294002 and wortmannin sensitive. However, induction of Akt/PKB activity by constitutive active PI 3-kinase is unaffected when dominant negative Rac1 is coexpressed, placing Rac1 upstream of PI 3-kinase in the signaling pathway. When analyzing the signaling hierarchy in the pathway leading to cytoskeleton rearrangements, we found that Rac1 acts downstream of PI 3-kinase, a finding that is in accordance with numerous studies in fibroblasts. Our results reveal a previously unrecognized role of the GTPase Rac1, acting upstream of PI 3-kinase in linking the TCR to Akt/PKB. This is the first report of a membrane receptor employing Rac1 as a downstream transducer for Akt/PKB activation.     

Phosphatidylinositol 3-kinase regulates Raf1 through Pak phosphorylation of serine 338

We have previously shown that inhibition of phosphatidylinositol (PI) 3-kinase severely attenuates the activation of extracellular signal-regulated kinase (Erk) following engagement of integrin/fibronectin receptors and that Raf is the critical target of PI 3-kinase regulation [1]. To investigate how PI 3-kinase regulates Raf, we examined sites on Raf1 required for regulation by PI 3-kinase and explored the mechanisms involved in this regulation. Serine 338 (Ser338), which was critical for fibronectin stimulation of Raf1, was phosphorylated in a PI 3-kinase-dependent manner following engagement of fibronectin receptors. In addition, fibronectin activation of a Raf1 mutant containing a phospho-mimic mutation (S338D) was independent of PI 3-kinase. Furthermore, integrin-induced activation of the serine/threonine kinase Pak-1, which has been shown to phosphorylate Raf1 Ser338, was also dependent on PI 3-kinase activity and expression of a kinase-inactive Pak-1 mutant blocked phosphorylation of Raf1 Ser338. These results indicate that PI 3-kinase regulates phosphorylation of Raf1 Ser338 through the serine/threonine kinase Pak. Thus, phosphorylation of Raf1 Ser338 through PI 3-kinase and Pak provides a co-stimulatory signal which together with Ras leads to strong activation of Raf1 kinase activity by integrins.     

Phosphatidylinositol 3-kinase is required for integrin-stimulated AKT and Raf-1/mitogen-activated protein kinase pathway activation.

Cell attachment to fibronectin stimulates the integrin-dependent interaction of p85-associated phosphatidylinositol (PI) 3-kinase with integrin-dependent focal adhesion kinase (FAK) as well as activation of the Ras/mitogen-activated protein (MAP) kinase pathway. However, it is not known if this PI 3-kinase-FAK interaction increases the synthesis of the 3-phosphorylated phosphoinositides (3-PPIs) or what role, if any, is played by activated PI 3-kinase in integrin signaling. We demonstrate here the integrin-dependent accumulation of the PI 3-kinase products, PI 3,4-bisphosphate [PI(3,4)P2] and PI(3,4,5)P3, as well as activation of AKT kinase, a serine/threonine kinase that can be stimulated by binding of PI(3,4)P2. The PI 3-kinase inhibitors wortmannin and LY294002 significantly decreased the integrin-induced accumulation of the 3-PPIs and activation of AKT kinase, without having significant effects on the levels of PI(4,5)P2 or tyrosine phosphorylation of paxillin. These inhibitors also reduced cell adhesion/spreading onto fibronectin but had no effect on attachment to polylysine. Interestingly, integrin-mediated Erk-2, Mek-1, and Raf-1 activation, but not Ras-GTP loading, was inhibited at least 80% by wortmannin and LY294002. In support of the pharmacologic results, fibronectin activation of Erk-2 and AKT kinases was completely inhibited by overexpression of a dominant interfering p85 subunit of PI 3-kinase. We conclude that integrin-mediated adhesion to fibronectin results in the accumulation of the PI 3-kinase products PI(3,4)P2 and PI(3,4,5)P3 as well as the PI 3-kinase-dependent activation of the kinases Raf-1, Mek-1, Erk-2, and AKT and that PI 3-kinase may function upstream of Raf-1 but downstream of Ras in integrin activation of Erk-2 MAP and AKT kinases.     

OxLDL induces mitogen-activated protein kinase activation mediated via PI3-kinase/Akt in vascular smooth muscle cells

Oxidized low-density lipoprotein (OxLDL) is a risk factor in atherosclerosis and stimulates multiple signaling pathways, including activation of phosphatidylinositol 3-kinase (PI3-K)/Akt and p42/p44 mitogen-activated protein kinase (MAPK), which are involved in mitogenesis of vascular smooth muscle cells (VSMCs). We therefore investigated the relationship between PI3-K/Akt and p42/p44 MAPK activation and cell proliferation induced by OxLDL. OxLDL stimulated Akt phosphorylation in a time- and concentration-dependent manner, as determined by Western blot analysis. Phosphorylation of Akt stimulated by OxLDL and epidermal growth factor (EGF) was attenuated by inhibitors of PI3-K (wortmannin and LY294002) and intracellular Ca2+ chelator (BAPTA/AM) plus EDTA. Pretreatment of VSMCs with pertussis toxin, cholera toxin, and forskolin for 24 h also attenuated the OxLDL-stimulated Akt phosphorylation. In addition, pretreatment of VSMCs with wortmannin or LY294002 inhibited OxLDL-stimulated p42/p44 MAPK phosphorylation and [3H]thymidine incorporation. Furthermore, treatment with U0126, an inhibitor of MAPK kinase (MEK)1/2, attenuated the p42/p44 MAPK phosphorylation, but had no effect on Akt activation in response to OxLDL and EGF. Overexpression of p85-DN or Akt-DN mutants attenuated MEK1/2 and p42/p44 MAPK phosphorylation stimulated by OxLDL and EGF. These results suggest that the mitogenic effect of OxLDL is, at least in part, mediated through activation of PI3-K/Akt/MEK/MAPK pathway in VSMCs.     

SDF-1 synergistically enhances IL-3-induced activation of the Raf-1/MEK/Erk signaling pathway through activation of Rac and its effector Pak kinases to promote hematopoiesis and chemotaxis

Stromal cell-derived factor 1 (SDF-1) cooperates with cytokines to promote hematopoiesis. Here we demonstrate that SDF-1 activates Erk synergistically with interleukin-3 (IL-3) in hematopoietic cells. Small GTPases Ras and Rac were prominently activated by IL-3 and SDF-1, respectively. In accordance with this, Raf-1 was significantly activated by IL-3 but not by SDF-1. SDF-1 strongly induced phosphorylation of Raf-1 on S338, the target site for the Rac effector Paks, and enhanced the IL-3-induced activation of Raf-1 and MEK. Furthermore, the synergistic activation of Erk was inhibited by expression of a dominant-negative mutant of Pak1 or that of Rac and was enhanced by an activated mutant of Pak1. SDF-1 and IL-3 also showed synergistic effects on expansion of hematopoietic cells and on induction of chemotaxis, which were both inhibited by the MEK inhibitor PD98059. These results suggest that SDF-1 synergistically enhances IL-3-induced Erk activation by up-regulating Raf-1 activity through the Rac effector Pak kinases to promote hematopoiesis.     

Interaction between active Pak1 and Raf-1 is necessary for phosphorylation and activation of Raf-1.

Activation of Raf-1 is a complex process in which phosphorylation of Ser(338)-Tyr(341) is a critical step. Previous studies have shown that Pak1/2 is implicated in both Ras-dependent and -independent activation of Raf-1 by phosphorylating Raf Ser(338). The present study explores the structural basis of Raf-1 phosphorylation by Pak1. We found that Pak directly associates with Raf-1 under both physiological and overexpressed conditions. The association is greatly stimulated by 4beta-12-O-tetradecanoylphorbol-13-acetate and nocodazole and by expression of the active mutants of Rac and Ras. The active forms of Pak generated by mutation of Thr(423) to Glu or truncation of the amino-terminal moiety exhibit a greater binding to Raf than the wild type, whereas the kinase-dead mutant Pak barely binds Raf. The extent of binding to Raf-1 is correlated with the ability of Pak to phosphorylate Raf and induce mitogen-activated protein kinase activation. Furthermore, the Raf-1 binding site is defined to the carboxyl terminus of the Pak catalytic domain. In addition, our results suggest that the amino-terminal regulatory region of Raf inhibits the interaction. Taken together, the results indicate that the interaction depends on the active conformations of Pak and Raf. They also argue that Pak1 is a physiological candidate for phosphorylation of Raf Ser(338) during the course of Raf activation.     

p21 activated kinase 5 activates Raf-1 and targets it to mitochondria

Raf-1 is an important effector of Ras mediated signaling and is a critical regulator of the ERK/MAPK pathway. Raf-1 activation is controlled in part by phosphorylation on multiple residues, including an obligate phosphorylation site at serine 338. Previously PAK1 and casein kinase II have been implicated as serine 338 kinases. To identify novel kinases that phosphorylate this site, we tested the ability of group II PAKs (PAKs 4-6) to control serine 338 phosphorylation. We observed that all group II PAKs were efficient serine 338 kinases, although only PAK1 and PAK5 significantly stimulated Raf-1 kinase activity. We also showed that PAK5 forms a tight complex with Raf-1 in the cell, but not A-Raf or B-Raf. Importantly, we also demonstrated that the association of Raf-1 with PAK5 targets a subpopulation of Raf-1 to mitochondria. These data indicate that PAK5 is a potent regulator of Raf-1 activity and may control Raf-1 dependent signaling at mitochondria.     

Cyclic AMP-dependent kinase regulates Raf-1 kinase mainly by phosphorylation of serine 259

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.     

Phosphorylation of Raf-1 by p21-activated kinase 1 and Src regulates Raf-1 autoinhibition

Exposure of cells to mitogens or growth factors stimulates Raf-1 activity through a complex mechanism that involves binding to active Ras, phosphorylation on multiple residues, and protein-protein interactions. Recently it was shown that the amino terminus of Raf-1 contains an autoregulatory domain that can inhibit its activity in Xenopus oocytes. In the present work we show that expression of the Raf-1 autoinhibitory domain blocks extracellular signal-regulated kinase 2 activation by the Raf-1 catalytic domain in mammalian cells. We also show that phosphorylation of Raf-1 on serine 338 by PAK1 and tyrosines 340 and 341 by Src relieves autoinhibition and that this occurs through a specific decrease in the binding of the Raf-1 regulatory domain to its catalytic domain. In addition, we demonstrate that phosphorylation of threonine 491 and serine 494, two phosphorylation sites in the catalytic domain that are required for Raf-1 activation, is unlikely to regulate autoinhibition. These results demonstrate that the autoinhibitory domain of Raf-1 is functional in mammalian cells and that its interaction with the Raf-1 catalytic domain is regulated by phosphorylation of serine 338 and tyrosines 340 and 341.     

p21-activated Kinase 1 (Pak1)-dependent phosphorylation of Raf-1 regulates its mitochondrial localization, phosphorylation of BAD, and Bcl-2 association

Raf-1 protects cells from apoptosis, independently of its signals to MEK and ERK, by translocating to the mitochondria where it binds Bcl-2 and displaces BAD. However, the answer to the question of how Raf-1 is normally lured to the mitochondria and becomes activated remains elusive. p21-activated protein kinases (Paks) are serine/threonine protein kinases that phosphorylate Raf-1 at Ser-338 and Ser-339. Here we elucidate the molecular mechanism through which Pak1 signals to BAD through a Raf-1-activated pathway. Upon phosphorylation by Pak1, Raf-1 translocates to mitochondria and phosphorylates BAD at Ser-112. Moreover, the mitochondrial translocation of Raf-1 and the interaction between Raf-1 and Bcl-2 are regulated by Raf-1 phosphorylation at Ser-338/Ser-339. Notably, we show that formation of a Raf-1-Bcl-2 complex coincides with loss of an interaction between Bcl-2 and BAD. These signals are specific for Pak1, because Src-activated Raf-1 only stimulates the MAP kinase cascade. Thus, our data identify the molecular connections of a Pak1-Raf-1-BAD pathway that is involved in cell survival signaling.