Regulation of Raf-1 by direct feedback phosphorylation

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.     

Oligonol, an oligomerized lychee fruit-derived polyphenol, activates the Ras/Raf-1/MEK1/2 cascade independent of the IL-6 signaling pathway in rat primary adipocytes

Oligonol is a lychee fruit-derived low-molecular form of polyphenol. In this study, the effect of Oligonol on the mitogen activated-protein kinase (MAPK) signaling pathway in primary adipocytes was investigated to examine the mechanism underlying the enhanced levels of phosphorylated extracellular-signaling regulatory kinase1/2 (ERK1/2) that accompany an in vitro increase in lipolysis. Oligonol significantly elevated the levels of activated Ras and the phosphorylation of Raf-1 and MAPK/ERK kinase1/2 (MEK1/2) with no increase in pan-Raf-1 and -MEK1/2 proteins. The increase in phosphorylation of Raf-1 and MEK1/2 with Oligonol was inhibited completely by pretreatment with GW5074, a selective Raf-1 inhibitor, or PD98059, a selective MEK1/2 inhibitor. IL-6 also activated the MAPK signaling pathway in adipocytes through the association with its receptor. IL-6-induced phosphorylation of Raf-1 and MEK1/2 was significantly inhibited by pretreatment with the IL-6 receptor antibody. Under such a condition, however, the levels of phosphorylated Raf-1 and MEK1/2 with Oligonol still remained significantly higher, and there was a significant decrease in secretion of IL-6 from adipocytes, compared with untreated control cells. These results suggest that Oligonol activates the Ras/Raf-1/MEK1/2 signaling pathway, independent of the IL-6 signaling pathway, leading to activation of ERK1/2 proteins in primary adipocytes.     

Erk1/2-dependent phosphorylation of Galpha-interacting protein stimulates its GTPase accelerating activity and autophagy in human colon cancer cells

Galpha-interacting protein (GAIP) is a regulator of G protein signaling (RGS) that accelerates the rate of GTP hydrolysis by the alpha-subunit of the trimeric G(i3) protein. Both proteins are part of a signaling pathway that controls lysosomal-autophagic catabolism in human colon cancer HT-29 cells. Here we show that GAIP is phosphorylated by an extracellular signal-regulated (Erk1/2) MAP kinase-dependent pathway sensitive to amino acids, MEK1/2 (PD098059), and protein kinase C (GF109203X) inhibitors. An in vitro phosphorylation assay demonstrates that Erk2-dependent phosphorylation of GAIP stimulates its GTPase-activating protein activity toward the Galpha(i3) protein (k = 0.187 +/- 0.001 s(-)(1), EC(50) = 1.12 +/- 0.10 microm) when compared with unphosphorylated recombinant GAIP (k = 0.145 +/- 0.003 s(-)(1), EC(50) = 3.16 +/- 0. 12 microm) or to GAIP phosphorylated by other Ser/Thr protein kinases (protein kinase C, casein kinase II). This stimulation and the phosphorylation of GAIP by Erk2 were abrogated when serine at position 151 in the RGS domain was substituted by an alanine residue using site-directed mutagenesis. Furthermore, the lysosomal-autophagic pathway was not stimulated in S151A-GAIP mutant-expressing cells when compared with wild-type GAIP-expressing cells. These results demonstrate that the GTPase-activating protein activity of GAIP is stimulated by Erk2 phosphorylation. They also suggested that Erk1/2 and GAIP are engaged in the signaling control of a major catabolic pathway in intestinal derived cells.     

Peptidoglycan induces nuclear factor-kappaB activation and cyclooxygenase-2 expression via Ras, Raf-1, and ERK in RAW 264.7 macrophages

In this study, we investigated the signaling pathway involved in cyclooxygenase-2 (COX-2) expression caused by peptidoglycan (PGN), a cell wall component of the Gram-positive bacterium Staphylococcus aureus, in RAW 264.7 macrophages. PGN caused dose- and time-dependent increases in COX-2 expression, which was attenuated by a Ras inhibitor (manumycin A), a Raf-1 inhibitor (GW 5074), and an MEK inhibitor (PD 098059). Treatment of RAW 264.7 macrophages with PGN caused time-dependent activations of Ras, Raf-1, and ERK. The PGN-induced increase in Ras activity was inhibited by manumycin A. Raf-1 phosphorylation at Ser-338 by PGN was inhibited by manumycin A and GW 5074. The PGN-induced increase in ERK activity was inhibited by manumycin A, GW 5074, and PD 098059. Stimulation of cells with PGN activated IkappaB kinase alpha/beta (IKKalpha/beta), IkappaBalpha phosphorylation, IkappaBalpha degradation, and kappaB-luciferase activity. Treatment of macrophages with an NF-kappaB inhibitor (pyrrolidine dithiocarbamate), an IkappaBalpha phosphorylation inhibitor (Bay 117082), and IkappaB protease inhibitors (l-1-tosylamido-2-phenylethyl chloromethyl ketone and calpain inhibitor I) all inhibited PGN-induced COX-2 expression. The PGN-mediated increase in the activities of IKKalpha/beta and kappaB-luciferase were also inhibited by the Ras dominant negative mutant (RasN17), manumycin A, GW 5074, and PD 098059. Further studies revealed that PGN induced the recruitment of p85alpha and Ras to Toll-like receptor 2 in a time-dependent manner. Our data demonstrate for the first time that PGN activates the Ras/Raf-1/ERK pathway, which in turn initiates IKKalpha/beta and NF-kappaB activation, and ultimately induces COX-2 expression in RAW 264.7 macrophages.     

Cyclic stretch activates ERK1/2 via G proteins and EGFR in alveolar epithelial cells

Mechanical stimuli are transduced into intracellular signals in lung alveolar epithelial cells (AEC). We studied whether mitogen-activated protein kinase (MAPK) pathways are activated during cyclic stretch of AEC. Cyclic stretch induced a rapid (within 5 min) increase in extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in AEC. The inhibition of Na(+), L-type Ca(2+) and stretch-activated ion channels with amiloride, nifedipine, and gadolinium did not prevent the stretch-induced ERK1/2 activation. The inhibition of Grb2-SOS interaction with an SH3 binding sequence peptide, Ras with a farnesyl transferase inhibitor, and Raf-1 with forskolin did not affect the stretch-induced ERK1/2 phosphorylation. Moreover, cyclic stretch did not increase Ras activity, suggesting that stretch-induced ERK1/2 activation is independent of the classical receptor tyrosine kinase-MAPK pathway. Pertussis toxin and two specific epidermal growth factor receptor (EGFR) inhibitors (AG-1478 and PD-153035) prevented the stretch-induced ERK1/2 activation. Accordingly, in primary AEC, cyclic stretch activates ERK1/2 via G proteins and EGFR, in Na(+) and Ca(2+) influxes and Grb2-SOS-, Ras-, and Raf-1-independent pathways.     

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.     

Phorbol 12-myristate 13-acetate upregulates cyclooxygenase-2 expression in human pulmonary epithelial cells via Ras, Raf-1, ERK, and NF-kappaB, but not p38 MAPK, pathways

In this study, we investigated the signaling pathway involved in cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) release by phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, in human pulmonary epithelial cells (A549). PMA-induced COX-2 expression was attenuated by PKC inhibitors (Go 6976 and Ro 31-8220), a Ras inhibitor (manumycin A), a Raf-1 inhibitor (GW 5074), a MEK inhibitor (PD 098059), and an NF-kappaB inhibitor (PDTC), but not by a tyrosine kinase inhibitor (genistein) or a p38 MAPK inhibitor (SB 203580). PMA also caused the activation of Ras, Raf-1, and ERK1/2. PMA-induced activation of Ras and Raf-1 was inhibited by Ro 31-8220 and manumycin A. PMA-mediated activation of ERK1/2 was inhibited by Ro 31-8220, manumycin A, GW 5074, and PD 098059. Stimulation of cells with PMA caused IkappaBalpha phosphorylation, IkappaBalpha degradation, and the formation of a NF-kappaB-specific DNA-protein complex. The PMA-mediated increase in kappaB-luciferase activity was inhibited by Ro 31-8220, manumycin A, GW5074, PD 098059, and PDTC. Taken together, these results indicate that PMA might activate PKC to elicit activation of the Ras/Raf-1/ERK1/2 pathway, which in turn initiates NF-kappaB activation, and finally induces COX-2 expression and PGE2 release in A549 cells.     

Interactions of opioid and chemokine receptors: oligomerization of mu,kappa, and delta with CCR5 on immune cells

In this study, we examined the signaling pathways for extracellular signal-related protein kinase (ERK) activation by three structurally different peroxisome proliferator activated receptor-gamma (PPARgamma) agonists. In murine C2C12 myoblasts, treatment with 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), ciglitazone, and GW1929 leads to ERK1/2 phosphorylation in a time- and concentration-dependent manner. Consistent with ERK phosphorylation, mitogen activated protein/ERK kinase (MEK) phosphorylation as well as Raf-1 kinase activity are also accordingly stimulated, while the constitutive Ser259 phosphorylation of Raf-1 is decreased. The ERK phosphorylation induced by PPARgamma agonists is not blocked by the PKC inhibitors GF109203X and Ro31-8220, the PI3K inhibitor wortmannin, the Ras inhibitor FPTI, the negative mutant of Ras, or the PPARgamma antagonist bisphenol A diglycidil ether. Expression of PPARgamma2 without DNA binding domain or with a nonphosphorylatable mutant (S112A) fails to change ERK phosphorylation by 15d-PGJ(2). On the contrary, the ERK phosphorylation by PPARgamma agonists is inhibited by the MEK inhibitor PD98059, GSH, and permeable SOD mimetic MnTBAP. Chemiluminescence study reveals that these three PPARgamma agonists are able to induce superoxide anion production, with an efficacy similar to their action on ERK phosphorylation. Consistent with this notion, we also show that superoxide anion donor 2,3-dimethoxy-1,4-naphoquinone elicits ERK phosphorylation. In this study, we for the first time demonstrate a novel mechanism, independent of Ras activation but initiated by superoxide anion production, for PPARgamma agonists to trigger the Raf-MEK-ERK1/2 signaling pathway.     

Insulin reverses growth hormone-induced homologous desensitization

Growth hormone (GH) is secreted in a pulsatile pattern to promote body growth and metabolism. GH exerts its function by activating several signaling pathways, including JAK2/STAT and MEK/ERK. ERK1/2 activation by GH plays important roles in gene expression, cell proliferation, and growth. We previously reported that in rat H4IIE hepatoma cells after an initial GH exposure, a second GH exposure induces STAT5 phosphorylation but not ERK1/2 phosphorylation (Ji, S., Frank, S. J., and Messina, J. L. (2002) J. Biol. Chem. 277, 28384-28393). In this study the mechanisms underlying GH-induced homologous desensitization were investigated. A second GH exposure activated the signaling intermediates upstream of MEK/ERK, including JAK2, Ras, and Raf-1. This correlated with recovery of GH receptor levels, but was insufficient for GH-induced phosphorylation of MEK1/2 and ERK1/2. Insulin restored the ability of a second GH exposure to induce phosphorylation of MEK1/2 and ERK1/2 without altering GH receptor levels or GH-induced phosphorylation/activation of JAK2 and Raf-1. GH and insulin synergized in promoting cell proliferation. Further investigation suggested that insulin increased the amount of MEK bound to KSR (kinase suppressor of Ras) and restored GH-induced tyrosine phosphorylation of KSR. Previous GH exposure also induced desensitization of STAT1 and STAT3 phosphorylation, but this desensitization was not reversed by insulin. Thus, insulin-regulated resensitization of GH signaling may be necessary to reset the complete response to GH after a normal, physiologic pulse of GH.     

Distribution, levels and phosphorylation of Raf-1 in Alzheimer's disease

Extracellular signal-regulated kinase (ERK), a member of the mitogen-activated protein kinase pathway, has been increasingly implicated in the pathogenesis of Alzheimer's disease due to its critical role in brain function. While we previously demonstrated that ERK is activated in Alzheimer's disease, the upstream cascade leading to its activation had not been fully examined. In this study, we focused on Raf-1, one of the physiological activators of the ERK pathway. Raf-1 is activated by phosphorylation at Ser338 and Tyr340/341 and inhibited by phosphorylation at Ser259. Interestingly, phosphorylation at all three sites on Raf-1 was increased as evidenced by both immunocytochemistry and immunoblot analysis in Alzheimer's disease brains compared to age-matched controls. Both phospho-Raf-1 (Ser259) and phospho-Raf-1 (Ser338) were localized to intracytoplasmic granular structures, whereas phospho-Raf-1 (Tyr340/341) was localized to neurofibrillary tangles and granules in pyramidal neurons in Alzheimer's disease hippocampus. There is extensive overlap between phospho-Raf-1 (Ser338) and phospho-Mek1/2, the downstream effector of Raf-1, suggestive of a mechanistic link. Additionally, increased levels of Raf-1 are associated with Ras and MEK1 in Alzheimer's disease as evidenced by its coimmunoprecipitation with Ras and Mek1, respectively. Based on these findings, we speculate that Raf-1 is activated to effectively mediate Ras-dependent signals in Alzheimer's disease.     

Raf-1 kinase activation is uncoupled from downstream MEK/ERK pathway in cells treated with Src tyrosine kinase inhibitor PP2

Recently we demonstrated that PP2 (4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), a potent and selective inhibitor of the Src-family tyrosine kinase, markedly enhanced Ras-independent activation of Raf-1 by the combination of phorbol myristate acetate (PMA) and hydrogen peroxide (H(2)O(2)). We report here that Raf-1 knockdown cells were significantly more sensitive to treatment of PP2 than control cells. This PP2-induced growth inhibition was found to be linked to decreased ERK and p38 activity. Interestingly, the growth of Sprouty knockdown cells appeared to be inhibited at earlier time points of PP2 treatment when compared with control cells. Unexpectedly, siRNA-mediated knockdown of Spry2, which is known to modulate the Ras/Raf/MAPK signal through feedback regulation, resulted in decreased Raf-1 kinase activity. PP2 had limited effect on the ability of PMA/H(2)O(2) to induce significant phosphorylation of MEK/ERK proteins in both Spry2 knockdown and control cells, indicating that PP2-mediated activation of Raf-1 did not potentiate signaling through the downstream MEK/ERK pathway. Taken together our results suggest that Raf-1 signaling may be bypassed in PP2-treated cells by uncoupling from downstream MEK/ERK pathway.     

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.     

cAMP-dependent protein kinase (PKA) inhibits T cell activation by phosphorylating ser-43 of raf-1 in the MAPK/ERK pathway

cAMP-dependent protein kinase (PKA) has been suggested to interfere with T-cell activation by inhibiting interleukin (IL-2) receptor alpha-chain (CD25) expression and IL-2 production. The Ras/MAP kinase pathway has been found to be necessary for induction of the IL-2 production. In this study, we have scrutinized the Ras/MAP kinase pathway in Jurkat T-cells to attempt to identify any sites for PKA-mediated regulatory phosphorylations. Here we unambiguously demonstrate that PKA directly inhibits anti-CD3-induced MAP kinase activation. In vitro phosphorylation experiments showed that Raf-1 was extensively phosphorylated by PKA, while ERK2 and MEK were not. Phosphopeptide mapping identified Ser-43 of Raf-1 as the only site phosphorylated by PKA in the Ras/MAPK pathway. Transient transfection experiments demonstrated that mutations of Ser-43 of the Raf-1 kinase were rendered insensitive to cAMP-mediated inhibition.     

Contribution of the ERK5/MEK5 pathway to Ras/Raf signaling and growth control.

The activity of the catalytic domain of the orphan MAP kinase ERK5 is increased by Ras but not Raf-1 in cells, which suggests that ERK5 might mediate Raf-independent signaling by Ras. We found that Raf-1 does contribute to Ras activation of ERK5 but in a manner that does not correlate with Raf-1 catalytic activity. A clue to the mechanism of action of Raf-1 on ERK5 comes from the observation that endogenous Raf-1 binds to endogenous ERK5, suggesting the involvement of regulatory protein-protein interactions. This interaction is specific because Raf-1 binds only to ERK5 and not ERK2 or SAPK. Finally, we demonstrate the ERK5/MEK5 pathway is required for Raf-dependent cellular transformation and that a constitutively active form of MEK5, MEK5DD, synergizes with Raf to transform NIH 3T3 cells. These observations suggest that ERK5 plays a large role in Raf-1-mediated signal transduction.     

Disruption of Raf-1/heat shock protein 90 complex and Raf signaling by dexamethasone in mast cells

Antigen stimulation of mast cells via the IgE receptor, FcepsilonRI, results in the recruitment of the cytosolic tyrosine kinase, Syk, and the activation of various signaling cascades. One of these, the extracellular signal-regulated kinase (ERK2) cascade, is inhibited by low concentrations of the immunosuppressant drug, dexamethasone, probably at a step prior to the activation of Raf-1 (Rider, L. G., Hirasawa, N., Santini, F., and Beaven, M. A. (1996) J. Immunol. 157, 2374-2380). We now show that treatment of cultured RBL-2H3 mast cells with nanomolar concentrations of dexamethasone causes dissociation of the Raf-1.heat shock protein 90 (Hsp90) complex. Raf-1 bereft of this protein fails to associate with the membrane or Ras in antigen-stimulated cells. Upstream events such as the Syk-dependent phosphorylation of Shc, the engagement of Shc with the adapter protein, Grb2, and the activation of Ras itself are unaffected. Interestingly, the counterpart of Raf-1 in the c-Jun N-terminal kinase (JNK) cascade, MEKK-1 (mitogen-activated protein kinase/ERK kinase), is similarly associated with Hsp90, and this association as well as the activation of MEKK-1 are disrupted by dexamethasone treatment. Disruption of the ERK and JNK cascades at the level of Raf-1 and MEKK-1 could account for the inhibitory action of dexamethasone on the generation of inflammatory mediators in stimulated mast cells.     

Transmembrane signaling pathway mediates oxidized low-density lipoprotein-induced expression of plasminogen activator inhibitor-1 in vascular endothelial cells

Atherosclerotic cardiovascular disease is the number one cause of death for adults in Western society. Plasminogen activator inhibitor-1 (PAI-1), the major physiological inhibitor of plasminogen activators, has been implicated in both thrombogenesis and atherogenesis. Previous studies demonstrated that copper-oxidized low-density lipoprotein (C-oLDL) stimulated production of PAI-1 in vascular endothelial cells (EC). The present study examined the involvement of lectin-like oxidized LDL receptor-1 (LOX-1) and Ras/Raf-1/ERK1/2 pathway in the upregulation of PAI-1 in cultured EC induced by oxidized LDLs. The results demonstrated that C-oLDL or FeSO(4)-oxidized LDL (F-oLDL) increased the expression of PAI-1 or LOX-1 in human umbilical vein EC (HUVEC) or coronary artery EC (HCAEC). Treatment with C-oLDL significantly increased the levels of H-Ras mRNA, protein, and the translocation of H-Ras to membrane fraction in EC. LOX-1 blocking antibody, Ras farnesylation inhibitor (FTI-277), or small interference RNA against H-Ras significantly reduced C-oLDL or LDL-induced expression of H-Ras and PAI-1 in EC. Incubation with C-oLDL or F-oLDL increased the phosphorylation of Raf-1 and ERK1/2 in EC compared with LDL or vehicle. Treatment with Raf-1 inhibitor blocked Raf-1 phosphorylation and the elevation of PAI-1 mRNA level in EC induced by C-oLDL or LDL. Treatment with PD-98059, an ERK1/2 inhibitor, blocked C-oLDL or LDL-induced ERK1/2 phosphorylation or PAI-1 expression in EC. The results suggest that LOX-1, H-Ras, and Raf-1/ERK1/2 are implicated in PAI-1 expression induced by oxidized LDLs or LDL in cultured EC.     

Raf-1激酶与肿瘤治疗

自Raf激酶被证明为逆转录病毒致癌基因的产物以来,逐渐成为人们研究的热点。研究表明,Raf激酶既是Ras的效应物,又能作为ERK信号通路中的重要组分,成为活化的Ras和ERK之间的一个重要纽带。Ras-Raf-MEK-ERK信号通路参与了细胞增殖、分化和凋亡等生物学过程。作为这一信号通路上的节点蛋白,Raf激酶在肿瘤发生过程中起着关键作用。Raf家族成员Raf-1（cRaf）在调控细胞运动和凋亡过程中发挥关键作用,它既可以通过抑制促凋亡激酶ASK1和MST2活性来抑制细胞凋亡,也可以通过激活Rok-α的活性来促进细胞迁移。该文主要综述了Raf-1激酶的调控机制及其在肿瘤发生过程中的作用,同时也总结了以Raf-1为靶点的肿瘤治疗的最新进展。