Mitogen-activated protein kinase cascade in human normal and tumoral parathyroid cells

The calcium-sensing receptor (CaR) activation has recently been shown to modulate the ERK1 and ERK2 cascade in different cell lines. The present study investigated this pathway in human normal and tumoral parathyroid cells. In cells from normal parathyroids and almost all hyperplasia increasing extracellular calcium concentrations (Ca(o)(2+)) induced a significant activation of ERK1 and -2, the percent stimulation over basal activity (at 0.5 mM Ca(o)(2+)) being 545 +/- 140 and 800 +/- 205 in normal cells and 290 +/- 71 and 350 +/- 73 in hyperplasia at 1 and 2 mM Ca(o)(2+), respectively. This effect was mediated by CaR because it was mimicked by the receptor agonist gadolinium and neomycin. Basal and Ca(o)(2+)-stimulated ERK1 and -2 activity was nearly abolished by the PKC inhibitor calphostin C, and PKA changes did not affect ERK1 and -2 activity. PI3K blockade by wortmannin, known to prevent G protein betagamma subunit effect on ERK1 and -2, induced a 30% reduction of the Ca(o)(2+)-stimulated ERK1 and -2 activity. Adenomatous cells showed high PKC-dependent ERK1 and -2 activity in resting conditions that was unresponsive to high Ca(o)(2+). A role of MAPK on PTH secretion was suggested by the finding that PD98059, a specific MEK inhibitor, abolished the inhibitory effect of 1.5 mM Ca(o)(2+) on PTH release from normal parathyroid cells. In conclusion, these data first demonstrate that CaR activation, through the PKC pathway and, to a lesser extent, PI3K, increases ERK1 and -2 activity in normal parathyroid cells and this cascade seems to be involved in the modulation of PTH secretion by Ca(o)(2+). Interestingly, this signaling pathway is disrupted in parathyroid tumors.     

Activation of the extracellular calcium-sensing receptor initiates insulin secretion from human islets of Langerhans: involvement of protein kinases

The extracellular calcium-sensing receptor (CaR) is usually associated with systemic Ca(2+) homeostasis, but the CaR is also expressed in many other tissues, including pancreatic islets of Langerhans. In the present study, we have used human islets and an insulin-secreting cell line (MIN6) to investigate the effects of CaR activation using the calcimimetic R-568, a CaR agonist that activates the CaR at physiological concentrations of extracellular Ca(2+). CaR activation initiated a marked but transient insulin secretory response from both human islets and MIN6 cells at a sub-stimulatory concentration of glucose, and further enhanced glucose-induced insulin secretion. CaR-induced insulin secretion was reduced by inhibitors of phospholipase C or calcium-calmodulin-dependent kinases, but not by a protein kinase C inhibitor. CaR activation was also associated with an activation of p42/44 mitogen-activated protein kinases (MAPK), and CaR-induced insulin secretion was reduced by an inhibitor of p42/44 MAPK activation. We suggest that the beta-cell CaR is activated by divalent cations co-released with insulin, and that this may be an important mechanism of intra-islet communication between beta-cells.     

Inhibition of the MAPK and PI3K pathways enhances UDCA-induced apoptosis in primary rodent hepatocytes

The mechanisms by which bile acids induce apoptosis in hepatocytes and the signaling pathways involved in the control of cell death are not understood fully. Here, we examined the impact of mitogen-activated protein kinase (MAPK) and phosphatidyl inositol 3-kinase (PI3K) signaling on the survival of primary hepatocytes exposed to bile acids. Treatment of hepatocytes with deoxycholic acid (DCA), chenodeoxycholic acid (CDCA) or ursodeoxycholic acid (UDCA) caused sustained MAPK activation that was dependent on activation of the epidermal growth factor receptor (EGFR). Activation of MAPK was partially blocked by inhibitors of PI3K. Inhibition of DCA-, CDCA-, and UDCA-stimulated MAPK activation resulted in approximately 20%, approximately 35%, and approximately 55% apoptosis, respectively. The potentiation of DCA- and CDCA-induced apoptosis by MEK1/2 inhibitors correlated with cleavage of procaspase 3, which was blocked by inhibitors of caspase 8 (ile-Glu-Thr-Asp-p-nitroanilide [IETD]) and caspase 3 (DEVD). In contrast, the potentiation of UDCA-induced apoptosis weakly correlated with procaspase 3 cleavage, yet this effect was also blocked by IETD and DEVD. Incubation of hepatocytes with the serine protease inhibitor AEBSF reduced the death response of cells treated with UDCA and MEK1/2 inhibitor to that observed for DCA and MEK1/2 inhibitor. The apoptotic response was FAS receptor- and neutral sphingomyelinase-dependent and independent of FAS ligand expression, and neither chelation of intracellular and extracellular Ca(2+) nor down-regulation of PKC expression altered the apoptotic effects of bile acids. In conclusion, bile acid apoptosis is dependent on the production of ceramide and is counteracted by activation of the MAPK and PI3K pathways.     

Critical roles of c-Kit tyrosine residues 567 and 719 in stem cell factor-induced chemotaxis: contribution of src family kinase and PI3-kinase on calcium mobilization and cell migration

Stem cell factor (SCF) has crucial roles in proliferation, survival, and differentiation of hematopoietic stem cells and mast cells through binding to c-Kit receptor (KIT). Chemotaxis is another unique function of SCF. However, little is known about the intracellular signaling pathway of SCF/KIT-mediated cell migration. To investigate the signaling cascade, we made a series of 22 KIT mutants, in which tyrosine (Y) residue was substituted for phenylalanine (F) in the cytoplasmic domain, and introduced into BAF3 cells or 293T cells. On stimulation with SCF, BAF3 expressing KIT(WT)(WT) showed cell migration and Ca(2+) mobilization. Among 22 YF mutants, Y567F, Y569F, and Y719F showed significantly reduced cell migration and Ca(2+) mobilization compared to WT. In Y567F, Lyn activation on SCF stimulation decreased and C-terminal Src kinase (Csk) suppressed KIT-mediated Ca(2+) influx and cell migration, suggesting that Y567-mediated Src family kinase (SFK) activation leads to Ca(2+) influx and migration. Furthermore, we found that p38 mitogen-activated protein kinase (p38 MAPK) and Erk1/2 were also regulated by Y567/SFK and involved in cell migration, and that p38 MAPK induced Ca(2+) influx, thereby leading to Erk1/2 activation. In Y719F, the binding of phosphatidylinositol 3'-kinase (PI3K) to KIT was lost and KIT-mediated cell migration and Ca(2+) mobilization were suppressed by PI3K chemical inhibitors or dominant-negative PI3K, suggesting the involvement of Y719-mediated PI3K pathway in cell migration. Combination of Csk and the PI3K inhibitor synergistically reduced cell migration, suggesting the cooperation of SFK and PI3K. Taken together, these results indicate that 2 major KIT signaling pathways lead to cell migration, one is Y567-SFK-p38 MAPK-Ca(2+) influx-Erk and the other is Y719-PI3K-Ca(2+) influx.     

Mitogenic action of calcium-sensing receptor on rat calvarial osteoblasts

The parathyroid calcium-sensing receptor (CaR) plays a nonredundant role in systemic calcium homeostasis. In bone, Ca(2+)(o), a major extracellular factor in the bone microenvironment during bone remodeling, could potentially serve as an extracellular first messenger, acting via the CaR, that stimulates the proliferation of preosteoblasts and their differentiation to osteoblasts (OBs). Primary digests of rat calvarial OBs express the CaR as assessed by RT-PCR, Northern, and Western blot analysis, and immunocolocalization of the CaR with the OB marker cbfa-1. Real-time PCR revealed a significant increase in CaR mRNA in 5- and 7-d cultures compared with 3-d cultures post harvesting. High Ca(2+)(o) did not affect the expression of CaR mRNA during this time but up-regulated cyclin D (D1, D2, and D3) genes, which are involved in transition from the G1 to the S phase of the cell cycle, as well as the early oncogenes, c-fos and early growth response-1; high Ca(2+)(o) did not, however, alter IGF-I expression, a mitogenic factor for OBs. The high Ca(2+)(o)-dependent increase in the proliferation of OBs was attenuated after transduction with a dominant-negative CaR (R185Q), confirming that the effect of high Ca(2+)(o) is CaR mediated. Stimulation of proliferation by the CaR involves the Jun-terminal kinase (JNK) pathway, as high Ca(2+)(o) stimulated the phosphorylation of JNK in a CaR-mediated manner, and the JNK inhibitor SP600125 abolished CaR-induced proliferation. Our data, therefore, show that the parathyroid/kidney CaR expressed in rat calvarial OBs exerts a mitogenic effect that involves activation of the JNK pathway and up-regulation of several mitogenic genes.     

Insulin receptor substrate-1-mediated enhancement of growth hormone-induced mitogen-activated protein kinase activation

Interaction of GH with the cell-surface GH receptor (GHR) causes activation of the GHR-associated tyrosine kinase, JAK2, and consequent triggering of signaling cascades including the STAT, Ras/Raf/MEK1/MAP kinase, and insulin receptor substrate-1(IRS-1)/PI3kinase pathways. We previously showed that IRS- and GHR-deficient 32D cells that stably express the rabbit GHR and rat IRS-1 (32D-rbGHR-IRS-1) exhibited markedly enhanced GH-induced proliferation and MAP kinase (ERK1 and ERK2) activation compared with cells expressing only the GHR (32D-rbGHR). We now examine biochemical mechanism(s) by which IRS-1 augments GH-induced MAP kinase activation. Time-course experiments revealed a similarly transient (maximal at 15 min) GH-induced ERK1 and ERK2 activation in both 32D-rbGHR and 32D-rbGHR-IRS-1 cells, but, consistent with our prior findings, substantially greater activation was seen in the IRS-1-containing cells. In both cells, GH-induced MAP kinase activation was markedly blunted by the MEK1 inhibitor, PD98059, but not by the PKC inhibitor, GF109203X. Interestingly, pretreatment with the PI3K inhibitor, wortmannin (EC50 approximately 10 nM), significantly reduced GH-induced MAP kinase activation in both 32D-rbGHR and 32D-rbGHR-IRS-1 cells. This same pattern in both cells of IRS-1-dependent augmentation and IRS-1-independent wortmannin sensitivity was also observed for GH-induced activation of Akt and MEK1 (using state-specific antibody blotting for both), despite the lack of difference in GHR, JAK2, SHP-2, p85, Akt, Ras, Raf-1, MEK1, ERK1, or ERK2 abundance between the two cells. A different PI3K inhibitor, LY294002 (50 microM), substantially inhibited (roughly 72%) GH-induced MAP kinase activation in 32D-rbGHR-IRS-1 cells, but only marginally (and statistically insignificantly) inhibited GH-induced MAP kinase activation in 32D-rbGHR cells. Because GH-induced Akt activation was completely inhibited in both cells by the same concentration of LY294002, these findings indicate that the wortmannin sensitivity of both the IRS-1-independent and -dependent GH-induced MAP kinase activation may reflect the activity of another wortmannin-sensitive target(s) in addition to PI3K in mediation of GH-induced MAP kinase activation in these cells. Notably, GH-induced STAT5 tyrosine phosphorylation, unlike Akt or MAPK activation, did not differ between the cells. Finally, while GH promoted accumulation of activated Ras in both cells, both basal and GH-induced activated Ras levels were greater in cells expressing IRS-1 than in 32D-rbGHR cells. These data indicate that while GH induces tyrosine phosphorylation of STAT5 and activation of the Ras/Raf/MEK1/MAPK and PI3K pathways, IRS-1 expression augments the latter two more than the former.     

Physiological changes in extracellular calcium concentration directly control osteoblast function in the absence of calciotropic hormones

We investigated the direct effects of changes in free ionized extracellular calcium concentrations ([Ca2+]o) on osteoblast function and the involvement of the calcium-sensing receptor (CaR) in mediating these responses. CaR mRNA and protein were detected in osteoblast models, freshly isolated fetal rat calvarial cells and murine clonal osteoblastic 2T3 cells, and in freshly frozen, undecalcified preparations of human mandible and rat femur. In fetal rat calvarial cells, elevating [Ca2+]o and treatment with gadolinium, a nonpermeant CaR agonist, resulted in phosphorylation of the extracellular signal-regulated kinases 1 and 2, Akt, and glycogensynthase kinase 3beta, consistent with signals of cell survival and proliferation. In agreement, cell number was increased under these conditions. Expression of the osteoblast differentiation markers core binding factor alpha1, osteocalcin, osteopontin, and collagen I mRNAs was increased by high [Ca2+]o, as was mineralized nodule formation. Alkaline phosphatase activity was maximal for [Ca2+]o between 1.2 and 1.8 mM. Inhibition of CaR by NPS 89636 blocked responses to the CaR agonists. In conclusion, we show that small deviations of [Ca2+]o from physiological values have a profound impact on bone cell fate, by means of the CaR and independently of systemic calciotropic peptides.     

Transforming growth factor-beta stimulates articular chondrocyte cell growth through p44/42 MAP kinase (ERK) activation

Transforming growth factor-beta1 (TGF-beta1) stimulates articular chondrocyte cell proliferation and extracellular matrix formation. We reported previously that immediate and transient expression of c-fos mRNA through protein kinase C activation is required for the mitogenic effect of TGF-beta1 on cultured rat articular chondrocytes (CRAC). In gel kinase assays using myelin basic protein (MBP) showed that total cell lysates from cells treated with TGF-beta1 caused rapid phosphorylation of MBP, which suggests the involvement of mitogen-activated protein kinase (MAPK) activation. To identify specific MAPK pathways activated by TGF-beta1, we performed in vitro kinase assays using specific substrates. TGF-beta1 induced a rapid activation of extracellular signal regulated kinase (ERK) with a peak at 5 min, which decreased to basal levels within 240 min after TGF-beta1 stimulation. In contrast, the c-jun N-terminal kinase activity increased only about 2.5-fold after 240 min of stimulation and p38 MAPK activity did not change significantly. ERK activation by TGF-beta1 was also confirmed by in vivo phosphorylation assays of Elk1. However, a specific MEK1 inhibitor, PD98059, significantly decreased TGF-beta1 induced Elk1 phosphorylation in a dose-dependent manner. Furthermore, PD98059 reduced the TGF-beta1-induced cell growth by 40%. These results indicate that TGF-beta1 specifically activates MEK1 and subsequent ERK pathways in CRAC, and that the activation of this MAPK pathway plays a role in the mitogenic response to TGF-beta1.     

Potentiation of mitogenic activity of platelet-derived growth factor by physiological concentrations of insulin via the MAP kinase cascade in rat A10 vascular smooth muscle cells

Hyperinsulinemia has been shown to be associated with diabetic angiopathy. Migration and proliferation of vascular smooth muscle cells (VSMC) are the processes required for the development of atherosclerosis. In this study, we attempted to determine whether insulin affects mitogenic signaling induced by platelet-derived growth factor (PDGF) in a rat VSMC cell line (A10 cells). PDGF stimulated DNA synthesis which was totally dependent on Ras, because transfection of dominant negative Ras resulted in complete loss of PDGF-stimulated DNA synthesis. Initiation of DNA synthesis was preceded by activation of Raf-1, MEK and MAP kinases (Erk 1 and Erk2). Treatment of the cells with PD98059, an inhibitor of MAPK kinase (MEK) attenuated but did not abolish PDGF-stimulated DNA synthesis, suggesting that MAPK is required but not essential for DNA synthesis. PDGF also stimulated phosphorylation of protein kinase B (Akt/PKB) and p70 S6Kinase (p70S6K) in a wortmannin-sensitive manner. Rapamycin, an inhibitor of p70S6K, markedly suppressed DNA synthesis. Low concentrations of insulin (1-10 nmol/l) alone showed little mitogenic activity and no significant effect on MAPK activity. However, the presence of insulin enhanced both DNA synthesis and MAPK activation by PDGF. The enhancing effect of insulin was not seen in cells treated with PD98059. Insulin was without effect on PDGF-stimulated activations of protein kinase B (Akt/PKB) and p70S6K. We conclude that insulin, at pathophysiologically relevant concentrations, potentiates the PDGF-stimulated DNA synthesis, at least in part, by potentiating activation of the MAPK cascade. These results are consistent with the notion that hyperinsulinemia is a risk factor for the development of atherosclerosis.     

Protein kinase C phosphorylates the inositol 1,4,5-trisphosphate receptor type 2 and decreases the mobilization of Ca2+in pancreatoma AR4-2J cells

In non-excitable cells, the inositol 1,4,5-trisphosphate receptor channel, which plays a major (IP(3)R) is an intracellular Ca(2+) role in Ca(2+) signalling. Three isoforms of IP(3)R have been identified (IP(3)R-1, IP(3)R-2 and IP(3)R-3) and most cell types express different proportions of each isoform. The differences between the pharmacological and functional properties of the various isoforms of IP(3)R are poorly understood. AR4-2J cells, which express almost exclusively (~86%) the IP(3)R-2, represent an interesting model to study this particular isoform. Here, we investigated a regulatory mechanism by which protein kinase C (PKC) influences IP(3)R-2-mediated Ca(2+) release. Using an immunoprecipitation approach, we confirmed that AR4-2J cells express almost exclusively the IP(3)R-2 isoform. Using an in vitro phosphorylation assay, we showed that the immunopurified IP(3)R-2 was efficiently phosphorylated by exogenous PKC. In intact AR4-2J cells metabolically labelled with (32)Pi, we showed that phorbol-12-myristate-13-acetate (PMA) and Ca(2+) mobilizing agonists cause the phosphorylation of IP(3)R-2. In saponin-permeabilized AR4-2J cells, IP(3)-induced Ca(2+) release was reduced after a pre-treatment with PMA or with exogenous PKC. PMA also reduced the Ca(2+) response of intact AR4-2J cells stimulated with carbachol and epidermal growth factor, two agonists that use different receptor types to activate phospholipase C. These results demonstrate that PKC decreases the Ca(2+)mobilizing activity of IP(3)R-2 and thus exerts a negative feedback on the agonists-induced Ca(2+) response of AR4-2J cells.     

Protein kinase Czeta mediated Raf-1/extracellular-regulated kinase activation by daunorubicin

In light of the emerging concept of a protective function of the mitogen-activated protein kinase (MAPK) pathway under stress conditions, we investigated the influence of the anthracycline daunorubicin (DNR) on MAPK signaling and its possible contribution to DNR-induced cytotoxicity. We show that DNR increased phosphorylation of extracellular-regulated kinases (ERKs) and stimulated activities of both Raf-1 and extracellular-regulated kinase 1 (ERK1) within 10 to 30 minutes in U937 cells. ERK1 stimulation was completely blocked by either the mitogen-induced extracellular kinase (MEK) inhibitor PD98059 or the Raf-1 inhibitor 8-bromo-cAMP (cyclic adenosine monophosphate). However, only partial inhibition of Raf-1 and ERK1 stimulation was observed with the antioxidant N-acetylcysteine (N-Ac). Moreover, the xanthogenate compound D609 that inhibits DNR-induced phosphatidylcholine (PC) hydrolysis and subsequent diacylglycerol (DAG) production, as well as wortmannin that blocks phosphoinositide-3 kinase (PI3K) stimulation, only partially inhibited Raf-1 and ERK1 stimulation. We also observed that DNR stimulated protein kinase C zeta (PKCzeta), an atypical PKC isoform, and that both D609 and wortmannin significantly inhibited DNR-triggered PKCzeta activation. Finally, we found that the expression of PKCzeta kinase-defective mutant resulted in the abrogation of DNR-induced ERK phosphorylation. Altogether, these results demonstrate that DNR activates the classical Raf-1/MEK/ERK pathway and that Raf-1 activation is mediated through complex signaling pathways that involve at least 2 contributors: PC-derived DAG and PI3K products that converge toward PKCzeta. Moreover, we show that both Raf-1 and MEK inhibitors, as well as PKCzeta inhibition, sensitized cells to DNR-induced cytotoxicity.     

Mixed-lineage kinase 3 regulates B-Raf through maintenance of the B-Raf/Raf-1 complex and inhibition by the NF2 tumor suppressor protein

The Ras --> Raf --> MEK1/2 --> extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway couples mitogenic signals to cell proliferation. B-Raf and Raf-1 function within an oligomer wherein they are regulated in part by mutual transactivation. The MAPK kinase kinase (MAP3K) mixed-lineage kinase 3 (MLK3) is required for mitogen activation of B-Raf and cell proliferation. Here we show that the kinase activity of MLK3 is not required for support of B-Raf activation. Instead, MLK3 is a component of the B-Raf/Raf-1 complex and is required for maintenance of the integrity of this complex. We show that the activation of ERK and the proliferation of human schwannoma cells bearing a loss-of-function mutation in the neurofibromatosis 2 (NF2) gene require MLK3. We find that merlin, the product of NF2, blunts the activation of both ERK and c-Jun N-terminal kinase (JNK). Finally, we demonstrate that merlin and MLK3 can interact in situ and that merlin can disrupt the interactions between B-Raf and Raf-1 or those between MLK3 and either B-Raf or Raf-1. Thus, MLK3 is part of a multiprotein complex and is required for ERK activation. The levels of this complex may be negatively regulated by merlin.     

The chemotactic and mitogenic responses of vascular smooth muscle cells to insulin-like growth factor-I require the activation of ERK1/2

Insulin-like growth factors (IGFs) play an important role in regulating vascular smooth muscle cell (VSMC) proliferation and directed migration. IGFs exert these biological actions through the activation of the IGF-I receptor and its downstream signaling network. While the involvement of the IRS-PI3 kinase-Akt pathway in mediating the chemotactic and mitogenic actions of IGFs is clear, the role of the mitogen-activated protein kinase (MAPK) signaling pathway is still under debate. In this study, the role of ERK1 and 2 in mediating the chemotactic and mitogenic actions of IGF-I in cultured porcine VSMCs was investigated. IGF-I treatment caused a significant increase in the phosphorylation state, as well as the kinase activity, of ERK1 and 2. Compared to the strong and sustained MAPK activation induced by platelet-derived growth factor-BB, the IGF-I-induced MAPK activation was weaker and more transient. Specific inhibition of the MAPK activation by PD98059 or U0126, two selective MEK inhibitors, significantly inhibited IGF-I-stimulated cell proliferation, and reduced the number of cells that migrated towards IGF-I. The p38 MAPK inhibitor SB203580 had no such effect. Likewise, depletion of ERK1/2 using antisense oligonucleotides abolished the IGF-I-induced VSMC migration and proliferation. These results suggest that the chemotactic and mitogenic responses of VSMCs to IGF-I require the activation of ERK1 and 2.     

Extracellular signal-regulated kinase and p38 mitogen-activated protein kinase mediate macrophage proliferation induced by oxidized low-density lipoprotein

We previously reported that oxidized low-density lipoprotein (Ox-LDL)-induced expression of granulocyte/macrophage colony-stimulating factor (GM-CSF) via PKC, leading to activation of phosphatidylinositol-3 kinase (PI-3K), was important for macrophage proliferation [J Biol Chem 275 (2000) 5810]. The aim of the present study was to elucidate the role of extracellular-signal regulated kinase 1/2 (ERK1/2) and of p38 MAPK in Ox-LDL-induced macrophage proliferation. Ox-LDL-induced proliferation of mouse peritoneal macrophages assessed by [3H]thymidine incorporation and cell counting assays was significantly inhibited by MEK1/2 inhibitors, PD98059 or U0126, and p38 MAPK inhibitors, SB203580 or SB202190, respectively. Ox-LDL-induced GM-CSF production was inhibited by MEK1/2 inhibitors but not by p38 MAPK inhibitors in mRNA and protein levels, whereas recombinant GM-CSF-induced macrophage proliferation was inhibited by p38 MAPK inhibitors but enhanced by MEK1/2 inhibitors. Recombinant GM-CSF-induced PI-3K activation and Akt phosphorylation were significantly inhibited by SB203580 but enhanced by PD98059. Our results suggest that ERK1/2 is involved in Ox-LDL-induced macrophage proliferation in the signaling pathway before GM-CSF production, whereas p38 MAPK is involved after GM-CSF release. Thus, the importance of MAPKs in Ox-LDL-induced macrophage proliferation was confirmed and the control of MAPK cascade could be targeted as a potential treatment of atherosclerosis.     

Raf-1 promotes cell survival by antagonizing apoptosis signal-regulating kinase 1 through a MEK–ERK independent mechanism

The Ser/Thr kinase Raf-1 is a protooncogene product that is a central component in many signaling pathways involved in normal cell growth and oncogenic transformation. Upon activation, Raf-1 phosphorylates mitogen-activated protein kinase kinase (MEK), which in turn activates mitogen-activated protein kinase/extracellular signal-regulated kinases (MAPK/ERKs), leading to the propagation of signals. Depending on specific stimuli and cellular environment, the Raf-1--MEK--ERK cascade regulates diverse cellular processes such as proliferation, differentiation, and apoptosis. Here, we describe a MEK--ERK-independent prosurvival function of Raf-1. We found that Raf-1 interacts with the proapoptotic, stress-activated protein kinase ASK1 (apoptosis signal-regulating kinase 1) in vitro and in vivo. Deletion analysis localized the Raf-1 binding site to the N-terminal regulatory fragment of ASK1. This interaction allows Raf-1 to act independently of the MEK--ERK pathway to inhibit apoptosis. Furthermore, catalytically inactive forms of Raf-1 can mimic the wild-type effect, raising the possibility of a kinase-independent function of Raf-1. Thus, Raf-1 may promote cell survival through its protein-protein interactions in addition to its established MEK kinase function.