Tumor Promoter Arsenite Activates Extracellular Signal-Regulated Kinase through a Signaling Pathway Mediated by Epidermal Growth Factor Receptor and Shc

Although arsenite is an established carcinogen, the mechanisms underlying its tumor-promoting properties are poorly understood. Previously, we reported that arsenite treatment leads to the activation of the extracellular signal-regulated kinase (ERK) in rat PC12 cells through a Ras-dependent pathway. To identify potential mediators of the upstream signaling cascade, we examined the tyrosine phosphorylation profile in cells exposed to arsenite. Arsenite treatment rapidly stimulated tyrosine phosphorylation of several proteins in a Ras-independent manner, with a pattern similar to that seen in response to epidermal growth factor (EGF) treatment. Among these phosphorylated proteins were three isoforms of the proto-oncoprotein Shc as well as the EGF receptor (EGFR). Tyrosine phosphorylation of Shc allowed for enhanced interactions between Shc and Grb2 as identified by coimmunoprecipitation experiments. The arsenite-induced tyrosine phosphorylation of Shc, enhancement of Shc and Grb2 interactions, and activation of ERK were all drastically reduced by treatment of cells with either the general growth factor receptor poison suramin or the EGFR-selective inhibitor tyrphostin AG1478. Down-regulation of EGFR expression through pretreatment of cells with EGF also attenuated ERK activation and Shc tyrosine phosphorylation in response to arsenite treatment. These results demonstrate that the EGFR and Shc are critical mediators in the activation of the Ras/ERK signaling cascade by arsenite and suggest that arsenite acts as a tumor promoter largely by usurping this growth factor signaling pathway.     

The ERK signaling cascade inhibits gonadotropin-stimulated steroidogenesis

The response of granulosa cells to luteinizing hormone (LH) and follicle-stimulating hormone (FSH) is mediated mainly by cAMP/protein kinase A (PKA) signaling. Notably, the activity of the extracellular signal-regulated kinase (ERK) signaling cascade is elevated in response to these stimuli as well. We studied the involvement of the ERK cascade in LH- and FSH-induced steroidogenesis in two granulosa-derived cell lines, rLHR-4 and rFSHR-17, respectively. We found that stimulation of these cells with the appropriate gonadotropin induced ERK activation as well as progesterone production downstream of PKA. Inhibition of ERK activity enhanced gonadotropin-stimulated progesterone production, which was correlated with increased expression of the steroidogenic acute regulatory protein (StAR), a key regulator of progesterone synthesis. Therefore, it is likely that gonadotropin-stimulated progesterone formation is regulated by a pathway that includes PKA and StAR, and this process is down-regulated by ERK, due to attenuation of StAR expression. Our results suggest that activation of PKA signaling by gonadotropins not only induces steroidogenesis but also activates down-regulation machinery involving the ERK cascade. The activation of ERK by gonadotropins as well as by other agents may be a key mechanism for the modulation of gonadotropin-induced steroidogenesis.     

Monomeric and dimeric models of ERK2 in conjunction with studies on cellular localization,nuclear translocation,and <Emphasis Type="Italic">in vitro</Emphasis> analysis

Extracellular signal-regulated protein kinase 2 (ERK2) plays many vital roles in cellular signal regulation. Phosphorylation of ERK2 leads to propagation and execution of various extracellular stimuli, which influence cellular responses to stress. The final response of the ERK2 signaling pathway is determined by localization and duration of active ERK2 at specific target cell compartments through protein-protein interactions of ERK2 with various cytoplasmic and nuclear substrates, scaffold proteins, and anchoring counterparts. In this respect, dimerization of phosphorylated ERK2 has been suggested to be a part of crucial regulating mechanism in various protein-protein interactions. After the report of putative dimeric structure of active ERK2 (Canagarajah et al., 1997), dimeric model was employed to explain many in vivo and in vitro experimental results. But more recently, many reports have been presented questioning the validity of dimer hypothesis of active ERK2. In this review, we summarize the various in vitro and in vivo studies concerning the Monomeric or the dimeric forms of ERK2 and the validity of the dimer hypothesis.     

Kinase suppressor of Ras1 compartmentalizes hippocampal signal transduction and subserves synaptic plasticity and memory formation

The ERK/MAP kinase cascade is important for long-term memory formation and synaptic plasticity, with a myriad of upstream signals converging upon ERK activation. Despite this convergence of signaling, neurons routinely activate appropriate biological responses to different stimuli. Scaffolding proteins represent a mechanism to achieve compartmentalization of signaling and the appropriate targeting of ERK-dependent processes. We report that kinase suppressor of Ras (KSR1) functions biochemically in the hippocampus to scaffold the components of the ERK cascade, specifically regulating the cascade when a membrane fraction of ERK is activated via a PKC-dependent pathway but not via a cAMP/PKA-dependent pathway. Specificity of KSR1-dependent signaling also extends to specific downstream targets of ERK. Behaviorally and physiologically, we found that the absence of KSR1 leads to deficits in associative learning and theta burst stimulation-induced LTP. Our report provides novel insight into the endogenous scaffolding role of KSR1 in controlling kinase activation within the nervous system.     

Regulation of human immunodeficiency virus type 1 infectivity by the ERK mitogen-activated protein kinase signaling pathway

ERK1 and ERK2 mitogen-activated protein kinases (MAPK) play a critical role in regulation of cell proliferation and differentiation in response to mitogens and other extracellular stimuli. Mitogens and cytokines that activate MAPK in T cells have been shown to activate human immunodeficiency virus type 1 (HIV-1) replication. Little is known about the signal transduction pathways that activate HIV-1 replication in T cells upon activation by extracellular stimulation. Here, we report that activation of MAPK through the Ras/Raf/MEK signaling pathway enhances the infectivity of HIV-1 virions. Virus infectivity was enhanced by treatment of cells with MAPK stimulators, such as serum and phorbol myristate acetate, as well as by coexpression of constitutively activated Ras, Raf, or MEK (MAPK kinase) in the absence of extracellular stimulation. Treatment of cells with PD 098059, a specific inhibitor of MAPK activation, or with a MAPK antisense oligonucleotide reduced the infectivity of HIV-1 virions without significantly affecting virus production or the levels of virion-associated Gag and Env proteins. MAPK has been shown to regulate HIV-1 infectivity by phosphorylating Vif (X. Yang and D. Gabuzda, J. Biol. Chem. 273:29879-29887, 1998). However, MAPK activation enhanced virus infectivity in some cells lines that do not require Vif function. The HIV-1 Rev, Tat, p17(Gag), and Nef proteins were directly phosphorylated by MAPK in vitro, suggesting that other HIV-1 proteins are potential substrates for MAPK phosphorylation. These results suggest that activation of the ERK MAPK pathway plays a role in HIV-1 replication by enhancing the infectivity of HIV-1 virions through Vif-dependent as well as Vif-independent mechanisms. MAPK activation in producer cells may contribute to the activation of HIV-1 replication when T cells are activated by mitogens and other extracellular stimuli.     

The effect of concomitant stimulation with cholecystokinin and epidermal growth factor on extracellular signal-regulated kinase (ERK) activity in pancreatic acinar cells.

The transmission of extracellular proliferation and differentiation signals into their intracellular targets is mediated by a signaling cascade culminating in mitogen-activated protein kinase (MAPK) also known as ERK. In pancreatic acinar cells both cholecystokinin (CCK) and epidermal growth factor (EGF) are known to stimulate ERK. Regulatory interactions among individual receptor-coupled signaling cascades are critically important for establishing cellular responses in the face of multiple stimuli. The aim of our study was to evaluate the effect of concomitant stimulation of G protein-coupled receptors (GPCR) and EGF receptors on ERK activity in isolated pancreatic acinar cells. ERK activity was determined by means of Western-blotting, with the use of the antibody which recognizes active, tyrosine-phosphorylated kinase (pY-ERK). pY-ERK level was strongly elevated by 10 nM CCK-8, 100 microM carbachol (CAR), or 100 nM EGF. The addition of EGF to 60 min-lasting incubations of acini with CCK-8 or CAR caused abrupt decrease of pY-ERK level to 56 and 59% of control, respectively. Similar phenomenon was observed when short stimulation with CCK-8 or CAR was superimposed on the effect of EGF. After the addition of EGF to acini incubated previously with phorbol ester TPA, strong decrease in pY-ERK level was also observed. In conclusion, in pancreatic acinar cells, concomitant stimulation with CCK or CAR and EGF has strong inhibitory effect on ERK cascade. This inhibitory cross-talk may be mediated, at least partially, by protein kinase C (PKC). These mutual inhibitory interactions demonstrate novel mechanism for integration of multiple signals generated by activation of G-protein-coupled and growth factor receptors in pancreatic acinar cells.     

CD147 is a signaling receptor for cyclophilin B.

Cyclophilins A and B (CyPA and CyPB) are cyclosporin A binding proteins that can be secreted in response to inflammatory stimuli. We recently identified CD147 as a cell-surface receptor for CyPA and demonstrated that CD147 is an essential component in the CyPA-initiated signaling cascade that culminates in ERK activation and chemotaxis. Here we demonstrate that CD147 also serves as a receptor for CyPB. CyPB induced Ca(2+) flux and chemotaxis of CD147-transfected, but not control, CHO cells, and the chemotactic response of primary human neutrophils to CyPB was blocked by antibodies to CD147. These results suggest that CD147 serves as a receptor for extracellular cyclophilins.     

The role of beta-arrestins in the formyl peptide receptor-like 1 internalization and signaling

The N-formyl peptide receptor-like 1 (FPRL1) is a G protein-coupled receptor (GPCR) that transmits intracellular signals in response to a variety of agonists, many of them being clearly implicated in human pathology. beta-arrestins are adaptor proteins that uncouple GPCRs from G protein and regulate receptor internalization. They can also function as signal transducers through the scaffolding of signaling molecules, such as components of the extracellular signal-regulated kinase (ERK) cascade. We investigated the role of beta-arrestins in ligand-induced FPRL1 internalization and signaling. In HEK293 cells expressing FPRL1, fluorescence microscopy revealed that agonist-stimulated FPRL1 remained co-localized with beta-arrestins during endocytosis. Internalization of FPRL1, expressed in a mouse embryonic fibroblast (MEF) cell line lacking endogenous beta-arrestins, was highly compromised. This distinguishes FPRL1 from the prototypical formyl peptide receptor FPR that is efficiently internalized in the absence of beta-arrestins. In both HEK293 and MEF cells, FPRL1-mediated ERK1/2 activation was a rapid and transient event. The kinetics and extent of ERK1/2 activation were not significantly modified by beta-arrestin overexpression. The pattern of FPRL1-mediated ERK1/2 activation was similar whether cells express or not beta-arrestins. Furthermore, treatment of the FPRL1 expressing cells with pertussis toxin inhibited ERK1/2 activation in MEF and in HEK293 cells. These results led us to conclude that activation of ERK1/2 mediated by FPRL1 occurs primarily through G protein signaling. Since beta-arrestin-mediated signaling has been observed essentially for receptors coupled to G proteins other than G(i), this may be a characteristic of G(i) protein-coupled chemoattractant receptors.     

Calcium-mediated interactions regulate the subcellular localization of extracellular signal-regulated kinases

The subcellular localization of ERKs in cells, which is important for proper signaling, may be regulated through protein-protein interactions. We found that inactive ERK2 interacts with a large number of proteins through its cytosolic retention sequence/common docking domain, whereas the phospho-ERK2 interacts with only few substrates. Varying calcium concentrations significantly modified the repertoire of ERK2-interacting proteins, of which many were identified. The effect of calcium on ERK interactions also influenced the localization of ERKs, as calcium chelators enhanced nuclear translocation, whereas elevated calcium levels prevented it. This effect of calcium was apparent upon lysophosphatidic acid stimulation, where ERKs translocation was delayed compared with that induced by EGF in a calcium-dependent manner. In vitro translocation assay revealed that high calcium concentrations affect ERK translocation by preventing the shuttling machinery through the nuclear envelope, probably due to higher binding to nuclear pore proteins. These results are consistent with a model in which ERKs in quiescent cells are bound to several cytoplasmic proteins. Upon stimulation, ERKs are phosphorylated and released from cytoplasmic anchors to allow shuttling toward the nucleus. This translocation is delayed when calcium levels are increased, and this modifies the localization of ERKs and, therefore, also their spatiotemporal regulation. Thus, calcium regulates ERK localization, which is important for the compartmentalization of ERKs with their proper substrates and thereby their signaling specificity.     

Interaction with MEK causes nuclear export and downregulation of peroxisome proliferator-activated receptor gamma

The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) cascade plays a central role in intracellular signaling by many extracellular stimuli. One target of the ERK cascade is peroxisome proliferator-activated receptor gamma (PPARgamma), a nuclear receptor that promotes differentiation and apoptosis. It was previously demonstrated that PPARgamma activity is attenuated upon mitogenic stimulation due to phosphorylation of its Ser84 by ERKs. Here we show that stimulation by tetradecanoyl phorbol acetate (TPA) attenuates PPARgamma's activity in a MEK-dependent manner, even when Ser84 is mutated to Ala. To elucidate the mechanism of attenuation, we found that PPARgamma directly interacts with MEKs, which are the activators of ERKs, but not with ERKs themselves, both in vivo and in vitro. This interaction is facilitated by MEKs' phosphorylation and is mediated by the basic D domain of MEK1 and the AF2 domain of PPARgamma. Immunofluorescence microscopy and subcellular fractionation revealed that MEK1 exports PPARgamma from the nucleus, and this finding was supported by small interfering RNA knockdown of MEK1 and use of a cell-permeable interaction-blocking peptide, which prevented TPA-induced export of PPARgamma from the nucleus. Thus, we show here a novel mode of downregulation of PPARgamma by its MEK-dependent redistribution from the nucleus to the cytosol. This unanticipated role for the stimulation-induced nuclear shuttling of MEKs shows that MEKs can regulate additional signaling components besides the ERK cascade.     

Involvement of the ERK signaling cascade in protein kinase C-mediated cell cycle arrest in intestinal epithelial cells

We have reported previously that protein kinase C (PKC) signaling can mediate a program of cell cycle withdrawal in IEC-18 nontransformed intestinal crypt cells, involving rapid disappearance of cyclin D1, increased expression of Cip/Kip cyclin-dependent kinase inhibitors, and activation of the growth suppressor function of pocket proteins. In the current study, we present evidence to support a requisite role for PKC alpha in mediating these effects. Furthermore, analysis of the signaling events linking PKC/PKC alpha activation to changes in the cell cycle regulatory machinery implicate the Ras/Raf/MEK/ERK cascade. PKC/PKC alpha activity promoted GTP loading of Ras, activation of Raf-1, and phosphorylation/activation of ERK. ERK activation was found to be required for critical downstream effects of PKC/PKC alpha activation, including cyclin D1 down-regulation, p21(Waf1/Cip1) induction, and cell cycle arrest. PKC-induced ERK activation was strong and sustained relative to that produced by proliferative signals, and the growth inhibitory effects of PKC agonists were dominant over proliferative events when these opposing stimuli were administered simultaneously. PKC signaling promoted cytoplasmic and nuclear accumulation of ERK activity, whereas growth factor-induced phospho-ERK was localized only in the cytoplasm. Comparison of the effects of PKC agonists that differ in their ability to sustain PKC alpha activation and growth arrest in IEC-18 cells, together with the use of selective kinase inhibitors, indicated that the length of PKC-mediated cell cycle exit is dictated by the magnitude/duration of input signal (i.e. PKC alpha activity) and of activation of the ERK cascade. The extent/duration of phospho-ERK nuclear localization may also be important determinants of the duration of PKC agonist-induced growth arrest in this system. Taken together, the data point to PKC alpha and the Ras/Raf/MEK/ERK cascade as key regulators of cell cycle withdrawal in intestinal epithelial cells.     

Regulation of mitogen-activated protein kinases in cardiac myocytes through the small G protein Rac1

Small guanine nucleotide-binding proteins of the Ras and Rho (Rac, Cdc42, and Rho) families have been implicated in cardiac myocyte hypertrophy, and this may involve the extracellular signal-related kinase (ERK), c-Jun N-terminal kinase (JNK), and/or p38 mitogen-activated protein kinase (MAPK) cascades. In other systems, Rac and Cdc42 have been particularly implicated in the activation of JNKs and p38-MAPKs. We examined the activation of Rho family small G proteins and the regulation of MAPKs through Rac1 in cardiac myocytes. Endothelin 1 and phenylephrine (both hypertrophic agonists) induced rapid activation of endogenous Rac1, and endothelin 1 also promoted significant activation of RhoA. Toxin B (which inactivates Rho family proteins) attenuated the activation of JNKs by hyperosmotic shock or endothelin 1 but had no effect on p38-MAPK activation. Toxin B also inhibited the activation of the ERK cascade by these stimuli. In transfection experiments, dominant-negative N17Rac1 inhibited activation of ERK by endothelin 1, whereas activated V12Rac1 cooperated with c-Raf to activate ERK. Rac1 may stimulate the ERK cascade either by promoting the phosphorylation of c-Raf or by increasing MEK1 and/or -2 association with c-Raf to facilitate MEK1 and/or -2 activation. In cardiac myocytes, toxin B attenuated c-Raf(Ser-338) phosphorylation (50 to 70% inhibition), but this had no effect on c-Raf activity. However, toxin B decreased both the association of MEK1 and/or -2 with c-Raf and c-Raf-associated ERK-activating activity. V12Rac1 cooperated with c-Raf to increase expression of atrial natriuretic factor (ANF), whereas N17Rac1 inhibited endothelin 1-stimulated ANF expression, indicating that the synergy between Rac1 and c-Raf is potentially physiologically important. We conclude that activation of Rac1 by hypertrophic stimuli contributes to the hypertrophic response by modulating the ERK and/or possibly the JNK (but not the p38-MAPK) cascades.     

p14-MP1-MEK1 signaling regulates endosomal traffic and cellular proliferation during tissue homeostasis

The extracellular signal-regulated kinase (ERK) cascade regulates proliferation, differentiation, and survival in multicellular organisms. Scaffold proteins regulate intracellular signaling by providing critical spatial and temporal specificity. The scaffold protein MEK1 (mitogen-activated protein kinase and ERK kinase 1) partner (MP1) is localized to late endosomes by the adaptor protein p14. Using conditional gene disruption of p14 in mice, we now demonstrate that the p14-MP1-MEK1 signaling complex regulates late endosomal traffic and cellular proliferation. This function its essential for early embryogenesis and during tissue homeostasis, as revealed by epidermis-specific deletion of p14. These findings show that endosomal p14-MP1-MEK1 signaling has a specific and essential function in vivo and, therefore, indicate that regulation of late endosomal traffic by extracellular signals is required to maintain tissue homeostasis.     

Involvement of MAPK signaling molecules and Runx2 in the NELL1-induced osteoblastic differentiation

NELL1 is an extracellular protein inducing osteogenic differentiation and bone formation of osteoblastic cells. To elucidate the intracellular signaling cascade evoked by NELL1, we have shown that NELL1 protein transiently activates the MAPK signaling cascade, induces the phosphorylation of Runx2, and promotes the rapid intracellular accumulation of Tyr-phosphorylated proteins. Unlike BMP2, NELL1 protein does not activate the Smad signaling cascade. These findings suggest that upon binding to a specific receptor NELL1 transduces an osteogenic signal through activation of certain Tyr-kinases associated with the Ras-MAPK cascade, and finally leads to the osteogenic differentiation.     

ERK implication in cell cycle regulation

The Ras/Raf/MEK/ERK signaling cascade that integrates an extreme variety of extracellular stimuli into key biological responses controlling cell proliferation, differentiation or death is one of the most studied intracellular pathways. Here we present some evidences that have been accumulated over the last 15 years proving the requirement of ERK in the control of cell proliferation. In this review we focus (i) on the spatio-temporal control of ERK signaling, (ii) on the key cellular components linking extracellular signals to the induction and activation of cell cycle events controlling G1 to S-phase transition and (iii) on the role of ERK in the growth factor-independent G2/M phase of the cell cycle. As ERK pathway is often co-activated with the PI3 kinase signaling, we highlight some of the key points of convergence leading to a full activation of mTOR via ERK and AKT synergies. Finally, ERK and AKT targets being constitutively activated in so many human cancers, we briefly touched the cure issue of using more specific drugs in rationally selected cancer patients.     

The activity of the extracellular signal-regulated kinase 2 is regulated by differential phosphorylation in the activation loop

The mitogen-activated protein kinases (MAP kinases) play a central role in signaling pathways initiated by extracellular stimuli such as growth factors, cytokines, and various forms of environmental stress. Full activation of the MAP kinases requires dual phosphorylation of the Thr and Tyr residues in the TXY motif of the activation loop by MAP kinase kinases. Interestingly, down-regulation of MAP kinase activity can be initiated by multiple Ser/Thr phosphatases, Tyr-specific phosphatases, and dual-specificity phosphatases. This would inevitable lead to the formation of monophosphorylated MAP kinases. However, in much of the literature investigating MAP kinase signaling, there has been the implicit assumption that the monophosphorylated forms are inactive. Thus, the significance for the need of multiple phosphatases in regulating MAP kinase activity is not clear, and the biological functions of these monophosphorylated MAP kinases are currently unknown. We have prepared extracellular signal-regulated protein kinase 2 (ERK2) in all phosphorylated forms and kinetically characterized them using two proteins (the myelin basic protein and Elk-1) and ATP as substrates. Our results revealed that a single phosphorylation in the activation loop of ERK2 produces an intermediate activity state. Thus, the catalytic efficiencies of the monophosphorylated ERK2/pY and ERK2/pT (ERK2 phosphorylated on Tyr-185 and Thr-183, respectively) are approximately 2-3 orders of magnitude higher than that of the unphosphorylated ERK2 and are only 1-2 orders of magnitude lower than that of the fully active bisphosphorylated ERK2/pTpY. This raises the possibility that the monophosphorylated ERK2s may have distinct biological roles in vivo. Different phosphorylation states in the activation loop could be linked to graded effects on a single ERK2 function. Alternatively, they could be linked to distinct ERK2 functions. Although less active than the bisphosphorylated species, the monophosphorylated ERK2s may differentially phosphorylate pathway components.     

Modulation of Agrin-induced Acetylcholine Receptor Clustering by Extracellular Signal-regulated Kinases 1 and 2 in Cultured Myotubes

Agrin released by motoneurons induces and/or maintains acetylcholine receptor (AChR) clustering and other aspects of postsynaptic differentiation at the vertebrate neuromuscular junction. Agrin acts by binding and activating a receptor complex containing LDL receptor protein 4 (Lrp4) and muscle-specific kinase (MuSK). Two critical downstream components of this signaling cascade, Dox-7 and rapsyn, have been identified. However, additional intracellular essential elements remain unknown. Prior observations by others and us suggested antagonistic interactions between agrin and neuregulin-1 (Nrg-1) signaling in cultured myotubes and developing muscle fibers in vivo. A hallmark of Nrg-1 signaling in skeletal muscle cells is the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). ERK1/2 are also activated in most cells by phorbol 12-myristate 13-acetate, a classical inhibitor of agrin-induced AChR clustering in myotubes. Here, it was investigated whether agrin activates ERK1/2 directly and whether such activation modulates agrin-induced AChR clustering. Agrin induced a rapid but transient activation of ERK1/2 in myotubes that was Lrp4/MuSK-dependent. However, blocking this ERK1/2 activation did not prevent but potentiated AChR clustering induced by agrin. ERK1/2 activation was dispensable for Nrg-1-mediated inhibition of the AChR clustering activity of agrin, but was indispensable for such activity by phorbol 12-myristate 13-acetate. Together, these results suggest agrin-induced activation of ERK1/2 is a negative modulator of agrin signaling in skeletal muscle cells.     

The receptor for advanced glycation end-products (RAGE) directly binds to ERK by a D-domain-like docking site

The receptor for advanced glycation end-products (RAGE)-mediated cellular activation through the mitogen-activated protein kinase (MAPK) cascade, activation of NF-κB and Rho family small G-proteins, cdc42/Rac, is implicated in the pathogenesis of inflammatory disorders and tumor growth/metastasis. However, the precise molecular mechanisms for the initiation of cell signaling by RAGE remain to be elucidated. In this study, proteins which directly bind to the cytoplasmic C-terminus of RAGE were purified from rat lung extracts using an affinity chromatography technique and identified to be extracellular signal-regulated protein kinase-1 and -2 (ERK-1/2). Their interactions were confirmed by immunoprecipitation of ERK-1/2 from RAGE-expressing HT1080 cell extracts with anti-RAGE antibody. Furthermore, the augmentation of kinase activity of RAGE-bound ERK upon the stimulation of cells with amphoterin was demonstrated by determining the phosphorylation level of myelin basic protein, an ERK substrate. In vitro binding studies using a series of C-terminal deletion mutants of human RAGE revealed the importance of the membrane-proximal cytoplasmic region of RAGE for the direct ERK–RAGE interaction. This region contained a sequence similar to the D-domain, a ERK docking site which is conserved in some ERK substrates including MAPK-interacting kinase-1/2, mitogen- and stress-activated protein kinase-1, and ribosomal S6 kinase. These data suggest that ERK may play a role in RAGE signaling through direct interaction with RAGE.