Enhancement of fibroblast collagenase (matrix metalloproteinase-1) gene expression by ceramide is mediated by extracellular signal-regulated and stress-activated protein kinase pathways

Inflammatory cytokines tumor necrosis factor-alpha and interleukin-1 trigger the ceramide signaling pathway, initiated by neutral sphingomyelinase-elicited hydrolysis of cell membrane phospholipid sphingomyelin to ceramide, a new lipid second messenger. Here, we show that triggering the ceramide pathway by sphingomyelinase or C2- and C6-ceramide enhances collagenase-1 (matrix metalloproteinase-1; MMP-1) gene expression by fibroblasts. C2-ceramide activates three distinct mitogen-activated protein kinases (MAPKs) in dermal fibroblasts, i.e. extracellular signal-regulated kinase 1/2 (ERK1/2), stress-activated protein kinase/Jun N-terminal-kinase (SAPK/JNK), and p38. Stimulation of MMP-1 promoter activity by C2-ceramide is dependent on the presence of a functional AP-1 cis-element and is entirely inhibited by overexpression of MAPK inhibitor, dual specificity phosphatase CL100 (MAPK phosphatase-1). Activation of MMP-1 promoter by C2-ceramide is also effectively inhibited by kinase-deficient forms of ERK1/2 kinase (MEK1/2) activator Raf-1, ERK1 and ERK2, SAPK/JNK activator SEK1, or SAPKbeta. In addition, ceramide-dependent induction of MMP-1 expression is potently prevented by PD 98059, a selective inhibitor of MEK1 activation, and by specific p38 inhibitor SB 203580. These results show that triggering the ceramide signaling pathway activates MMP-1 gene expression via three distinct MAPK pathways, i.e. ERK1/2, SAPK/JNK, and p38, and suggest that targeted modulation of the ceramide signaling pathway may offer a novel therapeutic approach for inhibiting collagenolytic activity, e.g. in inflammatory disorders.     

Mitogen-activated protein kinase kinase 2 activation is essential for progression through the G2/M checkpoint arrest in cells exposed to ionizing radiation

An increasing body of evidence suggests that mitogen-induced activation of the RAF/ERK signaling pathway is functionally separate from the stress-induced activation of the SEK/JNK/p38 signaling pathway. In general, stress stimuli strongly activate the p38s and the JNKs while only weakly activating ERK1 and ERK2. However, a number of independent groups have now shown that the RAF/ERK signaling pathway is strongly activated by ionizing radiation. In this work, we examine this paradox. We show that both mitogen-activated protein (MAP) kinase kinase 1 (MEK1) and MAP kinase kinase 2 (MEK2) are activated by ionizing radiation. Blockage of this activation through the use of dominant negative MEK2 increases sensitivity of the cell to ionizing radiation and decreases the ability of a cell to recover from the G2/M cell cycle checkpoint arrest. Blocking MEK2 activation does not affect double-strand DNA break repair, however. Although MEK1 is activated to a lesser extent by ionizing radiation, expression of a dominant negative MEK1 does not affect radiation sensitivity of the cell, the G2/M checkpoint of the cell, or double-strand break repair. Because ionizing radiation leads to a different cell cycle arrest (G2/M arrest) than that typically seen with other stress stimuli, and because we have shown that MEK2 can affect G2/M checkpoint kinetics, these results provide an explanation for the observation that the MEKs can be strongly activated by ionizing radiation and only weakly activated by other stressful stimuli.     

Retinoic acid induced mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase-dependent MAP kinase activation needed to elicit HL-60 cell differentiation and growth arrest

Retinoic acid (RA) activated the extracellular signal-regulated kinase (ERK) 2 mitogen-activated protein kinase (MAPK) of HL-60 human myeloblastic leukemia cells before causing myeloid differentiation and cell cycle arrest associated with hypophosphorylation of the retinoblastoma (RB) tumor suppressor protein. ERK2 activation by mitogen-activated protein/ERK kinase (MEK) was necessary for RA-induced differentiation in studies using PD98059 to block MEK phosphorylation. G0 growth arrest and RB tumor suppressor protein hypophosphorylation (which is typically associated with induced differentiation and G0 arrest), two putatively RB-regulated processes, also depended on ERK2 activation by MEK. Activation of ERK2 by RA occurred within hours and persisted until the onset of RB hypophosphorylation, differentiation, and arrest. ERK2 activation was probably needed early, because delaying the addition of PD98059 relative to that of RA restored most of the RA-induced cellular response. In contrast to RA (which activates RA receptors (RARs) and retinoid X receptors in HL-60 cells with its metabolite retinoids), a retinoid that selectively binds RAR-gamma, which is not expressed in HL-60 cells, was relatively ineffective in causing ERK2 activation. This is consistent with the need for a nuclear retinoid receptor function in RA-induced ERK2 activation. RA reduced the amount of unphosphorylated RAR-alpha, whose activation is necessary for RA-induced differentiation and arrest. This shifted the ratio of phosphorylated:unphosphorylated RAR-alpha to predominantly the phosphorylated form. Unlike other steroid thyroid hormone receptors susceptible to phosphorylation and activation by MAPKs, RAR-alpha was not phosphorylated by the activated ERK2 MAPK. The results thus show that RA augments MEK-dependent ERK2 activation that is needed for subsequent RB hypophosphorylation, cell differentiation, and G0 arrest. The process seems to be nuclear receptor dependent and an early seminal component of RA signaling causing differentiation and growth arrest.     

Calmodulin inhibitor W13 induces sustained activation of ERK2 and expression of p21(cip1)

One of the major signaling pathways by which extracellular signals induce cell proliferation and differentiation involves the activation of extracellular signal-regulated kinases (ERKs). Because calmodulin is essential for quiescent cells to enter cell cycle, the role of calmodulin on ERK2 activation was studied in cultured fibroblasts. Serum, phorbol esters, or active Ras induced ERK2 activation in NIH 3T3 fibroblasts. This activation was not inhibited by calmodulin blockade. Surprisingly, inhibition of calmodulin prior to fetal bovine serum addition prolonged activation of ERK2. Furthermore, inactivation of calmodulin in serum-starved cells induced ERK2 phosphorylation that was dependent on MAP kinase kinase (MEK). Inactivation of calmodulin in serum-starved cells also induced activation of Ras, Raf, and MEK. On the contrary, tyrosine phosphorylation of tyrosine kinase receptors was not observed. These results indicate that calmodulin inhibits ERK2 activation pathway at the level of Ras. Calmodulin inhibition induced overexpression of p21(cip1) which was dependent on MEK activity. We propose that inhibition of Ras by calmodulin prevents the activation of ERK2 at low serum concentration. Thus, entering into the cell cycle after serum addition would imply the overcoming of the inhibitory effect of calmodulin and consequently ERK2 activation. Furthermore, down-regulation of Ras by calmodulin may be also important to determine the duration of ERK2 activation and to prevent a high p21(cip1) expression that would lead to an inhibition of cell proliferation.     

Constitutively active mutant of the mitogen-activated protein kinase kinase MEK1 induces epithelial dedifferentiation and growth inhibition in madin-darby canine kidney-C7 cells

Overexpression of a constitutively active mitogen-activated protein kinase kinase (MAPKK or MEK) induces neuronal differentiation in adrenal pheochromocytoma 12 cells but transformation in fibroblasts. In the present study, we used a constitutively active MAPK/extracellular signal-regulated kinase (ERK) kinase 1 (MEK1) mutant to investigate the function of the highly conserved MEK1-ERK2 signaling module in renal epithelial cell differentiation and proliferation. Stable expression of constitutively active MEK1 (CA-MEK1) in epithelial MDCK-C7 cells led to an increased basal and serum-stimulated ERK1 and ERK2 phosphorylation as well as ERK2 activation when compared with mock-transfected cells. In both mock-transfected and CA-MEK1-transfected MDCK-C7 cells, basal and serum-stimulated ERK1 and ERK2 phosphorylation was almost abolished by the synthetic MEK inhibitor PD098059. Increased ERK2 activation due to stable expression of CA-MEK1 in MDCK-C7 cells was associated with epithelial dedifferentiation as shown by both a dramatic alteration in cell morphology and an abolished cytokeratin expression but increased vimentin expression. In addition, we obtained a delayed and reduced serum-stimulated cell proliferation in CA-MEK1-transfected cells (4.6-fold increase in cell number/cm2 after 5 days of serum stimulation) as compared with mock-transfected controls (12.9-fold increase in cell number/cm2 after 5 days). This result was confirmed by flow cytometric DNA analysis showing that stable expression of CA-MEK1 decreased the proportion of MDCK-C7 cells moving from G0/G1 to G2/M as compared with both untransfected and mock-transfected cells. Taken together, our data demonstrate an association of increased basal and serum-stimulated activity of the MEK1-ERK2 signaling module with epithelial dedifferentiation and growth inhibition in MDCK-C7 cells. Thus, the MEK1-ERK2 signaling pathway could act as a negative regulator of epithelial differentiation thereby leading to an attenuation of MDCK-C7 cell proliferation.     

Inhibition of mitogen-activated protein kinase kinase blocks T cell proliferation but does not induce or prevent anergy

Three mitogen-activated protein kinase pathways are up-regulated during the activation of T lymphocytes, the extracellular signal-regulated kinase (ERK), Jun NH2-terminal kinase, and p38 mitogen-activated protein kinase pathways. To examine the effects of blocking the ERK pathway on T cell activation, we used the inhibitor U0126, which has been shown to specifically block mitogen-activated protein kinase/ERK kinase (MEK), the kinase upstream of ERK. This compound inhibited T cell proliferation in response to antigenic stimulation or cross-linked anti-CD3 plus anti-CD28 Abs, but had no effect on IL-2-induced proliferation. The block in T cell proliferation was mediated by down-regulating IL-2 mRNA levels. Blocking Ag-induced proliferation by inhibiting MEK did not induce anergy, unlike treatments that block entry into the cell cycle following antigenic stimulation. Surprisingly, induction of anergy in T cells exposed to TCR cross-linking in the absence of costimulation was also not affected by blocking MEK, unlike cyclosporin A treatment that blocks anergy induction. These results suggest that inhibition of MEK prevents T cell proliferation in the short term, but does not cause any long-term effects on either T cell activation or induction of anergy. These findings may help determine the viability of using mitogen-activated protein kinase inhibitors as immune suppressants.     

Hydrogen peroxide activation of multiple mitogen-activated protein kinases in an oligodendrocyte cell line: role of extracellular signal-regulated kinase in hydrogen peroxide-induced cell death

Oxidative stress is known to induce cell death in a wide variety of cell types, apparently by modulating intracellular signaling pathways. In this study, we have examined the activation of mitogen-activated protein kinase (MAPK) cascades in relation to oxidant-induced cell death in an oligodendrocyte cell line, central glia-4 (CG4). Exposure of CG4 cells to hydrogen peroxide (H2O2) resulted in an increased tyrosine phosphorylation of several protein species, including the abundantly expressed platelet-derived growth factor (PDGF) receptor and the activation of the three MAPK subgroups, i.e., extracellular signal-regulated kinase (ERK), p38 MAPK, and c-Jun N-terminal kinase (JNK). Dose-response studies showed differential sensitivities of PDGF receptor phosphorylation (>1 mM) and ERK/p38 MAPK (>0.5 mM) and JNK (>0.1 mM) activation by H2O2. The activation of ERK was inhibited by PD98059, a specific inhibitor of the upstream kinase, MAPK or ERK kinase (MEK). H2O2 also activated MAPK-activated protein kinase-2, and this activation was blocked by SB203580, a specific inhibitor of p38 MAPK. The oxidant-induced cell death was indicated by morphological changes, decreased 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction, and DNA fragmentation. These effects were suppressed dose-dependently by the MEK inhibitor PD98059. The results demonstrate that H2O2 induces the activation of multiple MAPKs in oligodendrocyte progenitors and that the activation of ERK is associated with oxidant-mediated cytotoxicity.     

Oxidative stress induces DNA fragmentation and caspase activation via the c-Jun NH2-terminal kinase pathway in H9c2 cardiac muscle cells

The aim of this study was to test the hypothesis that oxidative stress induces apoptosis in the H9c2 cardiac muscle cell line, and that signaling via mitogen-activated protein kinase (MAPK) pathways is involved. Three forms of oxidative stress were utilized: the superoxide generator menadione; hydrogen peroxide; or simulated ischemia followed by reperfusion. Relatively low concentrations of menadione (10 micrometer) or H2O2 (250 micrometer) caused maximal DNA fragmentation and caspase activation, both markers for apoptotic cell death, and preferential activation of the c-Jun NH 2-terminal kinase (JNK) and p38 MAPK pathways. In contrast, higher concentrations of menadione or H 2O2 caused less DNA fragmentation, more necrotic cell death and preferential activation of the extracellular signal-regulated kinase (ERK) pathway. Simulated ischemia alone did not induce DNA fragmentation or caspase activation and activated only the p38 MAPK pathway. However, ischemia plus reperfusion resulted in DNA fragmentation, caspase activation, necrotic cell death and activation of all three MAPK pathways. Selective inhibition of the ERK or p38 MAPK pathways (by PD98059 or SB-203580, respectively) had no effect on the extent of oxidative stress-induced DNA fragmentation or caspase activation. In contrast, inhibition of the JNK pathway by transfection of a dominant negative mutant of JNK markedly reduced the extent of DNA fragmentation and caspase activation induced by oxidative stress. In conclusion, these data suggest that the JNK pathway plays an important role in signaling oxidative stress-induced apoptosis of H9c2 cardiac muscle cells.     

Terminal complement complexes induce cell cycle entry in oligodendrocytes through mitogen activated protein kinase pathway

Sublytic complement attack through C5b-9 assembly induces oligodendrocytes (OLG) to express proto-oncogenes and to enter the cell cycle from resting G0/G1 phase to S phase. We have investigated whether cell cycle induction by C5b-9 is mediated by mitogen activated protein kinase (MAPK) pathway in OLG. C5b-9 but not C5b6 induced activation of both ERK1 and c-jun NH2 terminal kinases 1 (JNK1) in OLG. The increased ERK1 and JNK1 activities are transient, reaching a maximum around 20 min following exposure to C5b-9. Activation of Raf-1 and MEK1, upstream kinases of ERK1, was shown by increased Raf-1 kinase activity in anti-Raf-1 immunoprecipitates of OLG treated with C5b-9 and ERK1 activity that can be inhibited by PD098,059, a specific MEK1 inhibitor. Requirement for the ERK1 pathway in DNA synthesis was then evaluated using PD098,059. Enhanced DNA synthesis induced by serum complement was completely abolished when OLG were pretreated with PD098,059. On the other hand, c-fos mRNA expression induced by complement was inhibited only 50% by PD098,059, while the c-jun mRNA level was not affected by this MEK1 inhibitor. Interestingly, p70 S6 kinase, an important ribosomal kinase in mitogenesis, was also activated by C5b-9. These findings indicated that the MAPK pathways appears to play a major role in inducing OLG to enter the S phase of the cell cycle from the resting G1/G0 phase.     

Stimulation of Jun N-terminal kinase (JNK) by gonadotropin-releasing hormone in pituitary alpha T3-1 cell line is mediated by protein kinase C, c-Src, and CDC42

The signaling of ligands operating via heterotrimeric G proteins is mediated by a complex network that involves sequential phosphorylation events. Signaling by the G protein-coupled receptor GnRH was shown to include elevation of Ca2+ and activation of phospholipases, protein kinase C (PKC) and extra-cellular signal-regulated kinase (ERK). In this study, GnRH was shown to activate Jun N-Terminal Kinase (JNK)/SAPK in alpha T3-1 cells in a PKC- and tyrosine kinase-dependent manner. GnRH as well as tumor-promoting agent (TPA) also increased c-Src activity, which peaked at 2 min after GnRH stimulation and was sensitive both to PKC and to tyrosine kinase inhibitors. Coexpression of Csk, which serves as a Src-dominant interfering kinase, and constitutively active forms of Src, together with JNK, confirmed the involvement of c-Src downstream of PKC in the GnRH-JNK pathway. Coexpression of dominant negative and constitutively active forms of CDC42, Rac1, Ras, MEKK1, and MEK1 with JNK indicated that JNK activation by GnRH and TPA is mediated by CDC42 and MEKK1. Ras and MEK1, which are involved in a related mitogen-activated protein kinase (MAPK) pathway, did not affect JNK activation in alpha T3-1 cells. Taken together, our results suggest that GnRH stimulation of JNK activity is mediated by a unique pathway that includes sequential activation of PKC, c-Src, CDC42, and probably also MEKK1.     

Differential activation of ERK and JNK mitogen-activated protein kinases by Raf-1 and MEKK

Growth factors activate mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinases (ERKs) and Jun kinases (JNKs). Although the signaling cascade from growth factor receptors to ERKs is relatively well understood, the pathway leading to JNK activation is more obscure. Activation of JNK by epidermal growth factor (EGF) or nerve growth factor (NGF) was dependent on H-Ras activation, whereas JNK activation by tumor necrosis factor alpha (TNF-alpha) was Ras-independent. Ras activates two protein kinases, Raf-1 and MEK (MAPK, or ERK, kinase) kinase (MEKK). Raf-1 contributes directly to ERK activation but not to JNK activation, whereas MEKK participated in JNK activation but caused ERK activation only after overexpression. These results demonstrate the existence of two distinct Ras-dependent MAPK cascades--one initiated by Raf-1 leading to ERK activation, and the other initiated by MEKK leading to JNK activation.     

MEKK1 binds directly to the c-Jun N-terminal kinases/stress-activated protein kinases

Mitogen-activated protein (MAP) kinases mediate responses to a wide array of cellular stimuli. These cascades consist of a MAP kinase or extracellular signal-regulated kinase (ERK), activated by a MAP/ERK kinase (MEK), in turn activated by a MEK kinase (MEKK). MEKK1 has been shown to be a strong activator of the c-Jun N-terminal kinase/stress-actived protein kinase (JNK/SAPK) pathway. We report here that JNK/SAPK binds directly to the N-terminal, noncatalytic domain of MEKK1 in vitro and in transfected cells. Immobilized MEKK1-derived peptides extract JNK/SAPK selectively from cell lysates. MEKK1 coimmunoprecipitates with multiple JNK/SAPK isoforms in transfected cells. Expression of the N terminus of MEKK1 lacking the kinase domain increases activation of endogenous JNK/SAPK by MEKK1. The data are consistent with a model in which MEKK1-JNK/SAPK binding facilitates the receipt of signals from upstream inputs and localizes JNK/SAPK to intracellular targets of the pathway.     

Early activation of mitogen-activated protein kinase kinase, extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and c-Jun N-terminal kinase in response to binding of simian immunodeficiency virus to Jurkat T cells expressing CCR5 receptor

We have shown that the binding of simian immunodeficiency virus (SIV) to Jurkat T cells expressing CD4 receptor strongly induces mitogen-activated protein (MAP) kinase kinase (MEK) and extracellular signal-regulated kinases 1 and 2 (ERK1/2) and only weakly induces p38 MAP kinase and c-Jun N-terminal kinase (JNK). Similarly, T-tropic NL4-3 virus, which uses both CD4 and CXCR4 receptors for entry, stimulated in these cells the MEK/ERK MAP kinase (MAPK) pathway in a CD4 receptor-dependent manner (Popik and Pitha, 1998). In contrast, both macrophage-tropic SIVmac316 and T cell-tropic SIVmac239, which in addition to CD4 require CCR5 coreceptor for entry, significantly enhanced early MEK/ERK, p38 MAPK, and JNK signaling in Jurkat cells expressing constitutively or transiently the CCR5 receptor. Together, this study provides the evidence that viruses using CXCR4 or CCR5 receptors for entry may differentially use signaling properties of their specific coreceptors to stimulate MAP kinase cascades. In addition, although SIVmac239 and SIVmac316 use different structural domains of the CCR5 receptor for entry, both viruses stimulate early phosphorylation of MEK, ERK, p38, and JNK independently of their tropism and replication.     

The Shp-2 tyrosine phosphatase has opposite effects in mediating the activation of extracellular signal-regulated and c-Jun NH2-terminal mitogen-activated protein kinases

Shp-2 is a widely expressed cytoplasmic tyrosine phosphatase with two SH2 domains. A targeted mutant allele of the Shp-2 gene with a deletion of 65 amino acids in the NH2-terminal SH2 domain was created that leads to embryonic lethality at mid-gestation in homozygous mutant mice. To define the Shp-2 function in cell signaling, we have established mutant fibroblast cell lines, and have examined the effect of the Shp-2 mutation on extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) mitogen-activated protein (MAP) kinase pathways. Insulin-like growth factor (IGF)-I-induced ERK activation was completely abolished, while ERK activity upon platelet-derived growth factor and epidermal growth factor stimulation was significantly reduced and shortened in mutant cells. Stimulation of ERK by phorbol 12-myristate 13-acetate was not affected in mutant cells, but the phorbol 12-myristate 13-acetate-induced ERK activity decayed much faster compared with that in wild-type cells. In contrast, JNK activation upon heat shock was significantly enhanced in Shp-2 mutant cells. Based on these results, we conclude that Shp-2 plays differential positive regulatory roles in various mitogenic signaling pathways leading to ERK activation, and that Shp-2 is a negative effector in JNK activation by cellular stress. This is the first evidence that a tyrosine phosphatase has opposite effects in mediating the activation of ERK and JNK MAP kinases.     

Mitotic Raf-1 is stimulated independently of Ras and is active in the cytoplasm

Raf-1 is a Ser/Thr protein kinase that is involved in regulation of proliferation, differentiation, and apoptosis. Recently, we and others showed that Raf-1 is not only activated in mitogenic pathways leading to cell cycle entry but also during mitosis. Transient expression studies in COS cells now demonstrate that, in contrast to growth factor-dependent activation of Raf-1, mitotic activation of Raf-1 is Ras-independent. Dominant negative RasS17N does not interfere with mitotic activation of Raf-1, whereas epidermal growth factor-dependent stimulation of Raf-1 is inhibited. In addition, the Raf-1 mutant RafR89L, which cannot bind to activated Ras, is still stimulated in mitotic cells. Mitotic activation of Raf-1 seems to be partially dependent on tyrosine phosphorylation since the kinase activity of the Raf mutant RafYY340/341FF, which can no longer be activated by Src, is reduced in mitotic cells. Surprisingly, cell fractionation experiments showed that mitotic-activated Raf-1 is predominantly located in the cytoplasm in contrast to the mitogen-activated Raf-1 that is bound to the plasma membrane. In addition, mitotic activation of Raf-1 does not lead to stimulation of the mitogen-activated protein kinase kinase (MAPKK or MEK) and the extracellular signal-regulated protein kinase (ERK). These data demonstrate that in mitotic cells a Ras-independent mechanism results in a cytoplasmic active Raf-1 kinase which does not signal via the MEK/ERK pathway. These data demonstrate that in mitotic cells a Ras-independent mechanism results in a cytoplasmic active Raf-1 kinase which does not signal via the MEK/ERK pathway.     

The JNK pathway regulates the In vivo deletion of immature CD4(+)CD8(+) thymocytes

The extracellular signal-regulated kinase (ERK), the c-Jun NH2-terminal kinase (JNK), and p38 MAP kinase pathways are triggered upon ligation of the antigen-specific T cell receptor (TCR). During the development of T cells in the thymus, the ERK pathway is required for differentiation of CD4(-)CD8(-) into CD4(+)CD8(+) double positive (DP) thymocytes, positive selection of DP cells, and their maturation into CD4(+) cells. However, the ERK pathway is not required for negative selection. Here, we show that JNK is activated in DP thymocytes in vivo in response to signals that initiate negative selection. The activation of JNK in these cells appears to be mediated by the MAP kinase kinase MKK7 since high levels of MKK7 and low levels of Sek-1/MKK4 gene expression were detected in thymocytes. Using dominant negative JNK transgenic mice, we show that inhibition of the JNK pathway reduces the in vivo deletion of DP thymocytes. In addition, the increased resistance of DP thymocytes to cell death in these mice produces an accelerated reconstitution of normal thymic populations upon in vivo DP elimination. Together, these data indicate that the JNK pathway contributes to the deletion of DP thymocytes by apoptosis in response to TCR-derived and other thymic environment- mediated signals.     

Reductions in the activation of ERK and JNK are associated with decreased IL-2 production in T cells from elderly humans stimulated by the TCR/CD3 complex and costimulatory signals

T cells from elderly humans often display impaired IL-2 production, but the mechanisms are unknown. Because the activities of extracellular signal-regulated kinases (ERK) and c-Jun NH2-terminal kinases (JNK) are important for IL-2 production, the current study evaluated if aberrancies in the expression and activation of ERK2 or JNK might underlie decreased IL-2 production by human T cells during aging. The present results show that diminished ERK2 and JNK catalytic activities were commonly detected in T cells from elderly humans stimulated with anti-CD3 mAb OKT3 plus PMA. These reductions did not represent temporal shifts in activation or altered expression of ERK2 or JNK. In addition, the reductions of ERK2 activation in stimulated T cells from elderly individuals were accompanied by decreased Raf-1 kinase activation and could be observed without coexisting impairments in JNK activation. Stimulation of ERK2 activation in elderly T cells correlated with IL-2 production and decreased ERK2 activation was consistently associated with reduced IL-2 production. Although the age-related decreases in JNK activation were accompanied by reduced IL-2 production, substantial impairments of JNK activation were observed with diminished ERK2 activation. Moreover, anti-CD3/PMA-stimulated T cells from elderly individuals that displayed normal JNK activation and impaired ERK2 activation continued to demonstrate reduced IL-2 production. These findings show that impairments in the activation of ERK2 and JNK can accompany decreased IL-2 production by T cells from elderly humans and further suggest that aberrancies in TCR/CD3-dependent activation of the Raf-1/MEK/ERK2 cascade may be rate-limiting for the full induction of IL-2.