contributed equally to this work IEX-1 is an early response and NF-kB target gene implicated in the regulation of cellular viability. We show here that IEX-1 is a substrate for ERKs and that IEX-1 and ERK regulate each other's activities. IEX-1 was isolated by phosphorylation screening with active ERK2 and found subsequently phosphorylated in vivo upon ERK activation. IEX-1 interacts with phosphorylated ERKs but not with c-jun N-terminal kinase (JNK) or p38. Upon phosphorylation by ERKs, IEX-1 acquires the ability to inhibit cell death induced by various stimuli. In turn, IEX-1 potentiates ERK activation in response to various growth factors. By using various IEX-1 mutants in which the ERK phosphoacceptor and/or ERK docking sites were mutated, we show that the IEX-1 pro-survival effect is dependent on its phosphorylation state but not on its ability to potentiate ERK activation. Conversely, IEX-1-induced modulation of ERK activation requires ERK±IEX-1 association but is independent of IEX-1 phosphorylation. Thus, IEX-1 is a new type of ERK substrate that has a dual role in ERK signaling by acting both as an ERK downstream effector mediating survival and as a regulator of ERK activation.

Mycoplasma lipoproteins have been demonstrated to stimulate monocytic cells and induce proinflammatory cytokine secretion. In this paper, we show that a synthetic analog of the Mycoplasma fermentans membraneassociated lipopeptide macrophage-activating lipopeptide-2 (MALP-2) induces mRNA synthesis and protein secretion of interleukin-1␤ and tumor necrosis factor-␣ in human monocytes/macrophages and the murine macrophage cell line RAW 264.7, whereas the nonlipidated counterpart lacks this effect, underscoring the importance of protein acylation for cell activation. Synthetic MALP-2 (sMALP-2) induced the activation of MAPK family members extracellular signal regulated kinases 1 and 2, c-Jun NH 2 -terminal kinase, and p38 and induced NF-B and AP-1 transactivation in macrophages. Whereas the specific p38 inhibitor SB203580 abrogated both cytokine synthesis and NF-B and AP-1 transactivation in response to MALP-2, the selective MAPK/extracellular signal-regulated kinase-1 inhibitor PD-98059 decreased interleukin-1␤ and tumor necrosis factor-␣ production in response to sMALP-2 without affecting the transactivation of NF-B or AP-1. These results indicate that activation of MAPKs by sMALP-2 is a crucial event leading to the expression of proinflammatory cytokines. Our findings demonstrate that the synthetic analog of MALP-2 reproduces the macrophage stimulation activity found in different fractions of mycoplasmas. Given that MALP-2 has been recently shown to be expressed at the surface of M. fermentans as a molecular entity, sMALP-2 constitutes a valuable surrogate for investigating immunomodulation by these microorganisms and evaluating the role that this activity plays in the development of inflammatory diseases associated with mycoplasma infections.Mycoplasma fermentans, a human pathogen, is a potent activator of monocytes/macrophages. Macrophage activation by M. fermentans results in the secretion of numerous cytokines including interleukin (IL) 1 -1␤, IL-6, IL-8, IL-10, and tumor necrosis factor-␣ (TNF␣) (1-3). Other inflammation mediators such as NO have also been shown to be produced by macrophages in response to mycoplasmas (3). Mycoplasmas, the smallest self-replicating bacteria, are characterized by a wallless envelope (4); thus, they are LPS-free pathogens. We have previously demonstrated that mycoplasma membrane lipoproteins (lipid-associated membrane proteins; LAMPs) are responsible for human and murine macrophage activation (1, 2, 5).Although the signaling pathways triggered in macrophages by both M. fermentans LAMP and Gram-negative bacteria LPS are comparable, unlike LPS, M. fermentans LAMP activity does not require binding to CD14 or serum proteins (5). M. fermentans LAMP activates mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun NH 2 -terminal kinase (JNK), and p38 (5).MAPKs have been shown to be involved in M. fermentans LAMP-mediated cytokine induction, since selective inhibitors of these pathways have been shown to impair M. fermenta...