Kinase activity is known as the key biochemical property of MAPKs. Here, we report that ERK1/2 also utilizes its noncatalytic function to mediate certain signal transductions. Sustained activation of the Raf/MEK/ERK pathway induces growth arrest, accompanied by changes in cell cycle regulators (decreased retinoblastoma phosphorylation, E2F1 down-regulation, and/or p21 CIP1 up-regulation) and cell type-specific changes in morphology and expression of c-Myc or RET in the human tumor lines LNCaP, U251, and TT. Ablation of ERK1/2 by RNA interference abrogated all these effects. However, active site-disabled ERK mutants (ERK1-K71R, ERK2-K52R, and ERK2-D147A), which competitively inhibit activation of endogenous ERK1/2, could not block Raf/MEK-induced growth arrest as well as changes in the cell cycle regulators, although they effectively blocked phosphorylation of the ERK1/2 catalytic activity readouts, p90 RSK and ELK1, as well as the cell type-specific changes. Because this indicated a potential noncatalytic ERK1/2 function, we generated stable lines of the tumor cells in which both ERK1 and ERK2 were significantly knocked down, and we further investigated the possibility using rat-derived kinase-deficient ERK mutants (ERK2-K52R and ERK2-T183A/Y185F) that were not targeted by human small hairpin RNA. Indeed, ERK2-K52R selectively restored Raf-induced growth inhibitory signaling in ERK1/2-depleted cells, as manifested by regained cellular ability to undergo growth arrest and to control the cell cycle regulators without affecting c-Myc and morphology. However, ERK2-T183A/Y185F was less effective, indicating the requirement of TEY site phosphorylation. Our study suggests that functions of ERK1/2 other than its "canonical" kinase activity are also involved in the pathway-mediated growth arrest signaling. ERK12 and its homologue ERK2, the MAPK components of the Raf/MEK/ERK cascade of Ras signaling, are ubiquitously expressed serine/threonine kinases with more than 160 substrates identified to date (1). ERK1/2 interacts with a wide variety of proteins (2, 3). Upon phosphorylation by MEK1/2, the only known activator of ERK1/2, ERK1/2 phosphorylates transcription factors, other kinases, phosphatases, cytoskeletal proteins, scaffolds, receptors, and signaling components that mediate diverse cellular processes. Although kinase activity of ERK1/2 is central in activation or inactivation of these ERK targets, it was also reported that ERK, in an in vitro reaction, can mediate noncatalytic activation of DNA topoisomerase II␣, suggesting that ERK1/2 also has noncatalytic function (4). Nonetheless, the possibility that ERK1/2 has functions other than kinase has not yet been clearly addressed in cells.Many studies have shown that ERK1/2 signaling is pivotal in controlling cell survival and cell cycle progression (5). Constitutive activation of the MAPK cascade is also a central signature of many cancers with dysregulated Ras/Raf signaling (6, 7). Paradoxically, sustained activation of the Ras/Raf pathway induces growth arrest in p...

Actinomyces naeslundii is a pioneer of the oral cavity and forms a biofilm on the tooth's surface. Most bacteria require iron for survival and in pathogenic bacteria iron availability regulates virulence gene expression. Metal-dependent repressors control gene expression involved in metal transport and uptake including siderophores. Siderophores are small molecules synthesized by bacteria and fungi to acquire iron. The A. naeslundii genome was searched for a gene encoding a metal-dependent repressor. Actinomyces metal-dependent repressor or amdR was identified. The AmdR protein was examined for its ability to bind to the promoter sequence of a gene encoding the siderophore uptake (sid gene). According to gel shift assays, AmdR binds to the sid gene promoter sequences. In the authors' model, when iron is available AmdR binds to the sid promoter and represses sid gene expression. To further explore the role of AmdR, an amdR-defective strain of A. naeslundii was constructed and biofilm formation and siderophore production were evaluated. When iron is removed from the medium A. naeslundii increases biofilm and siderophore production. However, amdR-defective A. naeslundii is less sensitive to metal ion concentrations in the growth medium.