ETS-domain transcription-factor networks represent a model for how combinatorial gene expression is achieved. These transcription factors interact with a multitude of co-regulatory partners to elicit gene-specific responses and drive distinct biological processes. These proteins are controlled by a complex series of inter and intramolecular interactions, and signalling pathways impinge on these proteins to further regulate their action.

Transcription of the proto-oncogene c-fos is stimulated rapidly and transiently by serum growth factors and mitogens. Critical for this response is the serum-response element which is bound in vivo in a ternary complex containing the transcription factors p67SRF and p62TCF (ref. 2). Disruption of the ternary complex correlates with impaired induction by serum and phorbol ester. Mitogen-activated protein (MAP) kinase is a serine/threonine kinase which is activated 1-5 minutes after treatment of cells with mitogens and growth factors that induce re-entry into the cell cycle, making MAP kinase a candidate for the transmission of proliferative signals. Here we show that p62TCF is phosphorylated by MAP kinase in vitro and that phosphorylation results in enhanced ternary complex formation. Serum-starved Swiss 3T3 cells treated with epidermal growth factor, which induces MAP kinase in these cells, are induced to express c-fos and yield p62TCF active in ternary complex formation. In contrast, treatment of Swiss 3T3 cells with insulin, which does not activate MAP kinase under these conditions, does not lead to enhanced ternary complex formation nor does it induce c-fos transcription. Our results link the expression of the human c-fos proto-oncogene to signal transduction pathways known to be activated before its own induction.

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The transcription factors Elk-1 and SAP-1 bind together with serum response factor to the serum response element present in the c-fos promoter and mediate increased gene expression. The ERK, JNK, and p38 groups of mitogen-activated protein (MAP) kinases phosphorylate and activate Elk-1 in response to a variety of extracellular stimuli. In contrast, SAP-1 is activated by ERK and p38 MAP kinases but not by JNK. The proinflammatory cytokine interleukin-1 (IL-1) activates JNK and p38 MAP kinases and induces the transcriptional activity of Elk-1 and SAP-1. These effects of IL-1 appear to be mediated by Rho family GTPases. Transcription of the prototypic immediate-early gene c-fos is rapidly induced in cells treated with a wide variety of extracellular stimuli, including growth factors, cytokines, UV radiation, and phorbol ester. The serum response element (SRE) is an important regulatory sequence located in the c-fos promoter (75) and is a target for a number of signal transduction pathways (7,8,81). The SRE is recognized by a complex of serum response factor (SRF) and a ternary complex factor (TCF) (76).SRF is phosphorylated in vitro by casein kinase II, the ERKactivated S6 kinase p90 rsk , and a DNA-activated protein kinase (8). However, the role of these kinases in the transcriptional activation of SRF remains unclear. Calcium activates SRFdependent transcription independently of TCF, by a mechanism involving Ca 2ϩ /calmodulin-dependent protein kinases (51). A recent study has provided evidence that members of the Ras-related Rho family of GTPases are involved in signaling pathways that lead to SRF-mediated activation of the c-fos SRE independently of complex formation with TCFs (27). This family of GTPases includes Cdc42, Rac1, and RhoA. These GTPases play a critical role in regulating the actin cytoskeleton and other physiological processes (60). RhoA is important for SRF-mediated induction of the c-fos SRE by serum, lysophosphatidic acid, and aluminum fluoride (27). Activated mutants of Cdc42 and Rac1 have been proposed to stimulate SRFmediated transcription independently of TCFs (27). The signaling mechanisms involved in these events await elucidation.TCF proteins belong to a subgroup of the Ets domain family (33) that includes Elk-1 (28), SAP-1 (13), and SAP-2 (21, 44, 54). The primary sequences of these proteins show significant similarity in three distinct regions (76). The N-terminal 93 amino acids comprise the Ets domain, which mediates DNA binding (13,32,70). The more centrally located 21-amino-acid B box is required for ternary complex formation and has been demonstrated to mediate direct protein-protein contacts between Elk-1 and SRF (69). The COOH-terminal activation domain contains several conserved mitogen-activated protein (MAP) kinase phosphorylation sites (76). Phosphorylation of the TCF proteins Elk-1 and SAP-1 on sites within the Cterminal activation domain results in activated SRE-dependent gene expression (7,8,81).MAP kinases are proline-directed Ser/Thr protein kinases that are regulated by ...

The phosphorylation of transcription factors by mitogen-activated protein kinases (MAP) is a pivotal event in the cellular response to the activation of MAP kinase signal transduction pathways. Mitogenic and stress stimuli activate different pathways and lead to the activation of distinct groups of target proteins. Elk-1 is targeted by three distinct MAP kinase pathways. In this study, we demonstrate that the MAP kinase ERK2 is targeted to Elk-1 by a domain which is distinct from, and located N-terminally to, its phosphoacceptor motifs. Targeting via this domain is essential for the efficient and rapid phosphorylation of Elk-1 in vitro and full and rapid activation in vivo. Specific residues involved in ERK targeting have been identified. Our data indicate that the targeting of different classes of MAP kinases to their nuclear substrates may be a common mechanism to increase the specificity and efficiency of this signal transduction pathway.

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The ETS domain transcription factor Elk-1 is a direct target of the MAP kinase pathways. Phosphorylation of the Elk-1 transcriptional activation domain by MAP kinases triggers its activation. However, Elk-1 also contains two domains with repressive activities. One of these, the R motif, appears to function by suppressing the activity of the activation domain. Here, we demonstrate that SUMO modification of the R motif is required for this repressive activity. A dynamic interplay exists between the activating ERK MAP kinase pathway and the repressive SUMO pathway. ERK pathway activation leads to both phosphorylation of Elk-1 and loss of SUMO conjugation and, hence, to the loss of the repressive activity of the R motif. Thus, the reciprocal regulation of the activation and repressive activities are coupled by MAP kinase modification of Elk-1.