A key event in the response of cells to proliferative signals is the rapid, transient induction of the c-fos proto-oncogene, which is mediated through the serum response element (SRE) in the fos promoter. Genomic footprinting and transfection experiments suggest that this activation occurs through a ternary complex that includes the serum response factor (SRF) and the ternary complex factor p62. Interaction of p62TCF with the SRF-SRE binary complex requires a CAGGA tract immediately upstream of the SRE. Proteins of the ets proto-oncogene family bind to similar sequences and we have found that a member of this family, Elk-1, forms SRF-dependent ternary complexes with the SRE. Elk-1 and p62TCF have the same DNA sequence requirements and antibodies against Elk-1 block the binding of both proteins. Furthermore, we show that like p62TCF, Elk-1 forms complexes with the yeast SRF-homologue MCM1 but not with yeast ARG80. But ARG80 mutants that convey interaction with p62TCF can also form complexes with Elk-1. The similarity, or even identity, between Elk-1 and p62TCF suggests a novel regulatory role for Ets proteins that is effected through interaction with other proteins, such as SRF. Furthermore, the possible involvement of an Ets protein in the control of c-fos has interesting implications for proto-oncogene cooperation in cellular growth control.

In cell culture systems, the TCF Elk-1 represents a convergence point for extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) subclasses of mitogen-activated protein kinase (MAPK) cascades. Its phosphorylation strongly potentiates its ability to activate transcription of the c-fos promoter through a ternary complex assembled on the c-fos serum response element. In rat brain postmitotic neurons, Elk-1 is strongly expressed (V. Sgambato, P. Vanhoutte, C. Pagès, M. Rogard, R. A. Hipskind, M. J. Besson, and J. Caboche, J. Neurosci. 18:214-226, 1998). However, its physiological role in these postmitotic neurons remains to be established. To investigate biochemically the signaling pathways targeting Elk-1 and c-fos in mature neurons, we used a semi-in vivo system composed of brain slices stimulated with the excitatory neurotransmitter glutamate. Glutamate treatment leads to a robust, progressive activation of the ERK and JNK/SAPK MAPK cascades. This corresponds kinetically to a significant increase in Ser383-phosphorylated Elk-1 and the appearance of c-fos mRNA. Glutamate also causes increased levels of Ser133-phosphorylated cyclic AMP-responsive element-binding protein (CREB) but only transiently relative to Elk-1 and c-fos. ERK and Elk-1 phosphorylation are blocked by the MAPK kinase inhibitor PD98059, indicating the primary role of the ERK cascade in mediating glutamate signaling to Elk-1 in the rat striatum in vivo. Glutamate-mediated CREB phosphorylation is also inhibited by PD98059 treatment. Interestingly, KN62, which interferes with calcium-calmodulin kinase (CaM-K) activity, leads to a reduction of glutamate-induced ERK activation and of CREB phosphorylation. These data indicate that ERK functions as a common component in two signaling pathways (ERK/Elk-1 and ERK/?/CREB) converging on the c-fos promoter in postmitotic neuronal cells and that CaM-Ks act as positive regulators of these pathways.

Vaccinia virus has been used as a vector to express foreign genes for the production of functional and posttranslationally modified proteins. A procedure is described here that allows the rapid native purification of vacciniaexpressed proteins fused to an amino-terminal tag of six histidines. Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni2+ nitrilotriacetic acid (Ni2+-NTA)-agarose and histidine-tagged proteins are selectively eluted with imidaole-containing buffers. In the case of the human serum response factor (SRF), a transcription factor involved in the regulation of the c-fos protooncogene, the vaccinia-expressed histidine-tagged SRF (SRF-6His) could be purified solely by this step to >95% purity. SRF-6His was shown to resemble authentic SRF by functional criteria: it was transported to the nucleus, bound specifically the c-fos serum response element, interacted with the p62TCF protein to form a ternary complex, and stimulated in vitro transcription from the serum response element. Thus, the combination of vaccinia virus expression and affinity purification by Ni2+|NTA chromatography promises to be useful for the production of proteins in a functional and posttranslationally modified form.

Akt is classically described as a prosurvival serine/threonine kinase activated in response to trophic factors. After activation by phosphoinositide 3-kinase (PI3-kinase), it can translocate to the nucleus where it promotes specific genetic programs by catalyzing phosphorylation of transcription factors. We report here that both dopamine (DA) D1 (SKF38393) and D2 (quinpirole) agonist treatments rapidly increase, in primary striatal neurons in culture, phosphorylation levels of Akt on Thr 308 , a residue that is critically involved in its kinase activity. These treatments also activate the extracellular signal-regulated kinase (ERK) pathway in the same population of striatal neurons. Induction of active, phospho-Thr 308 Akt by dopamine D1 and D2 agonists is insensitive to wortmannin and thus PI3-kinase independent, in contrast to growth factor-induced Akt activity. D1-and D2-induced phospho-Thr 308 Akt is decreased by the mitogen-activated protein kinase kinase (MEK) inhibitor, U0126, as well as by overexpression of a dominant-negative version of MEK, thus implicating the Ras/ERK signaling cascade in this process. Furthermore, overexpression of a mutant form of Akt that cannot be activated impaired cAMP response elementbinding protein (CREB) phosphorylation induced by SKF38393 and quinpirole treatments. Activation of Akt on Thr 308 was also found in vivo in striatal neurons after acute administration of cocaine, a psychostimulant that strongly increases DA transmission. Thus, multiple intracellular pathways can transduce signals from dopamine receptors to CREB in striatal neurons, one of these being Akt. We propose that this signaling pathway plays a pivotal role in DA-induced regulation of gene expression and long-term neuronal adaptation in the striatum.

Inhibitors of protein synthesis, such as anisomycin and cycloheximide, lead to superinduction of immediateearly genes. We demonstrate that these two drugs activate intracellular signaling pathways involving both the mitogen-activated protein kinase (