The proteins encoded by the proto-oncogenes c-fos and c-jun (Fos and Jun, respectively) form a heterodimeric complex that regulates transcription by interacting with the DNA-regulatory element known as the activator protein 1 (AP-1) binding site. Fos and Jun are members of a family of related transcription factors that dimerize via a leucine zipper structure and interact with DNA through a bipartite domain formed between regions of each protein that are rich in basic amino acids. Here we have defined other domains in the Fos-Jun heterodimer that contribute to transcriptional function in vitro. Although DNA-binding specificity is mediated by the leucine zipper and basic regions, Jun also contains a proline- and glutamine-rich region that functions as an ancillary DNA-binding domain but does not contribute directly to transcriptional activation. Transcriptional stimulation in vitro was associated with two regions in Fos and a single N-terminal activation domain in Jun. These activator regions were capable of operating independently; however, they appear to function cooperatively in the heterodimeric complex. The activity of these domains was modulated by inhibitory regions in Fos and Jun that repressed transcription in vitro. In the context of the heterodimer, the Jun activation domain was the major contributor to transcriptional stimulation and the inhibitory regions in Fos were the major contributors to transcriptional repression in vitro. Potentially, the inhibitory domains could serve a regulatory function in vivo. Thus, transcriptional regulation by the Fos-Jun heterodimer results from a complex integration of multiple activator and regulatory domains.
The protein products of the fos and jun protooncogenes interact cooperatively in the form of a heterodimer with the activator protein 1 (AP-1) regulatory element. To characterize the properties of these proteins, we have expressed polypeptides comprised of the dimerization and DNA-binding domains of Fos and Jun in Escherichia coli. The mini-Fos (wbFos) and the mini-Jun (wbJun) proteins were purified to apparent homogeneity by using a nickel affinity chromatography procedure. Purified wbFos and wbJun associated rapidly in vitro and interacted cooperatively with the human metallothionein IIA AP-1-binding site. However, efficient DNA binding of wbJun and wbFos-wbJun complexes required an additional activity present in nuclear extracts. This activity was sensitive to alkylating agents and could be partially mimicked by the presence of reducing and stabilizing agents. DNase I footprinting experiments demonstrated that Jun homodimeric complexes and Fos-Jun heterodimeric complexes interacted with the same site on the human metallothionein IIA gene. Moreover, UV-crosslinking studies demonstrated that Fos and Jun contact DNA directly and that both proteins interacted equivalently with either strand of the AP-1-binding site.
A procedure is described for the isolation of the nervous system-specific protein designated 14-3-2 from rat brain. The methods utilized are salt precipitation, DEAE-cellulose ion exchange chromatography, Sephadex G-150 gel filtration, and column isoelectric focusing. The native 14-3-2 protein has an isoelectric point of 4.7 in the absence of denaturing agents and 5.0 in the presence of 2.0 M urea. The protein, as isolated, appears homogeneous since it migrates as a single band on Tris-glycine (pH 8.9), sodium dodecyl sulfate (pH 7.2), and 8 M urea (pH 4.0) polyacrylamide gels. Sedimentation velocity and equilibrium data indicate a homogeneous component of molecular weight 78,000. Sedimentation of 14-3-2 in 6 M guanidine HCl containing 0.02% glutathione yielded a molecular weight of 39,000, indicating the dimeric nature of the protein as isolated. The rat brain protein seems to be composed of one subunit type, since polyacrylamide gel electrophoresis in 8 M urea yields a single protein component. Sodium dodecyl sulfate gel electrophoresis of rat brain 14-3-2 produced one sharp band with a relative mobility corresponding to a molecular weight of 48,000. Specific anti-14-3-2 serum has been prepared from both New Zealand white rabbits and goats. Rat 14-3-2 is very similar in amino acid composition to the beef brain protein and to antigen alpha. The antigenic properties of rat and beef 14-3-2 are also similar, since beef 14-3-2 antiserum reacts well with rat 14-3-2 and vice versa. Electrophoretic mobilities of denatured rat and beef 14-3-2 (0.1% sodium dodecyl sulfate and 8 M urea) are identical. Despite these similarities the two proteins are completely resolved on Tris-glycine gels. The sedimentation behavior of the beef and rat proteins are also different, indicating a difference in the association state and conformation of the two preparations.
Cellular immediate-early genes are rapidly induced by a diverse range of agents and conditions. Since many cIE genes encode known or potential transcription factors, they are believed to couple extracellular stimuli to long-lasting alterations in cellular phenotype through the regulation of gene transcription. In addition, the localization of the products of cIE genes has been used as a method for determining the cellular sites of action of particular agents in the nervous system. However, the methods of analysis are tedious, and the results may be ambiguous because of cross-reaction of reagents with related proteins. To further the utility of this approach, a bacterial gene encoding beta-galactosidase (lac Z) has been fused, in frame, into the fourth exon of c-fos, and this fos-lac Z fusion gene has been introduced into the germ line of mice. We have analyzed the expression of beta-galactosidase (under the control of the c-fos promoter) in the developing and adult nervous systems of these transgenic mice. As far as can be determined, the constitutive and stimulated expression of the transgene accurately reflects the expression of cognate c-fos in both cultured cells and the intact animal. This study has also revealed novel sites of constitutive and induced expression of c-fos that were overlooked using conventional analysis. In particular, constitutive expression of c-fos is associated with cells that are entering terminal differentiation and are destined to die. In addition, induced expression of the transgene in adult brain mirrors the pattern of neurotoxicity elicited by kainic acid.(ABSTRACT TRUNCATED AT 250 WORDS)
Oxidation of ribosomal protein L12 with hydrogen peroxide converts the three methionine residues to methionine sulfoxide. The oxidized protein has a decreased ability to bind to ribosomes, interact with ribosomal protein L10, be precipitated by L12 antiserum, and serve as substrate for the acetylating enzyme that converts L12 to L7. Full activity of L12 is regained when the protein is reduced with 2-mercaptoethanol. Sedimentation equilibrium analysis shows that oxidation of the methionine residues in L12 causes the conversion of the protein from the dimer to the monomer form, and the results indicate that the dimer is the active form of the protein in the above reactions.
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