An autonomous N-terminal transactivation domain in Fos protein plays a crucial role in transformation.

To date, three functional domains have been defined in c-Fos and v-Fos proteins and have been shown to play a role in transactivation: the leucine zipper mediating hetero-dimerization, the basic DNA contact site, and a C-terminally located transactivation domain (C-TA) harbouring the HOB1 and HOB2 motifs. While the bZip region, consisting of the leucine zipper and the DNA contact site, is indispensable for transformation, the C-TA domain is not required and is actually altered by internal deletions in the FBR-MuSV. We now show that the N-terminal regions of c-Fos and v-Fos contain a second transactivation domain (N-TA). A functionally crucial motif within the N-TA domain, termed NTM, was pinpointed to a approximately 25 amino acid stretch around positions 60-84 which is highly conserved in FosB. Analysis of LexA fusion proteins showed that the N-TA domains of both c-Fos and FosB function in an autonomous fashion in both fibroblasts and yeast. Most importantly, deletion of the NTM motif impairs the transforming properties of v-Fos. Apart from the bZip region, the N-TA domain is the only functional domain required for transformation by v-Fos, at least when its expression is driven by the strong FBR-MuSV-LTR promoter.     

Differential phosphorylation of c-Jun and JunD in response to the epidermal growth factor is determined by the structure of MAPK targeting sequences

MAPK phosphorylation of various substrates is mediated by the presence of docking sites, including the D domain and the DEF motif. Depending on the number and sequences of these domains, substrates are phosphorylated by specific subsets of MAPKs. For example, a D domain targets JNK to c-Jun, whereas a DEF motif is required for ERK phosphorylation of c-Fos. JunD, in contrast, contains both D and DEF domains. Here we show that these motifs mediate JunD phosphorylation in response to either ERK or JNK activation. An intact D domain is required for phosphorylation and activation of JunD by both subtypes of MAPK. The DEF motif acts together with the D domain to elicit efficient phosphorylation of JunD in response to the epidermal growth factor (EGF) but has no function on JunD phosphorylation and activation by JNK signaling. Furthermore, we show that conversion of a c-Jun sequence to a canonical DEF domain, as it is present in JunD, elicits c-Jun activation in response to EGF. Our results suggest that evolution of a particular modular system of MAPK targeting sequences has determined a differential response of JunD and c-Jun to ERK activation.     

Mitogen stimulation of T-cells increases c-Fos and c-Jun protein levels, AP-1 binding and AP-1 transcriptional activity.

We have analysed the effect of mitogenic lectins on c-Fos and c-Jun protein levels as well as on activator protein-1 (AP-1) binding and enhancer activity in Jurkat T-cells. Both c-Fos and c-Jun protein levels were increased after Con A and PHA stimulation. Since T-cell stimulation increases both intracellular Ca2+ and cAMP levels and activates protein kinase C (PKC), the possible involvement of these intracellular messengers in c-Fos and c-Jun induction was tested. PMA, which directly activates PKC, mimicked the effect of the lectins on c-Fos and c-Jun, but elevation of either intracellular Ca2+ or cAMP levels had little or no effect. The mitogen-induced increase of c-Fos and c-Jun immunoreactivity was inhibited by H-7, a kinase inhibitor with relatively high specificity for PKC, and less efficiently by H-8, a structurally related kinase inhibitor less active on PKC, but more active on cyclic nucleotide-dependent kinases. Con A stimulation was found to increase both binding of AP-1 to the AP-1 consensus sequence, TRE, and AP-1 enhancer activity, in Jurkat cells. PMA was also found to increase the AP-1 enhancer activity, whereas elevation of Ca2+ or cAMP had only minor effects. We conclude that stimulation with mitogenic lectins is sufficient to increase both c-Fos and c-Jun protein levels, AP-1 binding and AP-1 enhancer activity in Jurkat cells and that they act via mechanisms that could involve the activation of PKC.     

Role of the insertion domain and the zinc-finger motif of Escherichia coli UvrA in damage recognition and ATP hydrolysis

UvrA is the initial DNA damage-sensing protein in bacterial nucleotide excision repair. Each protomer of the UvrA dimer contains two ATPase domains, that belong to the family of ATP-binding cassette domains. Three structural domains are inserted in these ATPase domains: the insertion domain (ID) and UvrB binding domain (in ATP domain I) and the zinc-finger motif (in ATP domain II). In this paper we analyze the function of the ID and the zinc finger motif in damage specific binding of Escherichia coli UvrA. We show that the ID is not essential for damage discrimination, but it does stabilize UvrA on the DNA, most likely by forming a clamp around the DNA helix. We present evidence that two conserved arginine residues in the ID contact the phosphate backbone of the DNA, leading to strand separation after the ATPase-driven movement of the ID's. Remarkably, deletion of the ID generated a phenotype in which UV-survival strongly depends on the presence of photolyase, indicating that UvrA and photolyase form a ternary complex on a CPD-lesion. The zinc-finger motif is shown to be important for the transfer of the damage recognition signal to the ATPase of UvrA. In the absence of this domain the coupling between DNA binding and ATP hydrolysis is completely lost. Mutation of the phenylalanine residue in the tip of the zinc-finger domain resulted in a protein in which the ATPase was already triggered when binding to an undamaged site. As the zinc-finger motif is connected to the DNA binding regions on the surface of UvrA, this strongly suggests that damage-specific binding to these regions results in a rearrangement of the zinc-finger motif, which in its turn activates the ATPase. We present a model how damage recognition is transmitted to activate ATP hydrolysis in ATP binding domain I of the protein.     

Expression of different Jun and Fos proteins during the G0-to-G1 transition in mouse fibroblasts: in vitro and in vivo associations.

We have characterized the expression of c-Jun, JunB, JunD, c-Fos, and FosB proteins following serum stimulation of quiescent Swiss 3T3 cells by immunoprecipitation analyses. The synthesis of the three Jun proteins rapidly increases following stimulation, remaining at a significant level for at least 8 h. JunB protein presents the highest expression of all. FosB, like c-Fos, is transiently induced. Pulse-chase experiments show that all of the proteins except JunD are short-lived. We have shown that c-Fos and FosB form complexes in vivo with the different Jun proteins and that JunB complexes are predominant. In vitro association and competition experiments show that the affinities between the different Fos and Jun proteins are similar. This finding, together with the in vivo observations described above, suggests that the proportion of the different Jun/Fos heterodimers is governed by the concentration of the different components. The Fos and Jun proteins are phosphoproteins, and some remain relatively highly phosphorylated in their heterodimeric form.     

Identification of GATA2 and AP-1 Activator elements within the enhancer VNTR occurring in intron 5 of the human <Emphasis Type="Italic">SIRT3</Emphasis> gene

Human SIRT3 gene contains an intronic VNTR enhancer. A T > C transition occurring in the second repeat of each VNTR allele implies the presence/absence of a putative GATA binding motif. A partially overlapping AP-1 site, not affected by the transition, was also identified. Aims of the present study were: 1) to verify if GATA and AP-1 sites could bind GATA2 and c-Jun/c-Fos factors, respectively; 2) to investigate whether such sites modulate the enhancer activity of the SIRT3-VNTR alleles. DAPA assay proved that GATA2 and c-Jun/c-Fos factors are able to bind the corresponding sites. Moreover, co-transfection experiments showed that the over-expression of GATA2 and c-Jun/c-Fos factors boosts the VNTR enhancer activity in an allelic-specific way. Furthermore, we established that GATA2 and c-Jun/c-Fos act additively in modulating the SIRT3-VNTR enhancer function. Therefore, GATA2 and AP-1 are functional sites and the T S> C transition of the second VNTR repeat affects their activity.