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. 1998 Mar;18(3):1312-21.
doi: 10.1128/MCB.18.3.1312.

Bone-specific expression of the alpha chain of the nascent polypeptide-associated complex, a coactivator potentiating c-Jun-mediated transcription

A Moreau  1 , W V Yotov, F H Glorieux, R St-Arnaud
Affiliations

Bone-specific expression of the alpha chain of the nascent polypeptide-associated complex, a coactivator potentiating c-Jun-mediated transcription

A Moreau et al. Mol Cell Biol. 1998 Mar.

Abstract

The alpha chain of the nascent polypeptide-associated complex (alpha-NAC) coactivator was shown to potentiate the activity of the homodimeric c-Jun activator, while transcription mediated by the c-Fos/c-Jun heterodimer was unaffected. The use of deletion mutants in pull-down assays revealed that alpha-NAC interacted with amino acids 1 to 89 of the c-Jun protein and that the coactivator could interact with both the unphosphorylated and the serine 73-phosphorylated form of c-Jun. N-terminal-deleted c-Jun protein failed to interact with alpha-NAC in mammalian two-hybrid assays, while mutant c-Jun proteins lacking the leucine zipper or the basic domain retained interaction with alpha-NAC in vivo. Kinetics studies with purified c-Jun homodimer and recombinant alpha-NAC proteins allowed determination of the mechanism of coactivation by alpha-NAC: the coactivator stabilized the AP-1 complex formed by the c-Jun homodimer on its DNA recognition sequence through an eightfold reduction in the dissociation constant (kd) of the complex. This effect of alpha-NAC was specific, because alpha-NAC could not stabilize the interactions of JunB or Sp1 with their cognate binding sites. Interestingly, the expression of alpha-NAC was first detected at 14.5 to 15 days postconception, concomitantly with the onset of ossification during embryogenesis. The alpha-NAC protein was specifically expressed in differentiated osteoblasts at the centers of ossification. Thus, the alpha-NAC gene product exhibits the properties of a developmentally regulated, bone-specific transcriptional coactivator.

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Figures

FIG. 1
FIG. 1
Expression pattern of α-NAC mRNA during mouse embryogenesis. Whole mouse embryo mRNA was probed with the full-length α-NAC cDNA in a Northern blot assay. The membrane was subsequently stripped and rehybridized with a probe directed against the S28 ribosomal protein.
FIG. 2
FIG. 2
The α-NAC protein is specifically expressed in mineralizing bone during development. Cryosections of embryos at various developmental ages were probed with the anti-α-NAC antibody and fluorescein isothiocyanate-conjugated secondary antibody. (A, C, and E) Phase-contrast photomicrographs of the toluidine blue-stained sections. (B, D, and F) Indirect fluorescence photomicrographs. (A and B) Sagittal section from a 12-day-p.c. embryo. No detectable signal could be observed in any field examined. (C and D) Parasagittal section from a 14.5-day-p.c. embryo showing ossification of the ribs and expression of α-NAC in differentiated osteoblasts. (E and F) Longitudinal section of the ulna from a 14.5-day-p.c. embryo. The ossification center is in the midshaft region and corresponds to the area of expression of α-NAC. Par, panniculus carnosus (cutaneous muscle of the trunk); int, intercostal muscles; mu, muscle; per, periosteum; ul, ulna.
FIG. 3
FIG. 3
α-NAC interacts with c-Jun to potentiate c-Jun-mediated transcription. (A) Transient transfection assays with expression vectors for c-fos, c-jun, and α-NAC, alone or in combination. The expression level detected in cells transfected with the reporter alone (AP-1-tk-luc) was arbitrarily ascribed a value of 1. Results are expressed as mean fold induction ± standard error of four independent transfections. (B) Pull-down protein-protein interaction assays. [35S]methionine-labeled in vitro-translated proteins, identified above each panel, were incubated with the fusion GST-α-NAC protein or the free GST moiety. Input represents 1/10 of the total input of in vitro-translated protein. M, molecular mass (kilodaltons) markers.
FIG. 4
FIG. 4
α-NAC interacts with the N terminus of c-Jun. Pull-down protein interaction assays were performed with the phosphorylated or unphosphorylated GST-jun(1–89) fusion protein. Crude cell extracts from P19 embryonal carcinoma cells were incubated with glutathione-Sepharose beads loaded with the GST fusion proteins or the negative control GST moiety. Following binding, centrifugation, and washes, the bound proteins were analyzed by immunoblotting with anti-α-NAC (lanes 4 to 6), anti-GST (lanes 7 to 9), or anti-phospho-Ser 73 (lanes 10 to 12) antibodies. Background staining was assessed with preimmune sera (lanes 1 to 3). M, molecular mass (kilodaltons) markers.
FIG. 5
FIG. 5
Binding between α-NAC and the phosphorylated form of c-Jun is a strong interaction. (A) Affinity chromatography on a GST-α-NAC column. The recombinant GST-α-NAC protein was immobilized on a glutathione-Sepharose column. A nuclear extract from serum-stimulated MC3T3-E1 osteoblastic cells was passed through the column, allowing specific interactions between α-NAC and nuclear proteins to occur. Bound proteins were eluted with a step gradient of salt and detected by Western blotting with a monoclonal antibody directed against the phosphorylated form of c-Jun. Note that bound phospho-c-Jun elutes at 0.4 to 0.5 M salt. (B) The same nuclear extract was passed through a control GST column. Molecular mass (kilodaltons) markers (M) are indicated to the left. In, input protein; F.T., flowthrough; lanes 1 and 2, Coomassie blue-stained molecular size markers and input material, respectively.
FIG. 6
FIG. 6
α-NAC binds the c-Jun N terminus in vivo. (A) Schematic representation of the mammalian two-hybrid assay. (B) ROS 17/2.8 osteoblastic cells were transfected with the expression vectors for the various proteins mentioned. The expression level detected in cells transfected with the reporter and the GAL4 NAC vector was arbitrarily ascribed a value of 1. The c-Jun and c-Fos proteins did not interact with the GAL4-DBD. The fusion protein GAL4 NAC, which is transcriptionally inert, did not bind c-Fos; however, the interaction of c-Jun with α-NAC tethers the c-Jun activation domain to the GAL4-dependent promoter and results in the expression of the reporter gene. (C) The mammalian two-hybrid assay was performed with c-Jun deletion mutants. The expression level detected in cells transfected with the reporter and the GAL4 NAC vector was arbitrarily ascribed a value of 1. F.L., full-length c-Jun protein; 1–191, a truncated form of c-Jun in which the C-terminal domain was deleted; Δ281–313, leucine zipper deletion mutant; Δ251–276, basic domain deletion mutant; Δ1–87, N-terminal deletion mutant.
FIG. 7
FIG. 7
α-NAC stabilizes the binding of c-Jun to the AP-1 site. Binding reactions and electrophoretic mobility shift assays were performed with purified c-Jun and α-NAC proteins. (A) On rate measurements. Samples were removed at intervals and analyzed for DNA binding. Bound probe was measured by cutting out the bands on the gel and counting in a gamma counter; calculations were done as described by Chodosh et al. (5). Note that the slope of the binding curve, which represents the on rate, is not affected by α-NAC. A, free c-Jun; AP, c-Jun–DNA complex; PT, total DNA. (B) Off rate measurements. The bound complexes were formed as described, and a 50-fold excess of unlabeled AP-1 or GAL4 oligonucleotides was subsequently added to the binding reaction mixture. Samples were removed at intervals and processed as described above. The slope allows calculation of kd, which is reduced in the presence of α-NAC, thus stabilizing the c-Jun AP-1 complex and the GAL4/VP-16 complex. (C) Off rate measurement of the JunB AP-1 complex: in vitro-translated JunB protein was used as described for c-Jun in panel B. Note that α-NAC does not stabilize the JunB AP-1 complex.
FIG. 8
FIG. 8
Model of the α-NAC potentiation of c-Jun-activated transcription. (Upper panel) The c-Jun homodimer binds the AP-1 site and interacts weakly with TBP. (Lower panel) The α-NAC coactivator interacts with the N-terminal domain of c-Jun. This interaction leads to a stabilization of the interaction of the c-Jun homodimer with the AP-1 site through a reduction in the dissociation rate (kd). Moreover, since α-NAC interacts strongly with both c-Jun and TBP, it strengthens the contacts between c-Jun and the basal transcriptional machinery, resulting in enhanced transcription from the c-Jun dimer (represented in the form of the larger arrow than that in the upper panel). For simplicity, only the phosphorylated form of c-Jun is depicted in the model; however, in vitro protein interaction assays demonstrated that α-NAC can also interact with the unphosphorylated form of c-Jun.
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References

    1. Angel P, Karin M. The role of Jun, Fos and the AP-1 complex in cell proliferation and transformation. Biochim Biophys Acta. 1991;1072:129–157. - PubMed
    1. Arias J, Alberts A S, Brindle P, Claret F X, Smeal T, Karin M, Feramisco J, Montminy M. Activation of cAMP and mitogen responsive genes relies on a common nuclear factor. Nature. 1994;370:226–229. - PubMed
    1. Bannister A J, Kouzarides T. CBP-induced stimulation of c-Fos activity is abrogated by E1A. EMBO J. 1995;14:4758–4762. - PMC - PubMed
    1. Briggs M R, Kadonaga J T, Bell S P, Tjian R. Purification and biochemical characterization of the promoter-specific transcription factor, Sp1. Science. 1986;234:47–52. - PubMed
    1. Candeliere G A, Prud’homme J, St-Arnaud R. Differential stimulation of Fos and Jun family members by calcitriol in osteoblastic cells. Mol Endocrinol. 1991;5:1780–1788. - PubMed

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