Biology Reference
In-Depth Information
to AP-1.
18
The cellular-derived jun product is the
presumed oncogene from avian sarcoma virus 17 and
has been shown to induce fibrosarcomas in
chickens.
19,20
The transcription factors c-Fos and c-Jun
form homodimeric and heterdimeric complexes
through their leucine zipper domains, which include 4
constitutively active oncogene v-Jun had a mutation
resulting in substitution of a Ser for a Cys residue
272, which allowed it to escape normal cellular regula-
tion.
18
This in turn suggested an important role for the
Cys residue found within the highly conserved KCR
sequence located within the DNA-binding domain of
both c-Fos (Cys 154) and c-Jun (Cys 272) (
Figure 11.3
).
21
When this Cys was substituted with Ser (KSR), oxida-
tion of c-Fos/c-Jun by diazenedicarboxylic acid bis
[N,N-dimethylamide] (diamide) no longer inhibited
its DNA-binding activity. Addition of DTT or a nuclear
factor was found to significantly enhance the DNA-
binding activity of oxidized c-Fos/c-Jun suggesting
that the Cys residues in the DNA-binding domain
are subject to redox regulation.
23
The basic environ-
ment of this Cys residue is thought to affect its oxida-
tion. Interestingly, stimulation by the nuclear factor
was found to be greater than that of DTT alone.
23
The nuclear redox factor was identified through
purification of the protein that stimulated the DNA-
binding activity of c-Fos/c-Jun as monitored by electro-
phoretic mobility shift assay (EMSA).
24
HeLa nuclear
extracts were purified by chromatographic separation
on heparin-Sepharose, DNA-cellulose, blue Sepharose,
mono S, Superose 12, and Superdex 75 columns. For
each chromatographic step, flow-through or fractions
that stimulated the DNA-binding activity of c-Fos/
c-Jun were applied to the next column. A 37 kDa
protein, purified 176-fold over the crude extract, was
identified as the nuclear redox factor and referred to
as Ref-1. Inclusion of thioredoxin further stimulated
the DNA-binding activity of c-Fos/c-Jun, presumably
5
leucine residues spaced 7 residues apart.
21
The DNA-
binding domains comprise basic regions in each
protein that bind cAMP responsive elements (CRE)
and serve as intermediary transcriptional regulators
in signal transduction processes. The preferred DNA-
binding sequence for c-Fos/c-Jun is 5'-TGAGTCA-3'.
Although, c-Jun/c-Fos does not bind in a preferred
orientation with respect to the DNA, the complex is
stabilized by specific hydrogen-bonding interactions
between the basic regions of c-Fos and c-Jun and the
DNA. Two different binding orientations were
observed in the crystal structure with an Arg in either
c-Fos or c-Jun hydrogen-bonded to G found in the
central base pair.
22
Both heterodimeric c-Fos/c-Jun
and c-Jun homodimers bind DNA, while c-Fos homo-
dimers do not. Formation of preferred homodimeric
and heterdimeric c-Fos/c-Jun species arises from
favorable electrostatic interactions within the Leucine
zipper region of the proteins based on the analysis of
the crystal structure of c-Fos/c-Jun bound to DNA.
22
While the heterodimeric c-Fos/c-Jun and homodimeric
c-Jun/c-Jun form favorable electrostatic interactions
stabilizing these dimeric interactions, charge repulsion
in c-Fos prevents formation of a stable dimeric species.
In part, the idea that c-Jun might be regulated by
a redox mechanism came from the finding that the
e
FIGURE 11.3
Oxidized AP-1 (c-Jun/c-Fos) is reduced by the nuclear redox factor, apurinic/apyrimidinic endonuclease (APE1). A ribbon
rendering is shown of the DNA-binding domains of c-Jun and c-Fos in a putative oxidized state involving a disulfide bond between the Cys
residues within the DNA-binding domain of each protein. Reduction by APE1 greatly stimulates the DNA-binding activity of AP-1. (Please refer
to color plate section).
