Transcription factors are the proteins responsible for binding to DNA to either stimulate or inhibit transcription.  Cooperative interaction between transcription regulatory proteins requires a juxtaposition of contact surfaces that are either part of or tightly bound to the DNA binding domain (Kerppola 2001).  The Jun family of cellular transcription factors is comprised of three members: c-Jun, Jun B, and Jun D.  These transcription factors are involved in responses of cells to extracellular signaling agents, including growth factors and cytokines.  The Jun proteins show nearly identical amino acid sequences in their dimerizing/DNA-binding bZIP domains and little similarity anywhere else in their sequences.  The transcription factor c-Jun is a bZIP protein (basic leucine zipper) that binds to specific DNA sequence either as a homodimer with another c-Jun or as a heterodimer with other bZIP proteins such as the Fos family members, mainly c-Fos (Schaefer et. al. 2001).  Phosphorylation of c-Jun by c-Jun kinase (JNK) is known to increase the transcriptional activity of c-Jun.  Both c-Jun and JNK are implicated in the induction of programmed cell death, apoptosis (Xia 1998).

  c-Jun (red and yellow strands)  and c-Fos (orange and green strands) are both proto-oncogenes that are thought to be integral factors in cell differentiation, transformation, proliferation, and apoptosis.  The c-Fos and c-Jun genes encode proteins that can heterodimerize to form the complexes of the activator protein (AP)-1 family, accessory transcription factors that interact with DNA regulatory sequences known as 12-O-tetradecanoylphorbol-13-acetate response elements or AP-1 sites (Janssen et. al. 1997).  The bZIP domains of AP-1, c-Jun and c-Fos, dimerize forming continuous alpha helices that appear here as a Y shaped fork into which the major groove of the DNA binds (Sun et. al. 1997).  c-Fos and c-Jun bind palindromic AP-1 regulatory elements.  The c-Fos-c-Jun binding orientation is thought to be regulated by indirect recognition of differences in the DNA structure between the flanking sequences located on opposite sides of the AP-1 site (Kerppola 2001).  The c-Fos-c-Jun heterodimer binds specifically to the DNA sequence 5’TGAGTCA 3’ or the AP-1 site (Basuyaux 1997).  Analysis of the binding orientation of the Fos-Jun heterodimer at different Ap-1 sites indicates that the farther the base pairs are far from the central AP-1 recognition sequence, the more influence they have on the orientation of Fos-Jun binding (Kerppola 2001).  Evidence points to the c-jun component of AP-1 as being an integral feature for, hypertrophy and fetal heart gene expression.   c-Jun expression, along with JunB and NRF-1 expression, is related to transcriptional activation of cytochrome c and cytochrome c expression in the heart (Xia 1998).

The c-Jun protein weighs 3567.23 kDa and is 3622 base pairs long.  The gene/operon organization, pictured here, contains only one exon due to the fact that it is derived from a retrovirus and cloned into a vector.  The translation of the open reading frame can be seen here, it contains 332 residues.  The c-Jun-c-Fos heterodimer contains 4 a helices and oligomers of short identical helices.  c-Jun is also thought to be involved in pathways associated with Ca 2+ activated gene expression (Xia 1998) and to function as an androgen receptor coactivator by acting on residues 503-555 of the androgen receptor (Bubulya 2001).  The c-Jun protein is found in such organisms as yeast, bird, mammals, and amphibians.  The ortholog alignment shows the consensus between these organisms.
 
All bZIP transcription factor proteins contain a basic region.  This region can be seen as the ball and stick residues in c-Jun.   The homology can be seen in the paralog sequence alignment seen here.  bZIP domains induce DNA bending through charge interactions that involve the amino acid residues, which are adjacent to the basic region (Kerppola 1997).  While c-Fos possesses a cluster of positively charged amino acid residues, which are immediately adjacent to the basic region, c-Jun possesses negatively charged residues on the amino-terminal side of the basic region (Kerppola 2001).  Mutational analysis of bZIP domains show that DNA bending is proportional to the charge of the amino acid residues adjacent to the basic region, signifying that DNA bending is a result of electrostatic interaction (Kerppola 1997).  bZIP proteins’ basic region forms an extended a helix when it binds to DNA.  They have no other tertiary interactions within it to maintain a stable conformation, while most other DNA binding domains contain compact, globular modules to aid in stability.  Without these globular structures, a bZIP’s basic region may adopt different conformations along the DNA molecule (Kim et. al. 1993).

Another domain of bZIP proteins is the leucine zipper.  Every seventh residue is a leucine residue.  c-Jun (red and yellow strands)  and c-Fos (orange and green strands) leucine residues are seen at the right.

  c-Jun interacts with DNA by means of the leucine zipper, which forms a coiled-coil dimerization interface while the basic region forms an alpha helix that contacts base pairs within the major groove of the DNA recognition site.  The key residues of c-Jun that can increase DNA bending and binding affinity at the Ap-1 site are residues 241 through 252 (Kerppola 1997).  The leucine zipper positions a pair of basic-region a helices that pass through the major groove of the DNA.  Once the DNA specific complex forms, the unfolded basic region begins to fold into an a helix and DNA contact is made through a quintet of conserved basic region residues (Kim et. al. 1993).

Residues 252-281 in c-Jun are the basic subdomain, which is responsible for the sequence specific recognition site of DNA (Krebs 1995).  Other major residue regions include the transcription activation domain (residues 91-186), the transcription repression domain (residues 31-57), and the promoter region for T antigen-dependent DNA unwinding required for the initiation of polyoma virus DNA replication (residues 91-186) (Kerppola 1997).

There are some unique features to c-Jun’s structure that are no found in other bZIP proteins.  One of these features is an asymmetrical groove.  The asymmetric major groove occurs between c-Fos and c-Jun heterodimer DNA bases.  The asymmetry is due to the conserved arginine 279-side chain of the c-Jun strand (Glover 1995).  The c-Fos-c-JUN-DNA complex asymmetry in the coiled-coil and flexibility in its protein fork, could allow the heterodimer to recognize disparate binding surfaces presented on other transcription factors bound at adjacent sites on the DNA (Basuyaux 1997).

  Another key structural feature in c-Jun is a conserved arginine (285) residue located in the so-called “spacer region” (the junction between the basic domain and the leucine zipper) of c-Jun.   R285 is critical for cooperative NFAT (nuclear factor of activated T cells)/AP1 complex assembly on the DNA.  NFAT and Fos-Jun family members cooperatively bind to NFAT andAP-1 sequences and regulate an activated T cell’s cytokine gene expression (Diebold 1998).  A site-directed mutation of R285 to alanine resulted in virtually complete loss of cooperativity with NFAT, but had no effect on DNA recognition by AP1 alone (Sun 1997).  This arginine has been suggested to be necessary and sufficient for NFAT2 interaction, but Diehold reported that it had no effect on heterodimer binding orientation or bending cooperativity (Diebold 1998).  The Fos-Jun dimmer is oriented so that c-Jun binds to the half of the AP-1 site that is closest to NFAT (Chen et. al. 1998).

In conclusion c-Jun is an important transcription factor protein and oncogene that forms a heterodimer with c-Fos to form the AP-1 complex.  It is associated with T cell activation, fetal heart development, Ca 2+ gene expression, and androgen receptor activation.  The conserved R285, along with the leucine zipper domain, and the basic domain are extremely important for DNA binding and transcriptional regulation.