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The discovery of activating protein 1 (AP-1) took place in 1987 when researchers were investigating the human mellothionein (hMTIIA) gene. Upon analysis of the hMTIIA promoter, it was found that the gene contained five distinct control elements in the 5' flanking region that mediate specificity and regulation of transcription. Further analysis showed that when the hMTIIA gene is induced, a novel protein binds to the basal level enhancer sequence (BLE) of hMTIIA to a site within the 72 base pair (bp) repeats of the Simian virus 40 enhancer regions . This novel protein was AP-1 and since its discovery, the protein has gained a significant amount of attention for its multiple roles within the cell, including proliferation, inflammation, differentiation, cellular migration, wound healing, transformation, and cell death (apoptosis) . The dimeric transcription factor AP-1 belongs to a family of proteins which have a basic leucine zipper motif (bZIP) (Figure 1). The most studied subfamilies of proteins within the AP-1 family include Fos (c-Fos, FosB, Fra1 and Fra2) and Jun (c-Jun, JunB and JunD) while of lesser importance, some proteins from the ATF and CREB family of proteins are also part of AP-1 complexes . These families of proteins can interact with one another in many different dimeric combinations which determine its binding specificity as well as its affinity to a specific gene. This allows the regulation of many specific target genes from a small pool of proteins. The Jun subfamily of proteins are able to homodimerize or heterodimerize with Fos to form the AP-1 complex (Figure 2), whereas the Fos subfamily of proteins are unable to homodimerize. .
Figure 1: Structure of bZIP. GCN4 which is a DNA binding protein has a bZIP domain which binds DNA as shown. The basic regions of the leucine zipper interact with the acidic backbone of DNA to enable protein-DNA binding .
Figure 2: Structure of the AP-1 dimeric complex. Crystal structure of the heterodimeric bZIP transcription factor c-Fos-c-Jun bound to DNA .
Control and regulation of AP-1
Control and regulation of AP-1 can occur at both a transcriptional and post transcriptional level. AP-1 activity is induced by many stimuli including growth factors, cytokines, T cell activators, neurotransmitters, and UV radiation (Figure 3). Also, AP-1 is a transcription factor that mediates gene induction by the molecule, phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). The TPA Response element, TRE, is the recognition site for AP-1 .
Figure 3: Regulation of c-Jun and c-Fos transcription in response to extracellular stimuli. The genetic elements that modulate gene induction of c-Jun and c-Fos are shown along with their respective protein kinases. PKA, protein kinase A; CaMK, calmodulin-dependent protein kinase; SIE, Sis-inducible enhancer; CRE, cAMP response element; SRE, serum response element .
Trancription of the genes that encode AP-1 components is induced by extracellular factors. The regulation of c-Fos and c-Jun are well understood and serve as a good model for AP-1 transcriptional control.
There are four different cis elements that mediate c-Fos induction in response to extracellular stimuli . The cAMP response element (CRE) mediates c-Fos induction in response to neurotransmitters and polypeptide hormones, which by using either cAMP or Ca2+ as secondary messengers, activat either PKA or CamK . The SRE element mediates c-Fos induction by growth factors, cytokines, UV and other stimuli that activate MAPK. The SIE element mediates c-Fos induction by stimuli that activate the JAK family of protein kinases .
Activation of the ERK pathway leads to elevated AP-1 activity by increasing c-Fos production. c-Fos is then translocated to the nucleus, where it combines with pre-existing Jun proteins to form AP-1 heterodimers. This heterodimer is much more stable than the homodimers formed by Jun proteins resulting in an increased AP-1 activity due to increased stability of the complex .
The AP-1 components c-Jun, c-Fos, and ATF2 are regulated via phosphorylation. When c-Jun phosphorylation on sites located near its basic region, DNA binding by c-Jun homodimers is inhibited , whereas when c-Jun is phosphorylated by the JNK pathway at Ser-73 and Ser-63 at the transactivation domain of c-Jun, the homodimeric form is able to bind DNA. . Furthermore, it has been suggested that N-terminus phosphorylation of c-Jun results in the recruitment of CREB binding protein (CBP), a bZIP transcription factor which augments the transcriptional ability of c-Jun. CBP is thought to link the phosphorylated activation domains of CREB or c-Jun to the general transcription factors .
Role of AP-1 in cell proliferation, apoptosis and tumorigenesis
The role of AP-1 in cell proliferation was suggested based on observations of increased AP-1 activity in mitogenic cells and that different Fos and Jun proteins have varying expression patterns during the cell cycle . By overexpression or repressing various AP-1 components, it has been found that some AP-1 components are directly linked to cell cycle regulatory genes, summarized in Figure 4 . Research suggests that c-Fos, FosB and Fra-1 are not essential for cell cycle progression. Studies done with fibroblasts and embryonic stem cells lacking these components show no proliferation defect . In contrast, c-Jun is a positive regulator of cell proliferation, shown by studies with c-Jun deficient cells undergoing premature senescence in vitro . In the absence of c-Jun, there is elevated expression of tumor suppressor p53, as shown in Figure 4, which leads to increased activity of p21 and inhibits cyclin-dependent kinases which are required for S phase entry. Furthermore, c-Jun regulates the expression of cyclin D1 which is known to be a positive regulator of the cell cycle and reduced cyclin D1 expression in primary fibroblasts causes impaired proliferation .
JunB and JunD negatively regulate cell proliferation. This has been shown in fibroblasts derived from mice over expressing Jun B which display reduced proliferation. This is due to an extended G1 phase nd these cells undergo premature senescence. Also, it was found that in immortalized fibroblasts, over expression of JunD in these cells show increasing numbers of senescent cells, while JunD deficient fibroblasts show increased proliferation. This occurs because there are elevated levels of p16,a CDK inhibitor which inhibits cyclin D1/CDK4-6 kinase activity, thereby delaying G1 to S phase entry. JunB also antagonizes the expression of cyclin D1 thereby reducing expression of cyclin D1 in fibroblasts over expressing JunB .
Figure 4: Effects of AP-1 proteins on cell cycle regulation. The expression of certain cell cycle proteins associated with G1 to S phase entry are mediated by AP-1 proteins. c-Jun initiates G1 to S phase transition by increasing cyclin D1 activity and repressing p53 activity, which in turn reduces p21 levels. c-Fos and FosB have the same functions for S phase entry and the increase of cyclin D1 expression. JunB also inhibits G1 to S phase progression by inducing p16 which represses cyclin D1. JunD inhibits S phase entry and induces cell senescence by acting on the Ras/p53 pathway .
. Programmed cell death, or apoptosis can occur after cells are overly stressed, if there is significant DNA damage or if there is cytokine withdrawal . AP-1's role in apoptosis was proposed when it was observed that c-Jun and c-Fos expression is increased upon growth factor withdrawal in cytokine-dependent lymphoid cells. Early studies suggest that AP-1's function in the cell may be pro-apoptotic as researchers observed that when growth factor-deprived lymphoid cells are treated with anti sense oligonucleotides for c-Jun and c-Fos, the cells have an increased survival rate . Later studies show that c-Fos and c-Jun role in apoptosis depends on the cell type and apoptotic stimulus. Depending on either the cell type or stimulus, AP-1 can be pro-apoptotic or anti-apoptotic . c-Fos is found to be associated with apoptosis based on the observations that in embryonic Syrian hamster cells over expression of c-Fos induces cell death, and in vivo observations from fos-lacZ reporter mice which show constitutive c-Fos expression in the cell that are undergoing differentiation and apoptosis [27,32].
It appears as though c-Jun can act as a positive or a negative modulator of apoptosis. The suggestion that there is a pro-apoptotic function of c-Jun comes from an in vitro study in which researchers inhibit c-Jun activity by either mutating c-Jun in a dominant-negative fashion, or by using antibodies to neutralize c-Jun activity, and they found in both cases that the lack of c-Jun activity protects sympathetic neurons against NGF withdrawal-induced apoptosis . Over expression of c-Jun is sufficient to induce apoptosis in sympathetic neurons and fibroblasts, but as mentioned earlier certain conditions merit c-Jun anti-apoptotic activity. Depending on the cell, c-Jun expression may increase susceptibility to apoptosis such as the case mentioned earlier or the decrease susceptibility to apoptosis. Studies conducted by Karin in 1995 suggest that primary embryonic fibroblasts lacking c-Jun show enhanced sensitivity to UV-induced apoptosis, and c-Jun mutant foetuses has an increased number of apoptotic cells, suggesting that c-Jun in these cells is necessary to prevent apoptosis . Another AP-1 protein associated with a pro-apoptotic function is JunB. Research conducted in 1992 suggests that JunB is pro-apoptotic based on the observation that when JunB is inactivated in myeloid cells, there is a lack of apoptosis and there is an increase in expression of two anti-apoptotic genes, Bcl2 and Bclxl .
AP-1's role in apoptosis and cell proliferation also has a link to tumorigenesis which can be shown in figure 5. In in vivo studies, when c-Fos is over expressed it transforms chondroblast and osteoblast cells. Studies show that there is a significantly higher level of cyclin D1 in c-Fos induced osteosarcomas, which suggests that cell cycle regulators are modulated as a result of this transformation . Other studes show that FosB is able to transform fibroblasts in vitro, but cannot cause tumour formation . In comparison to other Fos proteins, the ability of Fra-1 and Fra-2 to transform cells significantly weaker, as shown by studies conducted from Bergers and colleagues in 1995. They observed that over expression of Fra-1 in fibroblasts does not lead to transformation while Fra-2 is only able to transform chicken but not rat embryo fibroblasts . Other studies conducted by Mendoza-Rodríguez and colleagues in 2003 suggest that c-Jun can lead to oncogenic transformation in mammalian cells in vitro when co-expressed with an activated oncogene such as Ras or Src. Although this transformation occurs in vitro, c-Jun over expression it does not occur in vivo. JunB or JunD expression in vitro inhibits oncogeneic transformation by Ras in immortalized fibroblasts, suggesting that these proteins are anti-oncogenes .
Figure 5: AP-1 functions in a multistep tumorigenesis. AP-1 protein components all play a certain role in tumorigenesis by regulating different aspects of proliferation, apoptosis, oncogenic transformation, angiogenesis and metastasis by controlling the expression of critical gene targets shown .
Effect of mutant AP-1 components on MEFs
Aberrant AP-1 proteins affect mouse embryo fibroblasts (MEFs) in numerous ways. One effect of aberrant c-Jun proteins is that c-Jun proteins lacking a trans-activation domain inhibits transformation of MEFs by RasV12 or v-Src, both of which are oncoproteins. However, these cells still exhibit tumorigenic activity as they still form tumors in animals, and biopsy of these tumors show that they have high levels of AP-1 activity resulting from increased expression of junD but not junB . These results reiterate the importance of AP-1 in proliferation, and also indicate that JunD, an antagonist for Ras transformation  can substitute for c-Jun in transformed cells.
In NIH 3T3 cells, researchers found that over-expression of JunD and JunB inhibit transformation of cells by Ras, implying that these factors are negative regulators of AP-1 or non-potent activators of AP-1 . In contrast, mutant MEFs with a c-jun or junD (-/-) mutation tend not to proliferate, but instead undergo premature senescence in vitro, thus suggesting that c-Jun and JunD are both needed for MEFs to proliferate [37,38]. Additional studies show that MEFs which lack JunD expression are highly susceptible to TNFα-induced apoptosis, because these cells express elevated levels of a protein involved in cell senescence, p19Arf .
Further Characterization of AP-1... Work done in lab
Through chromatin immune precipitation (CHIP) and electrophoretic mobility shift assay (EMSA), it was found that JunD and Fra-2 make up most of the AP-1 proteins in v-Src transformed cells, as v-Src induces expression of JunD and Fra-2 . By the use of retroviral vectors, the function of each AP-1 components were examined by down-regulating their activity by the use of shRNAi . As mentioned before when c-Jun is down-regulated in MEFs the cells enters premature senescence, and this holds true for chicken embryo fibroblasts (CEFs) which are transformed or normal. When JunD was down-regulated in v-Src transformed cells, there was significant a significant apoptotic response while in non-transformed cells, it would induce a significantly smaller apoptotic response, thus indicating that JunD is responsible for the pro-survival role of AP-1 . Fra-2 seems to initiate adipogenic conversion in v-Src transformed cells . These results indicate that c-Jun provides a block to senescence, JunD is required for cell survival and Fra-2 initiates adipogenic conversion in v-Src transformed cells.
Wang and colleagues constructed and expressed hybrid proteins that were made up of c-Jun or JunD trans-activation domains (TAD) fused to a Gal4 DNA binding domain (DBD), in order to examine the trans-activation potential of these factors. It was found that v-Src had little effect on the c-Jun TAD but stimulated the activity of the JunD-TAD . Upon further research, Wang and colleagues found that when a dominant negative mutant of SEK is over expressed causing inhibition of the SAPK/JNK pathway, JunD-TAD activity is partially reduced. Alternatively, much more suppression of AP-1 activity is met when v-Src transformed CEFs are treated with a specific inhibitor of MEK (PD98059). This result suggests that AP-1 is primarily dependent on the ERK pathway .
Since it is now known what pathway AP-1 is dependent on, we want to further characterize this finding determining what exactly the ERK pathway is acting on in the AP-1 dimer. The objective here is to determine if the ERK pathway acts on the JunD-TAD in v-Src transformed CEFs.
Objectives and Experimental Plan
Test to see if JunD-TAD is controlled by ERK pathway.
In order to test this, the following plan will be followed.
- After culturing enough plates of CEFs, they will be infected with temperature sensitive mutant, NY72-4 rous sarcoma virus (RSV) which is a group A virus. This retrovirus encodes src, which is a tyrosine kinase. This gene is oncogenic as it triggers uncontrolled growth in host cells.
- After the cells are infected, they will be transfected with the following DNA: rsv β-Gal plasmid DNA to assess transfection efficiency; JunD-TAD/Gal4 DBP which has the DNA encoding the JunD-TAD; and the Gal4-PJF CAT-TATA plasmid DNA which is used in part with the CAT assay and will ultimately give CAT activity.
-In total there will be nine CEF plates, three of the plates will be left to incubate at 41°C and six CEF plates will be left to incubate at 37°C. The purpose of this is that the CEFs growing at 41°C will grow like normal CEFs and will not have transformed. The CEFs growing at 37°C will be in the permissive temperature range for the virus to become active and produce the Src protein.
- After the transfection, the cells will be collected via the cell extraction protocol, and the transfection efficiency will be assessed by the β-Gal assay.
- The CAT activity will be assessed from the non-permissive CEFs, permissive CEFs and the CEFs in the presence of the MEK inhibitor. The CAT activity assessed from the non-permissive and permissive CEFs will serve as controls for the experiment. There should be low CAT activity in the non-permissive CEFs as there is no Src inducing JunD activity; there should be high CAT activity for the permissive CEFs as there is Src inducing JunD activity. The permissive CEFs in the presence of the MEK inhibitor will have an unknown effect depending on what is phosphorylating the JunD-TAD. If ERK is responsible for JunD-TAD phosphorylation then there will be low CAT activity, but if it is another pathway responsible for activating JunD, there will be high CAT activity.