1 Delineate the following signaling pathways 5pts each A Fro

1. Delineate the following signaling pathways (5pts each):

A) From the immunological synapse to the binding of fos and jun their enhancer element.

B) From the immunological synapse to the binding of NFAT to its enhancer element.

C) From the immunological synapse to the binding of Nfkb to its enhancer element.

* For each tell which proteins are involved, important signaling motifs, and the specific kinase. For example: TLR4 binds MAL and MyD88, this attracts the serine threonine kinases IRAK 1 and 4 which auto phosphorylates and in turn recruits and phosphorylates TRAF-6…etc *

Solution

A) Activating protein-1 is a large family of dimeric protein complexes mainly consisting of Fos (c-Fos, v-Fos, Fos B, Fra1, Fra2) and Jun (c-Jun, v-Jun, JunB, JunD). These proteins, which belong to the bZIP group of DNA-binding proteins, have leucine zippers through which they associate to form a variety of homo- and heterodimers that bind to common AP-1 sites (TRE-TGAC/GTCA) or (CRE-TGACTCA) in DNA. Both ATF (ATF2, ATF3, B-ATF, JDP1, JDP2) and Maf (c-MAF, MafA, MafB, Nr1) are also considered members of this family based on their dimerization potential with Fos or Jun. Jun-proteins, but not Fos-proteins, are known to undergo homo-dimerization. Hetero-dimerization of Fos with Jun is crucial for nuclear-cytoplasmic shuttling. Monomeric Fos and Jun shuttle actively but hetero-dimerization of both proteins inhibits their cytoplasmic shuttling. Surprisingly, this retro-transport inhibition is not caused by the binding of the AP-1 complex to DNA,

Levels of Fos and Jun proteins in T cells are either low or absent and are generally induced on signalling. The activity of AP-1 is regulated by mitogen-activated protein kinases (MAPK). Extra-cellular signal-regulated kinase (ERK) activation causes c-Fos induction, which results in increased synthesis of c-Fos and translocation to the nucleus. In the nucleus, it combines with pre-existing Jun proteins to form AP-1 dimers that are more stable than those formed by Jun proteins alone. It has been shown that ERK-1 is associated with the synapse after TCR stimulation and prevents docking of Src homology-2 (SH2) domain-containing phosphatase -1 (SHP-1) phosphatase.Transcription of c-Fos is regulated by ternary complex factors (Elk-1, SAP-1 and SAP-2) of which Elk-1 is phosphorylated by ERK. The c-Jun is expressed at low levels in unstimulated cells and its promoter is constitutively occupied by Jun-activating transcription factor (ATF2) dimer. Phosphorylation of c-Jun by Jun N-terminal kinases (JNKs) and of ATF2 by JNKs or p38MAPK stimulates their ability to activate transcription, thereby leading to c-Jun induction. As part of their negative regulation, AP-1 proteins are degraded in both ubiquitin-dependent and ubiquitin-independent manners. The GSK-3 can inhibit AP-1 transcriptional activity by producing inhibitory phosphorylation on Jun. The MAPK are negatively regulated by MAPK phosphatases, which are known to interact with the cytoplasmic tail of CD28 and are regulated by CD28 signalling.

RhoA is a small GTPase with a well-characterized role in cytoskeletal rearrangement. Upon activation, RhoA causes the formation of stress fibres in cells. In lymphocytes, this function of RhoA is critical for the proper homing of cells to areas of infection. Evidence has shown that RhoA is required for the process of leukocyte rolling and diapedesis or migration beneath the endothelial layer of cells. In addition, RhoA is involved in the assembly of the immunological synapse, an area on the cell surface where elements of the immune signalling apparatus congregate, allowing for proper activation of the T cell.

In some cells, RhoA has been shown to play a more direct role in signal transduction. Welsh et al. demonstrated a dependence on RhoA for ERK activity during the G1 phase of the cell cycle. Without ERK activation, there is no cyclin D induction, leading to a block in cell cycle progression. The specific role of RhoA in signalling pathways turned on during T cell activation has not been examined thoroughly, although reports have shown that the GTPase is able to activate the NFAT-binding partner AP-1 as well as NF-B. In addition, RhoA and its upstream activator G13 have been shown to increase the activity of phospholipase C (PLC), leading to increased levels of intracellular calcium.

The NFAT family of proteins was first discovered by identification of factors involved in the up-regulation of IL-2 in response to TCR stimulation. Since that time, NFAT proteins have been implicated in a wide variety of cellular processes including cardiac hypertrophy, learning and memory, and adipocyte differentiation. Immunologically relevant genes regulated by NFAT include IL-2, IL-4, IL-5, GM-CSF, TNF-, CD40 ligand (CD40L), and FasL. NFAT family proteins are regulated primarily through calcium levels in the cell. Upon stimulation, an increase in intracellular calcium turns on the serine/threonine phosphatase calcineurin, which then binds to NFAT and dephosphorylates the protein, allowing NFAT nuclear translocation. The immunosuppressive drugs FK506 and Cyclosporin A work through inhibition of calcineurin—preventing NFAT-dependent transcription. Studies have shown that p38MAPK, ERK, and JNK can potentially phosphorylate NFAT, inhibiting its translocation.

Other pathways have also been implicated in NFAT regulation at the level of nuclear translocation and DNA binding. Once the protein enters the nucleus, kinases, including glycogen synthase kinase 3, phosphorylate NFAT, preventing DNA binding and leading to nuclear export. Furthermore, NFAT proteins typically have a binding partner, which stabilizes their interaction with the DNA. Although several transcription factors can act in this capacity, the predominant protein is , a heterodimer comprised of c-jun and Fos, which are activated by MAPK pathways that can also be initiated upon TCR ligation. Fos is transcriptionally up-regulated through ERK, and JNK phosphorylates jun leading to its activation and nuclear translocation. Some studies have shown that NFAT plays a role in energy induction as well as activation in T cells. Stimulation of T cells with ionomycin alone activates NFAT in the absence of factors including AP-1 and leads to the up-regulation of “anergic factors,” which block activation instead of the up-regulation of IL-2 production and proliferation. In addition, recent reports have shown that alternate family members can replace Fos and jun in the formation of a heterodimer, which is still able to bind DNA cooperatively with NFAT but give the complex an inhibitory function.

Here, we demonstrate that activated RhoA is able to inhibit NFAT-dependent transcription from the HIV long-terminal repeat (LTR) and the IL-2 promoter. RhoA did not affect NFAT nuclear localization in response to Ca++-mediated activation. The RhoA-GTPase did, however, inhibit the transactivation potential of NFAT. In stimulated T cells, expression of activated RhoA also led to a decrease in the level of acetylated histone H3 at the IL-2 promoter but not the occupancy of NFAT at the IL-2 promoter. RhoA activation may, therefore, affect chromatin remodelling at the IL-2 promoter and the ability of NFAT to transactivate DNA.