Discussionp38 and ERK pathway were determined to be activated after LPS treatment. MAPK are activated by dual phosphorylation at the activation loop which changes the conformation shift. The phosphorylation in ERK pathway happens at specific motif that have sequence TEY. In p38 pathway, the phosphorylation sequence is TGY. Dual phosphorylation on Thr-183 and Tyr-185 in Erk2 and Thr-203 and Tyr-205 in Mapk3 shows activation of MAPK pathway. A conformational change enables full activation and interaction of protein with its substrates. ERK1/2 detaches from scaffold proteins making it accessible to Importin 7 (IPO7) for nuclear entry (Vougiouklakis et al., 2015). ERK accumulates in the nucleus after stimulus-induced phosphorylation and is essential for ERK-mediated processes, such as entry into S phase (Shindo et al., 2016).
The dual phosphorylation site of unphosphorylated form of ERK2 are contained within a loop structure. Tyr-185 is buried in a hydrophobic pocket whereas Thr-183 is exposed to the surface of the molecule. After phosphorylation of ERK2, the loop structure changes upon association with MEK. The exact interaction of MEK and ERK is still unknown but it is speculated that the loop structure is significant in determining MEK specificity. Amino acid residues located within the phosphorylation range may contribute to the MEK/ERK interaction and conformational mobility of ERK (Butch and Guan, 1996).
Dual phosphorylation on Thr-180 and Tyr-182 in MAPK14 is activated two kinases, MAP2K3/MKK3 or MAP2K6/MKK6 due to inflammatory cytokines and growth factors activates the enzyme. Phosphorylation of Tyr-182 alone is inactive whereas on Thr-180 alone is 10-20 less active than dual phosphorylation of MAPK14 (Zhang et al., 2008). The unphosphorylated state of p38, an ATP-binding side is located between N- and C-terminal domains. The activation site is a glycine-rich loop that connects both terminals domain and the catalytic loop. p38 has a docking site for activating kinases, substrate kinases and inactivating kinases. In the phosphorylated state, the substrate binding site opens up to stabilize the large negative charges produced by phosphate groups. The r.m.s.d for superposition of phosphorylated and unphosphorylated MAPK14 was 2.55A and when in either state, the activation loop is in ordered. Basic residues from both lobes carries out coordination of the activation-loop phosphates and leads to a more compact structure in the phosphorylated state. The difference in relative movement of MAPK14 in both state is 8°. In the unphosphorylated state, Met109 blocks the ATP-binding site however in the other state, the residue occupies a new pocket and the conformational rearrangement allows a flip in the peptide bond in the presence of adjacent Gly110 (Yurtsever et al., 2015).
MKK3 and MKK6 are highly selective for p38 MAPKs. MKK3 is known as a major p38 activator in mesangial cells whereas MKK6 is the predominant isoform in thymocytes.