PEA-15 C-Terminal Tail Allosterically Modulates Death-Effector Domain Conformation and Facilitates Protein–Protein Interactions

Abstract

Phosphoprotein enriched in astrocytes, 15 kDa (PEA-15) exerts its regulatory roles on several critical cellular pathways through protein–protein interactions depending on its phosphorylation states. It can either inhibit the extracellular signal-regulated kinase (ERK) activities when it is dephosphorylated or block the assembly of death-inducing signaling complex (DISC) and the subsequent activation of apoptotic initiator, caspase-8, when it is phosphorylated. Due to the important roles of PEA-15 in regulating these pathways that lead to opposite cellular outcomes (cell proliferation vs. cell death), we proposed a phosphostasis (phosphorylation homeostasis) model, in which the phosphorylation states of the protein are vigorously controlled and regulated to maintain a delicate balance. The phosphostasis gives rise to the protective cellular functions of PEA-15 to preserve optimum cellular conditions. In this article, using advanced multidimensional nuclear magnetic resonance (NMR) techniques combined with a novel chemical shift (CS)-Rosetta algorithm for de novo protein structural determination, we report a novel conformation of PEA-15 death-effector domain (DED) upon interacting with ERK2. This new conformation is modulated by the irregularly structured C-terminal tail when it first recognizes and binds to ERK2 at the d-peptide recruitment site (DRS) in an allosteric manner, and is facilitated by the rearrangement of the surface electrostatic and hydrogen-bonding interactions on the DED. In this ERK2-bound conformation, three of the six helices (α2, α3, and α4) comprising the DED reorient substantially in comparison to the free-form structure, exposing key residues on the other three helices that directly interact with ERK2 at the DEF-docking site (docking site for ERK, FxF) and the activation loop. Additionally, we provide evidence that the phosphorylation of the C-terminal tail leads to a distinct conformation of DED, allowing efficient interactions with Fas-associated death domain (FADD) protein at the DISC. Our results substantiate the allosteric regulatory roles of the C-terminal tail in modulating DED conformation and facilitating protein–protein interactions of PEA-15.

PEA-15 Engages in Allosteric Interactions Using a Common Scaffold in a Phosphorylation-Dependent Manner

“…When it comes to bind with FADD, little conformational change is needed to form the complex. This simulated PEA-15pp/FADD model agrees well with our initial NMR assessment on the complex, which demonstrated that the phosphorylation of PEA-15 C-terminal serine residues signi cantly modulate DED conformation, while binding to FADD does not seem to induce additional conformational change of PEA-15pp 17 . In an effort to partially assign the NMR spectra of PEA-15 S104D/S116D double mutant (PEA-15DD), mimicking the doubly phosphorylated state and having similar in vivo effects and binding speci city to FADD 25 , we identify that the residues experiencing the most chemical shift perturbations between the free PEA-15DD and the FADD-bound form are mostly located on helices α5 and α6, including Arg-71, which engages in direct interactions with FADD.…”

“…The backbone RMSD for PEA-15pp DED (before the kink) is only 0.803 Å between the free and FADD-bound structures. The simulation results of PEA-15pp in its free and FADD-bound form are consistent with our earlier NMR data, which indicated that phosphorylation at S104 and S116, mimicked by serine to aspartic acid mutation, on the C-terminal tail stimulates a conformational change at the DED, and FADD binding does not induce additional changes in DED conformation 17 . The NMR data and the current MD simulations both suggest that phosphorylation of C-terminal serine residues is enough to modulate the DED structure, converting the protein conformation better suited to bind with FADD.…”

“…In this MD study, together with our previous NMR studies 7,17,24 , we pointed to a very exible DED in PEA-15. PEA-15 DED adopts different conformations depending on its phosphorylation and binding status, and is allosterically controlled by its intrinsically disordered C-terminal tail.…”

Phosphorylation of PED/PEA‑15 at Ser116 and phosphorylation of p27 at Thr187 indicates a poor prognosis in hepatocellular carcinoma

“…This difference is primarily associated with the phosphorylation status of PED/PEA-15; in ovarian cancer, PED/PEA-15 is unphosphorylated ( 31 ). Therefore, PED/PEA-15 acts either as a tumor promoter or as a tumor suppressor, modulating cell proliferation or apoptosis depending on its phosphorylation status ( 11 ). Phosphorylated PED/PEA-15 is associated with increased ERK1/2 activity compared with that in non-tumorous cells, leading to increased cell growth and migration and, consequently, tumor promotion ( 32 ).…”

“…In addition, PED/PEA-15 regulates a range of cellular processes, including cell proliferation, apoptosis and migration in numerous types of cancer, such as breast and lung cancer ( 9 , 10 ). Notably, PED/PEA-15 acts as a tumor-promotor or a tumor-suppressor depending on its phosphorylation status ( 11 ). The unphosphorylated form binds extracellular signal receptor-activated kinase 1/2 (ERK1/2), suppressing its subsequent activation; by contrast, phosphorylation of PED/PEA-15 at Ser116 [PED/PEA-15(S116)] releases ERK1/2, resulting in its activation, which causes cell proliferation and migration, and thus, tumor promotion, including lung, breast and prostate cancer ( 9 , 10 , 12 ).…”