Assembly of ASK1-MKK4-JNK3 Complex

Abstract

Apoptosis signal-regulating kinase I (ASK1, also called MAP3K5) is a mitogen-activated kinase kinase kinase (MAP3K) that plays pivotal roles in cellular stress and immune response. Active ASK1 can directly phosphorylate downstream mitogen-activated kinase kinases (MKKs) from both stress-activated protein kinase (SAPK) pathways: c-jun N-terminal kinase (JNK). The structural of these three kinase complexes (JNK, MKK4, and ASK1) remains still unknown. For this reason, the present study has been designed to find out the assembly of ASK1-MKK4-JNK3 firstly using computational docking based on the Protein Frustratometer combined with the sequence identity calculations of Evolutionary Trace forms along with rigid docking (Cluspro) followed by experimental design. To verify the proposed computational model of ASK1-MKK4-JNK3 complex, we designed a series of maltose binding protein (MBP) fusion peptides, which contains different peptide fragments derived from MKK4: MBP-P1, MBP-P2, MBP-P3, MBP-P4 and MBP-P5. We then transformed into E-coli-BL21- DE3 to express those proteins via Isopropyl β-D-1-thiogalactopyranoside (IPTG) and then subsequently purified those proteins using Amylose-sepharose chromatography. We intend to develop direct pull-down assays to evaluate the interactions between these fusion peptides with ASK1 and JNK3, respectively to find out the binding interaction among these purified proteins.