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Molecular Mechanism of Apoptosis Signal‐Regulating Kinase I Oligomerization and Auto‐activation
Author(s) -
Rahman Adua,
Zhan Xuanzhi
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.662.15
Subject(s) - mechanism (biology) , microbiology and biotechnology , apoptosis , kinase , chemistry , signal transduction , signal (programming language) , biology , biochemistry , computer science , physics , quantum mechanics , programming language
Apoptosis Signal‐Regulating Kinase I (ASK1) is a member of the Mitogen Activated Protein Kinase (MAPK) pathway. Responding to various stimulations, ASK1 activates four MAPK kinase (MKK) substrates including MKK4/7 and MKK3/7, which phosphorylate the c – Jun N terminal Kinase (JNK) and p38, respectively. The dysfunction and misregulation of ASK1 lead to various diseases such as cancers, cardiovascular diseases, neurodegenerative disorders, inflammatory diseases and diabetes. The activation of ASK1 is highly regulated by many regulatory factors. It is widely believed that the oligomerization of ASK1 is essential for its activation. However, the molecular mechanism of ASK1 activation largely remains unclear. We recently found out that the purified ASK1 is capable of auto‐activating itself and it exists as a huge oligomer instead of a homodimer as previous studies suggested. To explore the mechanism of ASK1 oligomerization and auto‐activation, we have generated a series of ASK1 truncates and hoping to identify the responsible domains/motifs for its oligomerization and auto‐activation. We have purified two ASK1 truncates ‐ ASK1‐NK (ASK1 protein expressing N terminal and Kinase domains) and ASK1‐K (ASK1 protein expressing Kinase domain only). After performing gel filtration, it was found out that ASK1‐NK forms monomer and ASK1‐K forms dimer in native state. We are currently developing protocols of kinase assay for different ASK1 truncates to analyze its auto‐activation mechanism. In conclusion, this study will help us to unveil the detailed mechanism of ASK1 oligomerization which will surely enrich our knowledge in understanding the activation process of ASK1. Support or Funding Information This project is supported by Faculty Research Grant to Xuanzhi Zhan and Student Research Grant to Adua Rahman awarded by Tennessee Technological University.ASK1 mediated pathwayPurification of ASK1 truncates. (A) ASK1‐K domain was purified using Heparin and Q‐sepharose column chromatography. The purified ASK1‐K domain (~2μg) is shown in SDS‐PAGE; (B) ASK1‐NK domain was purified using Q – Sepharose and Phenyl – Sepharose column chromatography. The purified ASK1‐NK domain (~2μg) is shown in SDS‐PAGE.Sephacryl gel filtration chromatography of ASK1 truncates. (A) ASK1‐NK is observed as monomer (116KDa). (B) Predominant form of ASK1‐K is dimer (77 KDa). (C) The chromatogram of gel filtration standard and log of molecular weight vs. elution volume plot is generated to determine the molecular weights of the ASK1 truncates.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .