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Mapping domains of anti‐apoptotic Bcl‐xL required for interaction with cell cycle check point regulator Aven
Author(s) -
Datan Emmanuel,
Roelofs Brian,
Hardwick J. Marie
Publication year - 2010
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.24.1_supplement.518.1
Subject(s) - mutant , regulator , amino acid , hela , biology , homology (biology) , point mutation , cell cycle , phenotype , microbiology and biotechnology , genetics , apoptosis , cell , gene
Members of the Bcl‐2 family of proteins, known for their critical apoptotic functions, have often been shown to influence cellular processes based on knock‐out or over expression studies. However, the outcomes of these approach do not show how Bcl‐2 family members actually bring about such phenotypes or outcomes in over expressing, under expressing or knock‐out conditions. This void left by knock‐out/down or over expression studies is the avenue of protein‐protein interaction research. Mapping and crystallography has been the usual approach in protein‐protein interaction studies. In this study the interaction of a member of the Bcl‐2 family protein Bcl‐x L with cell cycle check point regulator Aven was mapped though point mutations in the BH homology domains of Bcl‐x L . After sequence verification and subsequent expression of the mutant constructs/plasmids with HA‐Aven in Hela cells, HA tagged Aven was then co‐immunoprecipitated with other complexing proteins. The mutant Bcl‐x L that was bound to Aven was identified through Western blots. Data suggests that amino acids 2, 18, 25, 131, 135, 136, 137, 181 are important for Bcl‐x L interaction with Aven. These amino acids belong to the BH4, BH1 and BH2 domains of Bcl‐xL. In addition to mapping out the required amino acids for interaction, results also show that the kind of Bcl‐x L mutant and it's concentration may affect or be affected by the stability of Aven in vitro. The results of this study should be verified in other conditions especially were Bcl‐x L and Aven are critical proteins.