The role of loop 7 in mediating calcineurin regulation.
Calcineurin (CN) is a heterodimer protein consisting of a 61 kDa catalytic subunit A and a 19 kDa regulatory subunit B. It plays a critical role in T-cell activation and is involved in many cellular processes. Regulation of CN is rather complex, including a number of factors such as divalent metal ions (primarily Ca(2+) and Mn(2+)), calmodulin (CaM) and autoinhibition (AI) segment. Previously, we reported that a loop 7 deletion mutant (V314) in subunit A exhibited high phosphatase activity, although the mechanism for the surprising activity enhancement and whether the activity change applies to other loop 7 residues were not known. In order to probe the role of loop 7, we have carried out extensive mutagenesis experiments, followed by systematic activity assays under a number of regulatory conditions. All mutants, including single deletion mutants Y315, N316 and double deletion mutant V314Y315, showed increased phosphatase activity. Significantly, activities of the mutants containing the V314 deletion, namely V314 and V314Y315, were no longer regulated by regulatory subunit B. These results, along with the structure analysis, suggest that loop 7 as a whole plays an important role in mediating CN's regulation through bridging the regulatory subunit and catalytic core and interaction with the AI segment of CN.
Xiang
Ben Q
BQ
Department of Biochemistry and Molecular Biology, Life Science Institute, Beijing Normal University, Beijing 100875, China.
Jia
Zongchao
Z
Xiao
Fang X
FX
Zhou
Ke
K
Liu
Ping
P
Wei
Qun
Q
D000595:Amino Acid Sequence; D019703:Calcineurin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017354:Point Mutation / Q000235:genetics; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins; D017384:Sequence Deletion / Q000235:genetics; D013329:Structure-Activity Relationship
D011994:Recombinant Proteins; D019703:Calcineurin