Contributions of myristoylation to calcineurin structure/function.
Calcineurin is a serine/threonine protein phosphatase composed of a catalytic subunit, calcineurin A (58 kDa), and a NH2-terminal myristoylated regulatory subunit, calcineurin B (19 kDa). In order to study the effect of myristoylation on calcineurin structure/function, a dual plasmid transfection system was used to generate myristoylated and nonmyristoylated calcineurin B. Both metabolic labeling of calcineurin B with radiolabeled myristic acid and electrospray mass spectral analysis confirmed that myristic acid was covalently and stoichiometrically linked to calcineurin B. Myristoyl and non-myristoyl calcineurin B were reconstituted with recombinant calcineurin A to form native-like heterodimers, and the properties of the two calcineurin forms were examined. Myristoylation had no effect on enzymatic activity, calcineurin-immunosuppressant/immunophilin interactions, or Ca2+ binding. Surprisingly, myristoylation also had no effect on calcineurin heterodimer association with phospholipid monolayers. Fatty acylation, however, significantly influenced the thermal stability of calcineurin, with an approximate 10 degrees C increase in t1/2 observed for myristoyl calcineurin when compared to the non-myristoyl form. Myristoylation of calcineurin B therefore appears to provide structural stability to the calcineurin heterodimer.
Kennedy
M T
MT
Section of Hematology Research and the Department of Biochemistry and Molecular Biology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905, USA.
Brockman
H
H
Rusnak
F
F
D000591:Amino Acid Isomerases / Q000494:pharmacology; D000818:Animals; D019703:Calcineurin; D002148:Calmodulin-Binding Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D002352:Carrier Proteins / Q000494:pharmacology; D021983:Cyclophilins; D016572:Cyclosporine / Q000494:pharmacology; D004795:Enzyme Stability; D006358:Hot Temperature; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism*; D019696:Peptidylprolyl Isomerase; D010743:Phospholipids / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship
D002148:Calmodulin-Binding Proteins; D002352:Carrier Proteins; D009227:Myristic Acids; D010743:Phospholipids; D011994:Recombinant Proteins; D019814:Myristic Acid; C081786:cyclophilin B; D016572:Cyclosporine; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D000591:Amino Acid Isomerases; D021983:Cyclophilins; D019696:Peptidylprolyl Isomerase