Work in this laboratory is aimed at elucidating the mechanism of stimulus- response coupling mediated by Ca2+ and calmodulin. The calmodulin- stimulated protein phosphatase, calcineurin, is used as a model system. Our ultimate goal is to elucidate the structure of the calcineurin/calmodulin complex in order to understand how calmodulin activates this important enzyme. Our efforts, this year, were focused on the role of the Ca2+-binding regulatory subunit of calcineurin, calcineurin B, in the regulation of the protein phosphatase activity. Ca2+ binding to calcineurin B was shown to be absolutely required for enzymatic activity as well as for the calmodulin stimulation of the enzyme. Myristoylated and unmyristoylated calcineurin B have been expressed in high yield in E. coli. The Ca2+ binding properties of the recombinant proteins are similar to those of native calcineurin B but substitution of the myristoylated calcineurin B of the native enzyme by unmyristoylated calcineurin B yields inactive enzyme. The three- dimensional structure of recombinant calcineurin B has been determined in collaboration with Ad Bax and Jacob Anglister (NIDDK). Recombinant calcineurin A-beta has also been expressed in high yield in E. coli. It is being recombined with myristoylated calcineurin B to reconstitute enzymatically active enzyme and to obtain homogenous preparations of calcineurin suitable for crystal structure determination.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Intramural Research (Z01)
Project #
1Z01CB005231-19
Application #
3774295
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
19
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Division of Cancer Biology and Diagnosis
Department
Type
DUNS #
City
State
Country
United States
Zip Code