We have described a disorder of cAMP metabolism in T lymphocytes of subjects with systemic lupus erythematosus (SLE) Our recent identification of defective cAMP -dependent phosphorylation of proteins in intact SLE T lymphocytes suggests a disorder of cAMP -dependent protein kinase (Protein kinase A) The objective of this proposal is to identify and characterize the abnormal protein kinase A function in SLE T lymphocytes by quantifying cAMP -dependent protein kinase activities and the amounts of the enzyme's constituent subunits, the types I (RI) and II (RII) regulatory and catalytic subunits (C subunit).
The specific aims of this proposal are (a) to compare protein kinase A activities in the nonparticulate and particulate fractions of T lymphocytes and T lymphocyte subsets from active and inactive SLE subjects with controls. cAMP -dependent protein kinase activity will be quantified by measuring the phosphorylation of histone and Kemptide in nonparticulate and particulate T cell extracts. (b) We will purify partially the cAMP -dependent protein kinases of SLE T lymphocytes by DEAE -cellulose chromatography in order to determine the presence of anomalous RI, RII and/or C subunits. Anomalous RI or RII subunits can exhibit altered cAMP binding while anomalous C subunits can possess reduced binding affinity for ATP. (c) The amounts of protein kinase A subunits will be quantified by ELISA and nitrocellulose immunolabeling to determine whether a deficiency of one or more subunits exists to explain altered protein kinase activities. Differences in the kinase activities and/or amounts of a kinase of a kinase subunit between T cell subsets will be quantified to determine whether a restricted kinase defect exists. Since the T lymphocyte plays an integral role in both cellular and humoral immunity, it is important to discover whether a defective cAMP pathway accounts, in part, for the aberrant immunoregulation in SLE. Thus, our longterm goal is to determine whether defective protein kinase A function results in abnormal suppressor cell activity. The identification of a protein kinase A defect should provide important insights into the pathogenesis of this autoimmune disorder.
Showing the most recent 10 out of 35 publications
