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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
2R01AR039501-04A3
Application #
2079546
Study Section
General Medicine A Subcommittee 2 (GMA)
Project Start
1994-09-01
Project End
1998-03-31
Budget Start
1994-09-01
Budget End
1995-08-31
Support Year
4
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Wake Forest University Health Sciences
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
041418799
City
Winston-Salem
State
NC
Country
United States
Zip Code
27106
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Khan, Islam U; Kammer, Gary M (2004) Protein kinase A and signal transduction in T lymphocytes: biochemical and molecular methods. Methods Mol Med 102:73-85
Kammer, Gary M; Laxminarayana, Dama; Khan, Islam U (2004) Mechanisms of deficient type I protein kinase A activity in lupus T lymphocytes. Int Rev Immunol 23:225-44
Nambiar, Madhusoodana P; Juang, Yuang-Taung; Krishnan, Sandeep et al. (2004) Dissecting the molecular mechanisms of TCR zeta chain downregulation and T cell signaling abnormalities in human systemic lupus erythematosus. Int Rev Immunol 23:245-63
Khan, Islam U; Tsokos, George C; Kammer, Gary M (2003) Abnormal B cell signal transduction in systemic lupus erythematosus. Curr Dir Autoimmun 6:89-104
Laxminarayana, Dama; Khan, Islam U; Kammer, Gary (2002) Transcript mutations of the alpha regulatory subunit of protein kinase A and up-regulation of the RNA-editing gene transcript in lupus T lymphocytes. Lancet 360:842-9
Kammer, Gary M; Tsokos, George C (2002) Abnormal T lymphocyte signal transduction in systemic lupus erythematosus. Curr Dir Autoimmun 5:131-50

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