Protein kinase A and signal transduction in T lymphocytes: biochemical and molecular methods.
Abnormal T-cell effector functions in systemic lupus erythematosus (SLE) are present and may be associated with disease immunopathogenesis. Our work has led to the characterization of a signaling defect, involving protein kinase A (PKA), leading to abnormal T-cell effector functions in SLE. PKA is a component of the adenylyl cyclase/cyclic adenosine monophosphate/PKA (AC/cAMP/PKA) pathway, a principal signal transduction system in T cells. The aim of this chapter is to provide a comprehensive, technical, step-by-step approach to studying PKA function in T cells. The methods detailed here are (a) chromatographic fractionation of PKA-I and PKA-II isozymes and PKA phosphotransferase activity in purified T cell populations, (b) Western immunoblotting to identify the presence of regulatory (R)-subunit proteins of PKA, and (c) isolation of RNA, and quantification of PKA R subunit-specific transcripts by competitive polymerase chain reaction. Although our emphasis in the chapter is T cells, these methods may be useful for investigation of signaling via PKA in other cell types as well.
Khan
Islam U
IU
Department of Internal Medicine, Wake Forst University Health Sciences, Winston-Salem, NC, USA.
Kammer
Gary M
GM
D015153:Blotting, Western; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D007158:Immunologic Techniques; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008180:Lupus Erythematosus, Systemic / Q000201:enzymology* / Q000235:genetics / Q000276:immunology*; D016133:Polymerase Chain Reaction; D015398:Signal Transduction / Q000276:immunology*; D013601:T-Lymphocytes / Q000201:enzymology* / Q000276:immunology*
D018076:DNA, Complementary; D007527:Isoenzymes; D017868:Cyclic AMP-Dependent Protein Kinases