Protein kinase A and signal transduction in T lymphocytes: biochemical and molecular methods.

Abstract

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.