Protein Kinase A and Signal Transduction in T Lymphocytes

Biochemical and Molecular Methods
  • Islam U. Khan
  • Gary M. Kammer
Part of the Methods in Molecular Medicine™ book series (MIMM, volume 102)

Summary

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.

Key Words

Cloning competitive PCR FPLC protein kinase A sequencing signal transduction SLE T cells Western blotting 

References

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Copyright information

© Humana Press Inc., Totowa, NJ 2004

Authors and Affiliations

  • Islam U. Khan
    • 1
  • Gary M. Kammer
    • 2
  1. 1.Section on Rheumatology and Clinical Immunology, Department of Internal MedicineWake Forest University Health SciencesWinston-Salem
  2. 2.Section on Rheumatology and Clinical Immunology, Department of Internal Medicine; Department of Microbiology and ImmunologyWake Forest University Health SciencesWinston-Salem

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