FLOW-CYTOMETRIC DETECTION OF THE RI-ALPHA SUBUNIT OF TYPE-I CAMP-DEPENDENT PROTEIN-KINASE IN HUMAN-CELLS

cAMP-dependent protein kinase (PKA) is composed of two genetically dis tinct catalytic (C) and regulatory (R) subunits. There are two differe nt classes of PKA, designated as type I and type II, which contain dis tinct R subunits (RI or RII, respectively) but share a common C subuni t. Enhanced expression of type I PKA has been correlated with cell pro liferation and neoplastic transformation. Detection of the different P KA subunits is usually performed by photoaffinity labeling with 8-N-3- P-32-cAMP or by radioimmunolabeling techniques. Both techniques are ti me consuming and require a high number of cells and the use of radioac tive reagents. Using the MCF-10A normal human mammary cell line infect ed with a recombinant retroviral vector containing the human RI alpha gene (MCF-10A RI alpha), we have developed a flow-cytometric assay to detect the intracellular content of RI alpha protein in human cells. M CF-10A and MCF-10A RI alpha cells were fixed in 1.5% paraformaldehyde at 37 degrees C for 15 min and permeabilized by methanol and acetone ( 1:1) at -20 degrees C for 5 min before staining with a specific IgG2a MoAb followed by a FITC-conjugate rabbit-anti mouse IgG. This procedur e was also successfully utilized to recognize RI alpha protein content in human peripheral blood lymphocytes. Flow-cytometric detection of t he RI alpha subunit in human cells is feasible and allows the study of the role of type I PKA in cell growth and neoplastic transformation. (C) 1994 Wiley-Liss, Inc.