Flow‐cytometric detection of the RIα subunit of type I cAMP‐dependent protein kinase in human cells

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Abstract
CAMP‐dependent protein kinase (PKA) is composed of two genetically distinct catalytic (C) and regulatory (R) subunits. There are two different classes of PKA designated as type I and type II, which contain distinct R subunits (RI or RII, respectively) but share a common C subunit. Enhanced expression of type I PKA has been correlated with cell proliferation and neoplastic transformation. Detection of the different PKA subunits is usually performed by photoaffinity labeling with 8–N3‐32P‐cAMP or by radioimmunolabeling techniques. Both techniques are time, consuming and require a high number of cells and the use of radioactive reagents. Using the MCF‐10A normal human mammary cell line infected with a recombinant retroviral vector contaming the human RIa gene (MCF‐10A RIα), we have developed a flow‐cytometric assay to detect the intracellular content of Met protein in human cells. MCF‐10A and MCF‐10A RIα cells were fixed in 1.5% paraformaldehyde at 37°C for 15 min and permeabilized by methanol and acetone (1:1) at ‐20°C for 5 min before staining with a specific IgG2a MoAb followed by a FITC‐conjugate rabbit‐anti mouse IgG. This procedure was also successfully utilized to recognize RIα protein content in human peripheral blood lymphocytes. Flow‐cytometric detection of the RIa subunit in human cells is feasible and allows the study of the role of type I PKA in cell growth and neoplastic transformation.