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 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 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. MCF-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 procedure was also successfully utilized to recognize RI alpha protein content in human peripheral blood lymphocytes. Flow-cytometric detection of the 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.
Pepe
S
S
Cattedra di Oncologia Medica, II Facoltà di Medicina e Chirurgia, Napoli, Italy.
Ruggiero
A
A
Tortora
G
G
Ciardiello
F
F
Garbi
C
C
Yokozaki
H
H
Cho-Chung
Y S
YS
Clair
T
T
Skalhegg
B S
BS
Bianco
A R
AR
D001940:Breast / Q000166:cytology; D002478:Cells, Cultured; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000032:analysis* / Q000096:biosynthesis; D004247:DNA / Q000032:analysis; D004790:Enzyme Induction; D005434:Flow Cytometry; D006801:Humans; D008214:Lymphocytes / Q000201:enzymology; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000096:biosynthesis; D014162:Transfection
D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; C517834:PRKAR1A protein, human; C517835:Prkar1a protein, mouse; D011993:Recombinant Fusion Proteins; D004247:DNA; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases