Localization of the catalytic subunit of cyclic AMPdependent protein kinase in cultured cells using a specific antibody

Michael P. Murtaugh, Alton L. Steiner, Peter J.A. Davies

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Abstract

We developed a specific antibody to the catalytic subunit (C-subunit) of cyclic AMP-dependent protein kinase and used it to localize C-subunit in cultured cells. C-subunit antigen was purified from bovine cardiac muscle and cross-linked to hemocyanin with glutaraldehyde. Immunized goat serum showed a low titer of antibody after boosting; it was enriched 100-fold by affinity chromatography on catalytic subunit-Sepharose. The antibody immunoprecipitated C-subunit from type I and type II holoenzyme and depleted enzymatic activity from solution. At 12.5 nM antigen, I µg antibody immunoprecipitated 10 ng of Csubunit. Immunoprecipitation of 35S-labeled cell extracts and 125l-antibody detection on nitrocellulose paper revealed that the antibody specifically reacts with C-subunit in Chinese hamster ovary (CHO) whole cell extracts. Using indirect immunofluorescence to localize Csubunit, we found a pattern of diffuse staining in the cytoplasm of CHO cells with little or no nuclear staining. A similar distribution of the enzyme was observed in Swiss 3T3 cells, bovine endothelial tracheal cells, human lung fibroblasts and NRK cells. Treatment of CHO cells with 8-bromo-cyclic AMP produced no change in the pattern or intensity of immunofluorescence. We conclude that the majority of C-subunit is localized in cytoplasm and that in cultured fibroblasts exposure to cyclic AMP analogues causes no apparent redistribution of catalytic subunit.

Original languageEnglish (US)
Pages (from-to)64-72
Number of pages9
JournalJournal of Cell Biology
Volume95
Issue number1
DOIs
StatePublished - Oct 1 1982

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Protein Kinases
Cultured Cells
Catalytic Domain
Antibodies
Cricetulus
Ovary
Cell Extracts
Cytoplasm
Fibroblasts
Cyclic AMP-Dependent Protein Kinase Catalytic Subunits
Swiss 3T3 Cells
Staining and Labeling
8-Bromo Cyclic Adenosine Monophosphate
Antigens
Hemocyanin
Holoenzymes
Collodion
Glutaral
Indirect Fluorescent Antibody Technique
Affinity Chromatography

Cite this

Localization of the catalytic subunit of cyclic AMPdependent protein kinase in cultured cells using a specific antibody. / Murtaugh, Michael P.; Steiner, Alton L.; Davies, Peter J.A.

In: Journal of Cell Biology, Vol. 95, No. 1, 01.10.1982, p. 64-72.

Research output: Contribution to journalArticle

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