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Published on: September 18, 2013

A structural gene mutation affecting the regulatory subunit of cyclic AMP-dependent protein kinase in mouse lymphoma

J Hochman, P A Insel, H R Bourne

    Proceedings of the National Academy of Sciences of the United States of America
    |December 1, 1975
    PubMed

    Insights

    A mutation in the regulatory subunit of cyclic adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase in S49 lymphoma cells alters enzyme activity and cAMP sensitivity. This structural mutation is crucial for cAMP-mediated growth regulation and enzyme induction.

    Area of Science:

    • Molecular Biology
    • Cell Biology
    • Biochemistry

    Background:

    • S49 mouse lymphoma cells are a model system for studying cyclic adenosine 3':5'-monophosphate (cAMP) signaling.
    • Exogenous cAMP elicits biological effects including enzyme induction, growth inhibition, and cytolysis in wild-type S49 cells.
    • A mutant cell line, kin.A, exhibits reduced sensitivity to these cAMP-mediated effects.

    Purpose of the Study:

    • To investigate the molecular basis for the reduced cAMP sensitivity in kin.A mutant S49 cells.
    • To determine the role of cAMP-dependent protein kinase in mediating cAMP's biological effects.
    • To identify the specific component of the cAMP-dependent protein kinase responsible for the altered phenotype.

    Main Methods:

    • Comparison of cAMP-dependent protein kinase activity and kinetics between wild-type and kin.A cells.
    • Purification of cAMP-dependent protein kinase holoenzyme from both cell lines.
    • Heterologous reconstitution experiments using separated regulatory (R) and catalytic (C) subunits.

    Main Results:

    • Kin.A cells showed a 10-fold greater apparent Ka for cAMP activation of protein kinase activity and increased thermolability compared to wild-type.
    • These altered properties were attributed to the regulatory (R) subunit of the cAMP-dependent protein kinase.
    • Reconstitution experiments confirmed that the R subunit of kin.A conferred altered cAMP affinity and heat sensitivity.

    Conclusions:

    • The kin.A mutation involves a structural alteration in the gene coding for the R subunit of cAMP-dependent protein kinase.
    • This structural mutation is responsible for the observed changes in enzyme kinetics and thermolability.
    • cAMP-dependent protein kinase is essential for mediating cAMP's regulatory effects on growth and enzyme induction in S49 cells.

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