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Oligopeptide Competition Assay for Phosphorylation Site Determination
09:16

Oligopeptide Competition Assay for Phosphorylation Site Determination

Published on: May 18, 2017

Phosphorylation of ribosomal protein S6 at multiple sites by a cyclic AMP-independent protein kinase from lymphoid

M J Donahue, R A Masaracchia

    The Journal of Biological Chemistry
    |January 10, 1984
    PubMed

    Insights

    A novel cyclic AMP-independent protein kinase, H4P kinase, phosphorylates the S 6 protein in 40 S ribosomal subunits. This enzyme

    Area of Science:

    • Molecular Biology
    • Biochemistry
    • Cellular Biology

    Background:

    • Ribosomes are crucial for protein synthesis.
    • Specific ribosomal proteins are targets for regulatory phosphorylation.
    • Insulin signaling involves post-translational modifications of ribosomal proteins.

    Purpose of the Study:

    • To characterize a cyclic AMP-independent protein kinase (H4P kinase) involved in ribosome phosphorylation.
    • To investigate the substrate specificity and kinetics of H4P kinase.
    • To explore the role of H4P kinase in the phosphorylation of ribosomal protein S 6.

    Main Methods:

    • Isolation and purification of H4P kinase from murine lymphosarcoma cells.
    • Enzyme activation assays using Mg2+-ATP and an endogenous converting enzyme.
    • Phosphorylation assays using ribosomal subunits and [32P]phosphate.
    • Kinetic analysis (Km, Vmax) of H4P kinase activity.
    • Comparison of phosphorylation stoichiometry with previous studies.

    Main Results:

    • H4P kinase, initially inactive, is activated by Mg2+-ATP and a converting enzyme.
    • Activated H4P kinase selectively phosphorylates ribosomal protein S 6 in the 40 S subunit.
    • Over 90% of phosphate transfer to 40 S subunits is incorporated into S 6.
    • Kinetic parameters (Km = 7.2 microM, Vmax = 50 nmol Pi/min/mg) were determined.
    • Exhaustive phosphorylation incorporated 3 mol phosphate/mol S 6, differing from prior reticulocyte studies.

    Conclusions:

    • H4P kinase is a distinct enzyme capable of multiple phosphorylations on S 6.
    • These findings support a role for H4P kinase in insulin-mediated S 6 phosphorylation.
    • The study provides insights into the regulation of protein synthesis via ribosomal modification.

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