Interaction with receptor for activated C-kinase 1 (RACK1) sensitizes the phosphodiesterase PDE4D5 towards hydrolysis of cAMP and activation by protein kinase C

Rebecca J. Bird, George S. Baillie, S. J. Yarwood

    Research output: Contribution to journalArticlepeer-review

    27 Citations (Scopus)

    Abstract

    We have previously identified the PKC (protein kinase C)-anchoring protein RACK1 (receptor for activated C-kinase 1), as a specific binding partner for the cAMP-specific phosphodiesterase PDE4D5, suggesting a potential site for cross-talk between the PKC and cAMP signalling pathways. In the present study we found that elevation of intracellular cAMP, with the β₂-adrenoceptor agonist isoproterenol (isoprenaline), led to activation of PDE4 enzymes in the particulate and soluble fractions of HEK (human embryonic kidney)-293 cells. In contrast activation of PDE4D5, with isoproterenol and the PKC activator PMA, was restricted to the particulate fraction, where it interacts with RACK1; however, RACK1 is dispensable for anchoring PDE4D5 to the particulate fraction. Kinetic studies demonstrated that RACK1 alters the conformation of particulate-associated PDE4D5 so that it more readily interacts with its substrate cAMP and with rolipram, a PDE4 inhibitor that specifically targets the active site of the enzyme. Interaction with RACK1 was also essential for PKC-dependent and ERK (extracellular-signal-regulated kinase)-independent phosphorylation (on Ser¹²⁶), and activation of PDE4D5 in response to PMA and isoproterenol, both of which trigger the recruitment of PKCα to RACK1. Together these results reveal novel signalling cross-talk, whereby RACK1 mediates PKC-dependent activation of PDE4D5 in the particulate fraction of HEK-293 cells in response to elevations in intracellular cAMP.

    Original languageEnglish
    Pages (from-to)207-216
    Number of pages10
    JournalBiochemical Journal
    Volume432
    Issue number1
    DOIs
    Publication statusPublished - 15 Nov 2010

    Keywords

    • Binding Sites
    • Blotting, Western
    • Cyclic AMP
    • Cyclic Nucleotide Phosphodiesterases, Type 3
    • Cyclic Nucleotide Phosphodiesterases, Type 4
    • Enzyme Activation
    • GTP-Binding Proteins
    • HEK293 Cells
    • Humans
    • Hydrolysis
    • Intracellular Space
    • Isoproterenol
    • Kinetics
    • Mutation
    • Neoplasm Proteins
    • Phosphodiesterase 4 Inhibitors
    • Phosphorylation
    • Protein Binding
    • Protein Kinase C
    • Protein Kinase C-alpha
    • Protein Transport
    • Receptors, Cell Surface
    • Rolipram
    • Tetradecanoylphorbol Acetate
    • Transfection

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