Interaction with receptor for activated C-kinase 1 (RACK1) sensitizes the phosphodiesterase PDE4D5 towards hydrolysis of cAMP and activation by protein kinase C.
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.
Bird
Rebecca J
RJ
Institute of Molecular Cell and Systems Biology, University of Glasgow, Scotland, UK.
Baillie
George S
GS
Yarwood
Stephen J
SJ
D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D000242:Cyclic AMP / Q000378:metabolism*; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3 / Q000235:genetics / Q000378:metabolism*; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D004789:Enzyme Activation / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D006868:Hydrolysis; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D009154:Mutation; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D058988:Phosphodiesterase 4 Inhibitors / Q000494:pharmacology; D010766:Phosphorylation; D011485:Protein Binding / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D020889:Rolipram / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D009363:Neoplasm Proteins; D058988:Phosphodiesterase 4 Inhibitors; C419368:RACK1 protein, human; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; D000242:Cyclic AMP; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; C517376:PDE4D protein, human; D019204:GTP-Binding Proteins; D020889:Rolipram; D007545:Isoproterenol; D013755:Tetradecanoylphorbol Acetate
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