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

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 ??-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 Ser¹²?), and activation of PDE4D5 in response to PMA and isoproterenol, both of which trigger the recruitment of PKC? 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.

SUBMITTER: Bird RJ

PROVIDER: S-EPMC2973232 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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

Bird Rebecca J RJ Baillie George S GS Yarwood Stephen J SJ

The Biochemical journal 20101101 1

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 (h ...[more]

PMID: 20819076

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Project description:Acrodysostosis represents a group of rare genetic disorders characterized by defective skeletal development and is often accompanied by intellectual disabilities. Mutations in the 3'5'cyclic AMP (cAMP)-dependent protein kinase (PKA) type I regulatory subunit isoform α (RIα) and phosphodiesterase (PDE) PDE4D have both been implicated in impaired PKA regulation in acrodysostosis. How mutations on PDEs and RIα interfere with the regulation of cAMP-PKA signaling is not understood. cAMP-PKA signaling can be described in two phases. In the activation phase, cAMP binding to RIα dissociates the free C-subunit (Catalytic subunit). PDEs hydrolyze cAMP bound to RIα, priming the cAMP-free RIα for reassociation with the C-subunit, thereby completing one PKA activation cycle. Signal termination is thus critical for resetting PKA to its basal state and promoting adaptation to hormonal hyperstimulation. This proceeds through formation of a transient signal termination RIα: PDE complex that facilitates cAMP channeling from the cAMP-binding domain of RIα to the catalytic site of PDE. Signal termination of cAMP-PKA proceeds in three steps: Step 1) Channeling: translocation of cAMP from the CNB of RIα to the PDE catalytic site for hydrolysis. Step 2) Processivity: binding of free cAMP from the cytosol at both CNBs of RIα. Step 3) Product (5'AMP) release from the PDE hydrolysis site through competitive displacement by a new molecule of cAMP that triggers subsequent activation cycles of PKA. We have identified the molecular basis for two acrodysostosis mutants, PDE (PDE8 T690P) and RIα (T207A), that both allosterically impair cAMP-PKA signal termination. A combination of amide hydrogen/deuterium exchange mass spectrometry (HDXMS) and fluorescence polarization (FP) reveals that PDE8 T690P and RIα T207A both blocked processive hydrolysis of cAMP by interfering with competitive displacement of product 5'AMP release from the nucleotide channel at the end of each round of cAMP hydrolysis. While T690P blocked product 5'AMP release from the PDE, T207A greatly slowed the release of the substrate from RIα. These results highlight the role of processivity in cAMP hydrolysis by RIα: PDE termination complexes for adaptation to cAMP from GPCR hyperstimulation. Impairment of the signal termination process provides an alternate molecular basis for acrodysostosis.

Dataset Information

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

Publications

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

Similar Datasets

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