Phosphodiesterase-4 gates the ability of protein kinase A to phosphorylate G-protein receptor kinase-2 and influence its translocation

2006 ◽  
Vol 34 (4) ◽  
pp. 474-475 ◽  
Author(s):  
M.D. Houslay ◽  
G.S. Baillie
Keyword(s):  
Protein Kinase ◽  
G Protein ◽  
Fluorescent Protein ◽  
Resting Cells ◽  
Receptor Kinase ◽  
Human Embryonic Kidney Cells ◽  
Phosphodiesterase 4 ◽  
Protein Receptor ◽  
Dependent Protein ◽  
Green Fluorescent

Challenge of the β2Ar (β2-adrenergic receptor) with isoprenaline in HEK-293β2 cells (human embryonic kidney cells stably overexpressing a FLAG- and green fluorescent protein-tagged β2Ar) results in the PKA (cAMP-dependent protein kinase) phosphorylation of GRK2 (G-protein receptor kinase-2). This response was enhanced when PDE4 (phosphodiesterase-4) activity was attenuated using either rolipram, a PDE4-selective inhibitor, or with siRNA (small interfering RNA) knockdown of both PDE4B and PDE4D. Rolipram also facilitated GRK2 recruitment to the membrane and phosphorylation of the β2Ar by GRK2 in response to isoprenaline challenge of cells. In resting cells, rolipram treatment alone is sufficient to promote PKA phosphorylation of GRK2, with consequential effects on GRK2 translocation and GRK2 phosphorylation of the β2Ar. Similar effects are observed in cardiac myocytes. We propose that PDE4 activity protects GRK2 from inappropriate phosphorylation by PKA in resting cells that might have occurred through fluctuations in basal cAMP levels. Thus PDE4 gates the action of PKA to phosphorylate GRK2.

scholarly journals Phosphodiesterase-4 influences the PKA phosphorylation status and membrane translocation of G-protein receptor kinase 2 (GRK2) in HEK-293β2 cells and cardiac myocytes

2006 ◽  
Vol 394 (2) ◽  
pp. 427-435 ◽  
Author(s):  
Xiang Li ◽  
Elaine Huston ◽  
Martin J. Lynch ◽  
Miles D. Houslay ◽  
George S. Baillie
Keyword(s):  
G Protein ◽  
Cardiac Myocytes ◽  
Fluorescent Protein ◽  
Selective Inhibition ◽  
Receptor Kinase ◽  
Membrane Translocation ◽  
Membrane Recruitment ◽  
Phosphodiesterase 4 ◽  
Protein Receptor ◽  
Adrenoceptor Agonist

Membrane-recruitment of GRK2 (G-protein receptor kinase 2) provides a fundamental step in the desensitization process controlling GPCRs (G-protein-coupled receptors), such as the β2AR (β2-adrenergic receptor). In the present paper, we show that challenge of HEK-293β2 [human embryonic kidney cells stably overexpressing the FLAG-tagged β2AR–GFP (green fluorescent protein)] cells with the β-adrenoceptor agonist, isoprenaline, causes GRK2 to become phosphorylated by PKA (cAMP-dependent protein kinase). This action is facilitated when cAMP-specific PDE4 (phosphodiesterase-4) activity is selectively inactivated, either chemically with rolipram or by siRNA (small interfering RNA)-mediated knockdown of PDE4B and PDE4D. PDE4-selective inhibition by rolipram facilitates the isoprenaline-induced membrane translocation of GRK2, phosphorylation of the β2AR by GRK2, membrane translocation of β-arrestin and internalization of β2ARs. PDE4-selective inhibition also enhances the ability of isoprenaline to trigger the PKA phosphorylation of GRK2 in cardiac myocytes. In the absence of isoprenaline, rolipram-induced inhibition of PDE4 activity in HEK-293β2 cells acts to stimulate PKA phosphorylation of GRK2, with consequential effects on GRK2 membrane recruitment and GRK2-mediated phosphorylation of the β2AR. We propose that a key role for PDE4 enzymes is: (i) to gate the action of PKA on GRK2, influencing the rate of GRK2 phosphorylation of the β2AR and consequential recruitment of β-arrestin subsequent to β-adrenoceptor agonist challenge, and (ii) to protect GRK2 from inappropriate membrane recruitment in unstimulated cells through its phosphorylation by PKA in response to fluctuations in basal levels of cAMP.

mAKAP: an A-kinase anchoring protein targeted to the nuclear membrane of differentiated myocytes

1999 ◽  
Vol 112 (16) ◽  
pp. 2725-2736 ◽  
Author(s):  
M.S. Kapiloff ◽  
R.V. Schillace ◽  
A.M. Westphal ◽  
J.D. Scott
Keyword(s):  
Protein Kinase ◽  
Nuclear Membrane ◽  
Fluorescent Protein ◽  
Dependent Protein Kinase ◽  
Repeat Sequences ◽  
Camp Dependent Protein Kinase ◽  
Anchoring Proteins ◽  
Anchoring Protein ◽  
Dependent Protein ◽  
Green Fluorescent

The compartmentalization of second messenger-activated protein kinases contributes to the fidelity of hormone-mediated signal transduction events. For example, the cAMP-dependent protein kinase is tethered at specific intracellular locations through association with A-kinase anchoring proteins (AKAPs). We now report the cloning of mAKAP, an anchoring protein found predominantly in heart, skeletal muscle and brain, and whose expression is induced in neonatal ventriculocytes by treatment with hypertrophic stimuli. mAKAP is targeted to the nuclear membrane of differentiated myocytes. Analysis of mAKAP-green fluorescent protein (GFP) fusion constructs revealed that nuclear membrane targeting is conferred by two regions of the protein, between residues 772–915 and 915–1065, which contain spectrin-like repeat sequences. Heterologous expression of the mAKAP targeting sequences displaced the endogenous anchoring protein from the nuclear membrane, demonstrating that mAKAP targeting is saturable. Collectively, these data suggest that a domain containing spectrin-like repeats mediates targeting of the anchoring protein mAKAP and the cAMP-dependent protein kinase holoenzyme to the nuclear membrane in response to differentiation signals.

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