Oxidative stress employs phosphatidyl inositol 3-kinase and ERK signalling pathways to activate cAMP phosphodiesterase-4D3 (PDE4D3) through multi-site phosphorylation at Ser239 and Ser579.
RAW macrophages, which express the PDE4D3 and PDE4D5 cAMP phosphodiesterase isoforms, exhibited increased PDE4 activity when challenged with H2O2 in a fashion that was negated by treatment with the cell permeant antioxidant, N-acetyl cysteine and by diphenyleneiodonium chloride, an inhibitor of NADPH oxidase. In Cos1 cells transfected to express PDE4D3, challenge with H2O2 caused a rapid increase in both the activity and phosphorylation of PDE4D3. Lysates from H2O2-treated COS cells caused the phosphorylation of purified, recombinant PDE4D3 at two sites. One was the established ERK phosphorylation site at Ser579, located at the extreme C-terminus of the catalytic unit, and the other was a novel site at Ser239, located at the extreme N-terminus of the catalytic unit. Double Ser239Ala:Ser579Ala mutation of PDE4D3 prevented its H2O2-dependent phosphorylation both in vitro and in intact COS cells. Phosphorylation of PDE4D3 at Ser579 was ablated by treating COS cells with the MEK inhibitor, PD98059, which also negated activation. The activity of the Ser239Ala:Ser579Ala double mutant, and the Ser579Ala single PDE4D3 mutant was unaffected by H2O2 challenge of COS cells, whilst the Ser239Ala mutant was inhibited. Wortmannin inhibited the H2O2-dependent phosphorylation of PDE4D3 in COS cells by around 50%, whilst it fully ablated phosphorylation at Ser239 as well as ablating activation of PDE4D3. Neither immunodepletion of p70S6 kinase nor siRNA-mediated knockdown of mTor inhibited the H2O2-dependent phosphorylation of PDE4D3 at Ser239. Activation of PDE4D3 by challenge with H2O2 was not additive with activation through protein kinase A (PKA)-mediated phosphorylation of PDE4D3. Challenge with H2O2 did not alter PKA-mediated phosphorylation of PDE4D3 at Ser54. H2O2 dependent phosphorylation of PDE4D3, at Ser239 and Ser579, did not alter the sensitivity of PDE4D3 to inhibition by the selective PDE4 inhibitor, rolipram. An unknown protein kinase acting downstream of phosphatidyl inositol 3-kinase phosphorylates PDE4D3 at Ser239. This switches the effect of phosphorylation by ERK at Ser579 from inhibition to activation. We propose that phosphorylation at Ser239 attenuates interaction between either UCR2 or the UCR1/UCR2 module and the PDE4 catalytic unit so as to re-programme the functional outcome effect of phosphorylation by ERK. We identify a novel process through which reactive oxygen species activate long PDE4 isoforms so as to reduce cAMP levels and thereby promote inflammatory responses.
Hill
Elaine V
EV
Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, UK.
Sheppard
Catherine L
CL
Cheung
York-Fong
YF
Gall
Irene
I
Krause
Eberhard
E
Houslay
Miles D
MD
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002460:Cell Line; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D004789:Enzyme Activation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D051379:Mice; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D012694:Serine / Q000737:chemistry* / Q000378:metabolism; D015398:Signal Transduction
D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D048049:Extracellular Signal-Regulated MAP Kinases; D008070:Lipopolysaccharides; D016877:Oxidants; D019869:Phosphatidylinositol 3-Kinases; D010726:Phosphodiesterase Inhibitors; D012694:Serine