An anchored PKA and PDE4 complex regulates subplasmalemmal cAMP dynamics.
The spatiotemporal regulation of cAMP can generate microdomains just beneath the plasma membrane where cAMP increases are larger and more dynamic than those seen globally. Real-time measurements of cAMP using mutant cyclic nucleotide-gated ion channel biosensors, pharmacological tools and RNA interference (RNAi) were employed to demonstrate a subplasmalemmal cAMP signaling module in living cells. Transient cAMP increases were observed upon stimulation of HEK293 cells with prostaglandin E1. However, pretreatment with selective inhibitors of type 4 phosphodiesterases (PDE4), protein kinase A (PKA) or PKA/A-kinase anchoring protein (AKAP) interaction blocked an immediate return of subplasmalemmal cAMP to basal levels. Knockdown of specific membrane-associated AKAPs using RNAi identified gravin (AKAP250) as the central organizer of the PDE4 complex. Co-immunoprecipitation confirmed that gravin maintains a signaling complex that includes PKA and PDE4D. We propose that gravin-associated PDE4D isoforms provide a means to rapidly terminate subplasmalemmal cAMP signals with concomitant effects on localized ion channels or enzyme activities.
Willoughby
Debbie
D
Department of Pharmacology, University of Cambridge, Cambridge, UK.
Wong
Wei
W
Schaack
Jerome
J
Scott
John D
JD
Cooper
Dermot M F
DM
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000235:genetics / Q000378:metabolism*; D054758:A Kinase Anchor Proteins; D000527:Alprostadil / Q000378:metabolism; D000818:Animals; D015374:Biosensing Techniques; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D054815:Cyclic Nucleotide-Gated Cation Channels; D006801:Humans; D007473:Ion Channels / Q000235:genetics / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D051379:Mice; D046912:Multiprotein Complexes; D018408:Patch-Clamp Techniques; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D051381:Rats; D011943:Receptors, Adrenergic, beta / Q000235:genetics / Q000378:metabolism; D015290:Second Messenger Systems / Q000502:physiology*
D054758:A Kinase Anchor Proteins; C491162:AKAP12 protein, human; D018797:Cell Cycle Proteins; D054815:Cyclic Nucleotide-Gated Cation Channels; D007473:Ion Channels; D007527:Isoenzymes; D046912:Multiprotein Complexes; D021122:Protein Subunits; D011943:Receptors, Adrenergic, beta; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; C517376:PDE4D protein, human; D000527:Alprostadil