Numerous distinct PKA-, or EPAC-based, signalling complexes allow selective phosphodiesterase 3 and phosphodiesterase 4 coordination of cell adhesion.
By activating two distinct classes of effector enzymes, namely Protein Kinases A [PKA] or Exchange Proteins Activated by cAMP [EPAC], the ubiquitous second messenger cAMP selectively coordinates numerous events simultaneously in virtually all cells. Studies focused on dissecting the manner by which cAMP simultaneously regulates multiple cellular events have shown that cAMP activates its effectors non-uniformly in cells and that this localized cAMP-mediated signalling is made possible, at least in part, by anchoring of cAMP effectors to selected subcellular structures. In the work described here, we report that HEK293T cells ["293T"] contain several PKA- and EPAC1-based signalling complexes. Interestingly, our data do not identify signalling complexes in which both PKA and EPAC are each present but rather are consistent with the idea that these two effectors operate in distinct complexes in these cells. Similarly, we report that while individual PKA- or EPAC-containing complexes can contain either phosphodiesterase 3B, [PDE3B] or phosphodiesterase 4D [PDE4D], they do not contain both these phosphodiesterases. Indeed, although PDE4D enzymes were identified in both PKA- and EPAC-based complexes, PDE3B was largely identified in EPAC-based complexes. Using a combination of approaches, we identified that integration of PDE3B into EPAC-based complexes occurred through its amino terminal fragment [PDE3B(AT)]. Consistent with the idea that integration of PDE3B within EPAC-based complexes was dynamic and regulated PDE3 inhibitor-mediated effects on cellular functions, expression of PDE3B(AT) competed with endogenous PDE3B for integration into EPAC-based complexes and antagonized PDE3 inhibitor-based cell adhesion. Our data support the concept that cells can contain several non-overlapping PKA- and EPAC-based signalling complexes and that these complexes may also represent sites within cells were the effects of family-selective PDE inhibitors could be integrated to affect cell functions, including adhesion.
Raymond
Daniel R
DR
Department of Pharmacology & Toxicology, Queen's University, Kingston, ON, Canada K7L 3N6.
Wilson
Lindsay S
LS
Carter
Rhonda L
RL
Maurice
Donald H
DH
D012368:4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000378:metabolism; D002448:Cell Adhesion / Q000187:drug effects; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D020536:Enzyme Activators / Q000494:pharmacology; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D046912:Multiprotein Complexes / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D058987:Phosphodiesterase 3 Inhibitors; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D015363:Quinolones / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection
C473217:8-(4-chloro-phenylthio)-2'-O-methyladenosine-3'-5'-cyclic monophosphate; D020536:Enzyme Activators; D020662:Guanine Nucleotide Exchange Factors; D046912:Multiprotein Complexes; D010446:Peptide Fragments; D058987:Phosphodiesterase 3 Inhibitors; D010726:Phosphodiesterase Inhibitors; D015363:Quinolones; C116122:RAPGEF3 protein, human; D005576:Colforsin; D012368:4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone; C050086:N(6)-benzoyl-cyclic AMP; C021294:cilostamide; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; C517291:PDE3B protein, human; C517376:PDE4D protein, human; D000262:Adenylyl Cyclases