PKA‐induced Interaction of Phosphorylated PDE3A (pPDE3A) and BIG1 (Brefeldin A‐inhibited guanine nucleotide exchange protein 1) in Cytosolic Fractions from Human Myocardium may be Important in Regulation of BIG1 Function

BIG1 and BIG2 proteins, which contain A‐kinase anchoring protein (AKAP) motifs, catalyze activation of class I ARFs (ADP‐ribosylation factors) by accelerating exchange of bound GDP for GTP (GEF activity). PKA‐catalyzed phosphorylation of BIG inhibits GEF activity. In Hela cells, PDE3A interacts with BIG proteins (PNAS 106, 6158, 2009). Three PDE3A isoforms (PDE3A1‐3), with identical sequences except for N‐terminal deletions of different lengths, are present in human myocardium. PDE3A1 is found exclusively in membranes; PDE3A2‐3, in both membrane and cytosolic fractions. Non‐phosphorylated cytosolic PDE3A2‐3 eluted from Superose 6 (S6) in LMW (low molecular weight) peaks(~800 kD). Incubation of cytosol with PKA and ATP induced assembly of macromolecular signalosomes that eluted from S6 as HMW (high molecular weight) (>3000 kD) complexes and contained pPDE3A (predominantly pPDE3A2) and proteins involved in cAMP signaling (i.e., PKA‐RII, PP2A, 14‐3‐3, BIG1). These molecules co‐immunoprecipitated with pPDE3A in HMW, but not with non‐phosphorylated PDE3A in LMW fractions. These data suggest that, in cardiomyocytes, pPDE3A is incorporated into BIG1‐organized signalosomes, and in these spatially restricted signalosomes, pPDE3A may modulate compartmentalized cAMP/PKA signaling and thereby contribute to regulation of BIG1 activity and ARF function.