Phosphorylation-dependent PDE3A and BIG1 interactions may be important in regulation of BIG1 function in cytosolic fractions of human myocardium

Wednesday, October 10, 2012 — Poster Session II
Noon – 2:00 p.m	Natcher Conference Center, Building 45	NHLBI	SIG-3

Authors

• F.A. Khan
• J Krall
• J Moss
• M. Vaughan
• M Movsesian
• V.C. Manganiello

Abstract

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 (Brefeldin A-inhibited guanine nucleotide exchange protein) 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.

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