β-Adrenergic regulation of cardiac type 2A protein phosphatase through phosphorylation of regulatory subunit B56δ at S573

Background: Type 2A protein phosphatase (PP2A) enzymes are serine/threonine phosphatases which comprise a scaffold A subunit, a regulatory B subunit and a catalytic C subunit, and have been implicated in the dephosphorylation of multiple cardiac phosphoproteins. B subunits determine subcellular targeting, substrate specificity and catalytic activity, and can themselves be regulated by post-translational modifications. We explored potential beta-adrenergic regulation of PP2A in cardiomyocytes through phosphorylation of the regulatory B subunit isoform B56 delta. Methods and results: Phosphate affinity SDS-PAGE and immunoblot analysis revealed increased phosphorylation of B56 delta in adult rat ventricular myocytes (ARVM) exposed to the beta-adrenergic receptor (beta AR) agonist isoprenaline (ISO). Phosphorylation of B56 delta occurred at S573, primarily through stimulation of the beta(1)AR subtype, and was dependent on PKA activity. The functional role of the phosphorylation was explored in ARVM transduced with adenoviruses expressing wild type (WT) or non-phosphorylatable (S573A) B56 delta, fused to GFP at the N-terminus. C subunit expression was increased in ARVM expressing GFP-B56 delta-WT or GFP-B56 delta-S573A, both of which co-immunoprecipitated with endogenous C and A subunits. PP2A activity in cell lysates was increased in response to ISO in ARVM expressing GFP-B56 delta-WT but not GFP-B56 delta-S573A. Immunoblot analysis of the phosphoproteome in ARVM expressing GFP-B56 delta-WT or GFP-B56 delta-S573A with antibodies detecting (i) phospho-serine/threonine residues in distinct kinase substrate motifs or (ii) specific phosphorylated residues of functional importance in selected proteins revealed a comparable phosphorylation profile in the absence or presence of ISO stimulation. Conclusions: In cardiomyocytes, beta AR stimulation induces PKA-mediated phosphorylation of the PP2A regulatory subunit isoform B56 delta at 5573, which increases associated PP2A catalytic activity. This is likely to regulate the phosphorylation status of specific B56 delta-PP2A substrates, which remain to be identified.