Bhasin N, Cunha SR, Mudannayake M, Gigena MS, Rogers TB, Mohler PJ. Molecular basis for PP2A regulatory subunit B56␣ targeting in cardiomyocytes. Am J Physiol Heart Circ Physiol 293: H109-H119, 2007. First published April 6, 2007; doi:10.1152/ajpheart.00059.2007.-Protein phosphatase 2A (PP2A) is a multifunctional protein phosphatase with critical roles in excitable cell signaling. In the heart, PP2A function is linked with modulation of -adrenergic signaling and has been suggested to regulate key ion channels and transporters including Na/Ca exchanger, ryanodine receptor, inositol 1,4,5-trisphosphate receptor, and Na/K ATPase. Although many of the functional roles and molecular targets for PP2A in heart are known, little is established regarding the cellular pathways that localize specific PP2A isoform activities to subcellular sites. We report that the PP2A regulatory subunit B56␣ is an in vivo binding partner for ankyrin-B, an adapter protein required for normal subcellular localization of the Na/Ca exchanger, Na/K ATPase, and inositol 1,4,5-trisphosphate receptor. Ankyrin-B and B56␣ are colocalized and coimmunoprecipitate in primary cardiomyocytes. Using multiple strategies, we identified the structural requirements on B56␣ for ankyrin-B association as a 13 residue motif in the B56␣ COOH terminus not present in other B56 family polypeptides. Finally, we report that reduced ankyrin-B expression in primary ankyrin-B ϩ/Ϫ cardiomyocytes results in disorganized distribution of B56␣ that can be rescued by exogenous expression of ankyrin-B. These new data implicate ankyrin-B as a critical targeting component for PP2A in heart and identify a new class of signaling proteins targeted by ankyrin polypeptides. cytoskeleton; trafficking; phosphatase EFFICIENT EXCITABLE CELL FUNCTION requires precisely orchestrated signaling pathways to modulate the function of diverse membrane, cytosolic, and nuclear proteins. Specifically, the collaboration between finely tuned protein phosphorylation/ dephosphorylation pathways is critical for neuronal communication and cardiac excitation-contraction coupling. Findings over the past decade clearly demonstrate that a local organization of kinase and phosphatase proteins with specific effector protein pathways facilitates efficient signaling. This local organization may occur by direct interaction of kinases and phosphatases with anchoring or scaffolding proteins, cytoskeleton, or even by direct interaction with target ion channels, transporters, or receptors (21,36,42,44,67). However, the cellular pathways underlying the biogenesis/targeting of kinases or phosphatases to specialized membrane protein complexes are not clearly resolved.Protein phosphatase 2A (PP2A) is a multifunctional serine/ threonine phosphatase with critical roles in ion channel/transporter regulation (3, 34, 41, 88), Wnt signaling (39, 63, 72, 84), transformation (2, 6, 27, 75), cell polarization (23), circadian rhythm (5, 68, 85), and cell survival and apoptosis (37,40,64,73,76,81). In heart, PP2A function is neces...