Proteomic studies of PP2A-B56gamma1 phosphatase complexes reveal phosphorylation-regulated partners in cardiac local signaling.

Defects of kinase-phosphatase signaling in cardiac myocytes contribute to human heart disease. The activity of one phosphatase, PP2A, is governed by B targeting subunits, including B56gamma1, expressed in heart cells. As the role of PP2A/B56gamma1 on the heart function remains largely unknown, this study sought to identify protein partners through unbiased, affinity purification-based proteomics combined with the functional validation.

Molecular basis for PP2A regulatory subunit B56alpha targeting in cardiomyocytes.

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 beta-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.

A B56 regulatory subunit of protein phosphatase 2A localizes to nuclear speckles in cardiomyocytes.

Protein phosphatase 2A (PP2A) is widely distributed in heart tissues, yet its precise cellular functions are poorly understood. This study is based on the notion that PP2A action is governed by interactions of the core enzyme with B targeting/regulatory subunits. The subcellular localizations of two B subunits, B56alpha and B56gamma1, were assessed using adenovirus-driven expression of epitope-tagged (hemagglutinin, HA) in cultured neonatal and adult rat ventricular myocytes.

Effects of PP1/PP2A inhibitor calyculin A on the E-C coupling cascade in murine ventricular myocytes.

Calyculin A was used to examine the importance of phosphatases in the modulation of cardiac contractile magnitude in the absence of any neural or humoral stimulation. Protein phosphatase (PP)1 and PP2A activity, twitch contractions, intracellular Ca(2+) concentration ([Ca(2+)](i)) transients, action potentials, membrane currents, and myofilament Ca(2+) sensitivity were measured in isolated mouse ventricular myocytes. Calyculin A (125 nM) inhibited PP1 and PP2A by 50% and 85%, respectively, whereas it doubled the twitch magnitude and increased twitch duration by 50% in field-stimulated cells.

The cardiac-specific nuclear delta(B) isoform of Ca2+/calmodulin-dependent protein kinase II induces hypertrophy and dilated cardiomyopathy associated with increased protein phosphatase 2A activity.

The delta isoform of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) predominates in the heart. To investigate the role of CaMKII in cardiac function, we made transgenic (TG) mice that express the nuclear delta(B) isoform of CaMKII. The expressed CaMKIIdelta(B) transgene was restricted to the myocardium and highly concentrated in the nucleus.