Changes in heart rate and contractility in response to sympathetic activation occur via activation of cAMP dependent protein kinase A (PKA) leading to phosphorylation of Flumatinib mesylate numerous substrates that alter Ca2+ cycling. PKA substrates regardless of the state of the complex. If true such a mechanism would require an abundance of both AKAP and its binding partners that is similar to that of the PKA substrates – but the cellular concentration of PKA is not nearly as high as PLB nor are the concentrations of AKAP7??γ or its other binding partners (Protein phosphatase 1 Inhibitor-1 and Phosphodiesterase 3A) [12 15 However it is usually clear Flumatinib mesylate that this AKAP plays an important role in the phosphorylation of PLB . Here we confirm that binding of PKA to AKAP7δ/γ is required for PLB phosphorylation and that deletion of the PKA binding Rabbit Polyclonal to Uba2. domain name around the AKAP7 results in a significant reduction in PLB phosphorylation. Importantly several human mutants of PLB which are known to exhibit decreased phosphorylation and are associated with dilated cardiomyopathy do not interact with AKAP7δ/γ further suggesting that this PLB/AKAP7δ/γ is necessary for phosphorylation. Therefore the question remains: how are these AKAP-binding requirements compatible with the efficient phosphorylation of large amounts of PLB? We hypothesized that this could be explained by our newly observed phosphorylation state-dependent binding of AKAP7δ/γ to PLB. Here we show that this high affinity association between AKAP7δ/γ and PLB is usually lost upon phosphorylation of PLB. A computation model of the detailed biochemical kinetics of the pathway showed that if state-dependent binding is included in the reaction network phosphorylation of high concentrations of PLB is possible at low concentrations of both AKAP7γ and PKA consistent with the observed results . Importantly our experimental findings and kinetic analysis provide a mechanistic hypothesis of AKAP7δ/γ complex signaling in cardiac myocytes that reconciles the problem with disparity of complex component concentrations. 2 Experimental Methods 2.1 Antibodies The following primary antibodies were utilized for immunoblotting: mouse monoclonal Phospholamban (Millipore; 1:1000 dilution) polyclonal phosphor-phospholaman serine 16 (Millipore; 1:500) mouse monoclonal GFP (Santa Cruz Biotechnology; 1:500 dilution) polyclonal mCherry (Thermo Scientific Pierce; Flumatinib mesylate 1:3000 dilution) monoclonal PKA RIIα subunit (Santa Cruz Biotechnology; 1:500). Immunoprecipitations were carried out using the following antibodies: polyclonal AKAP7 (Sigma; 5 μg) mouse monoclonal GFP (Santa Cruz Biotechnology; 5 μg) mouse monoclonal Phospholamban (Millipore; 3 μg) 2.2 Expression constructs The human phospholamban construct was obtained Origene and amended with EcoRI/BamHI restriction sites using PCR and subcloned into the peGFP-N1 Flumatinib mesylate vector. Mutant phospholamban constructions were made by site directed mutagenesis. 2.3 Cell Transfection and Immunoprecipitation HEK293 cells were transfected at 50-70% confluency in 60 mm plates using the calcium phosphate method with 6 μg of each plasmid DNA. Cells were treated with numerous drugs for the time given and cell lysate was collected in 0.5 ml HSE buffer (HEPES pH 7.4 150 mM NaCl 5 mM EDTA 1 Triton X-100 and protease inhibitors). Supernatants were incubated overnight at 4°C with Flumatinib mesylate the indicated antibody and 15 μl of prewashed protein A-or G-agarose. Following considerable washing captured proteins were solubilized in 2X sample buffer and analyzed by immunoblot. Rat heart extract was prepared as previously explained [20 21 Immunoprecipitating antibodies were added to 500 μl of extract along with 13 μl protein agarose. After an immediately incubation followed by considerable washing captured proteins were analyzed by immunoblot. 2.4 In vitro Phospholamban phosphorylation assays Various PLB peptides (1 μg) were incubated in kinase buffer (50 mM Tris-HCL pH 7.5 5 mM MgCl2) containing 100 μM ATP 5 μM [γ?32]ATP and Flumatinib mesylate 800 models of purified PKA catalytic subunit (NEB). After a 15 minute incubation at 30°C the reaction mixture was spotted onto phosphocellulose strips and washed five occasions in 75 mM phosphoric acid. Filters were air flow dried and counted. 2.5 Rat neonatal myocyte culture Myocytes were prepared from 2 day old Sprague-Dawley rats as previously explained. Cell were plated in Dulbecco’s Modified Eagle medium (DMEM)with 17% Media 199 1 penicillin/streptomycin answer 10 horse serum and 5% fetal bovine serum (FBS) at 125 ooo per cm2. After an immediately incubation in plating medium the myocytes were maintained in culture for up.