Cypher Is A Novel A-kinase Anchoring Protein In The Heart

Aims and SignificancePKA is known as a cAMP-dependent protein kinase, which acts diverse physiological functions including regulation of glycogen, sugar, and lipid metabolism.In cardiomyocytes, the cAMP/PKA signaling initiated by β-adrenergic receptors (β-ARs) is of central importance for all functions, including excitation-contraction coupling, metabolism, hypertrophy and survival. The PKA holoenzyme is a heterotetramer containing two catalytic (C) and two regulatory (R) subunits. Binding of the second-messenger cAMP to the regulatory subunits releases the catalytic subunits of PKA, which catalyze substrate to be phosphorylated at either the serine or threonine residues.In addition, the regulatory subunits regulate the cellular localization of PKA by binding to a special group of proteins:A kinase anchoring proteins (AKAPs). AKAP anchors the phosphorylation reaction at a specific position, and the AKAP guides kinase near the substrate, which improves the efficiency of phosphorylation of kinase substrates. To date over20AKAPs are found in the heart, and they regulate the physiological function of heart by forming a new regulating network. PKA-mediated phosphorylation induced by β-AR stimulation is also important for myofilament contraction and torsion that include cardiac Troponin I (cTnI), Myosin binding protein C (MyBP-C), Titin and Myosin light chain. The phosphorylation of cTnI and cMyBP-C leads to decreased calcium responsiveness and thereafter increases the myofibril relaxation rate. Some cardiac AKAPs have been shown to localize at sarcomeres, such as Synemin, cardiac Troponin T (cTnT), Myospryn and Myomegalin, but their functions are unclear.Cypher, also known as ZASP in human, a member of PDZ-LIM domain protein family, is a striated Z-line protein, which plays an important structural role in cardiac muscle to maintain the integrity of sarcomeres under the stress of contraction force. Cardiac-specific ablation of Cypher leads to a severe form of dilated cardiomyopathy with premature death. Till now,16Cypher/ZASP mutations are identified in patients with myopathies including dilated cardiomyopathy. However the pathologic mechanisms remain unknown. By analyzing the secondary structure of the Cypher lc amino-acid sequence, we found that the fragment200-217could form an amphipathic helix, a feature of AKAPs. The finding that Cypher is in the signaling complex expands our knowledge about the structural protein Cypher. It also is in line with the concept that sarcomeric Z-lines are not only structural borders for sarcomeres, but also signaling centers, especially for mechanic signaling.2MethodsBy analysis of bioinformatics there is a potential structure of amphipathic heliex in Cypher. Cypherr isforms and PKA regulatory subunit are co-expressed respectively in HEK293cells. By using the method of truncating mutation we identify the region combined with PKA subunits RⅡα. There are two classic sites that can be phosphorylated by PKA in Cypher. Variants are expressed in HEK293cells, and purified proteins are assayed for phosphorylation by phosph-(Ser/Thr) PKA substrate antibody. Neonatal mice were given isoproterenol at different time points before the hearts are dissected and lysates are analyzed for LTCC and its phosphorylation. Calcineurin enzymatic activities are detected respectively by co-immunoprecipitation and enzyme activity kit.3. Results3.1Cypher/ZASP interacted with RⅡα regulatory subunit of PKA. Using overexpression and immunoprecipitation, we identified that the CCSR of Cypher interacted specifically with the D/D domain of PKA RⅡα. Both proline substitutions and the deletion of AA200-217significantly decreased Cypher binding to PKARⅡα. Cypher interacted with the D/D domain of RⅡα.3.2Cardiomyopathy-associated mutation T206I (T203I in mouse) enhanced the Cypher-PKA interaction.3.3Cypher/ZASP was phosphorylated by PKA at Ser265and Ser296. The single mutation either S265A or S296A significantly decreased the phosphorylated Cypher in forskolin-stimulated HEK293cells, while the double-mutation S265/296A completely eliminated the phosphorylation.3.4Without stimulation, the expression of Cypher slightly increased the phosphorylated LTCC, while with forskolin stimulation the phosphorylated LTCC was increased further in HEK293cells. Disrupting the interaction between LTCC and Cypher by either deletion of the C-tail four residues of LTCC or deletion of the Cypher PDZ domain significantly impaired the PKA-mediated phosphorylation of the LTCC. Isoproterenol injection dramatically increased the phosphorylation of LTCC at Ser1928in WT mouse hearts, and this was blunted in Cypher-null hearts.3.5Cypher and CaN were in the same complex. And deletion of Cypher in mouse hearts did not change the expression level of CaN, while more CaM protein interacted with CaN indicating the enzymatic activity of CaN was enhanced.4. Conclusions4.14.1The interaction of Cypher and PKA regulatory subunit is based on the amphipathic helix of the Cypher and the D/D domain of the PKA regulatory subunit RⅡα. Thus Cypher is a typical AKAP. The molecular pathological mechanism of mutant T206I might enhance the interaction of Cypher and PKA regulatory subunit.4.2Cypher is a substrate for PKA. Two serine residues, Ser265and Ser296, can be phosphorylated by PKA.4.3Cypher facilitates PKA-mediated phosphorylation of L-type calcium channel, which is dependent on the interaction between the PDZ domain of Cypher and C-terminus of LTCC.4.4Cypher binds to Calcineurin, inhibiting Calcineurin enzymatic activity.