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Abstract
PKA signaling is important for the post-translational modification of proteins, especially those in cardiomyocytes involved in cardiac excitation-contraction coupling. PKA activity is spatially and temporally regulated through compartmentalization by protein kinase A anchoring proteins. Cypher/ZASP, a member of PDZ-LIM domain protein family, is a cytoskeletal protein that forms multiprotein complexes at sarcomeric Z-lines. It has been demonstrated that Cypher/ZASP plays a pivotal structural role in the structural integrity of sarcomeres, and several of its mutations are associated with myopathies including dilated cardiomyopathy. Here we show that Cypher/ZASP, interacting specifically with the type II regulatory subunit RIIα of PKA, acted as a typical protein kinase A anchoring protein in cardiomyocytes. In addition, we show that Cypher/ZASP itself was phosphorylated at Ser(265) and Ser(296) by PKA. Furthermore, the PDZ domain of Cypher/ZASP interacted with the L-type calcium channel through its C-terminal PDZ binding motif. Expression of Cypher/ZASP facilitated PKA-mediated phosphorylation of the L-type calcium channel in vitro. Additionally, the phosphorylation of the L-type calcium channel at Ser(1928) induced by isoproterenol was impaired in neonatal Cypher/ZASP-null cardiomyocytes. Moreover, Cypher/ZASP interacted with the Ser/Thr phosphatase calcineurin, which is a phosphatase for the L-type calcium channel. Taken together, our data strongly suggest that Cypher/ZASP not only plays a structural role for the sarcomeric integrity, but is also an important sarcomeric signaling scaffold in regulating the phosphorylation of channels or contractile proteins.
Highlights
Cypher/ZASP plays an essential structural role in cardiac muscle
Cypher/ZASP interacted with the Ser/Thr phosphatase calcineurin, which is a phosphatase for the L-type calcium channel
By analyzing the secondary structure of the Cypher1c amino acid sequence (AF114378_1), we found that the fragment AA200 –217 could potentially form an amphipathic helix (Fig. 1A), a feature of A-kinase anchoring proteins (AKAPs)
Summary
Cypher/ZASP plays an essential structural role in cardiac muscle. Results: Cypher/ZASP interacted with PKARII␣ and calcineurin. Conclusion: Cypher/ZASP is a novel AKAP acting as a sarcomeric signaling center for potential phosphorylation regulation the function of channels and myofilament proteins. We show that Cypher/ZASP, interacting with the type II regulatory subunit RII␣ of PKA, acted as a typical protein kinase A anchoring protein in cardiomyocytes. Cypher/ZASP is a striated Z-line protein, which plays an important structural role in cardiac muscle in maintaining the integrity of sarcomeres under the stress of contraction force [17,18,19,20]. We report that the Z-line protein Cypher/ZASP is a typical type II AKAP that interacts with the RII␣ regulatory subunit of PKA and the Ser/Thr phosphatase CaN, making Cypher/ZASP-PKA-CaN a signaling center for sarcomeric proteins or channels such as the L-type calcium channel (LTCC)
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