Cypher/ZASP is a novel A-kinase anchoring protein

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.

Keywords: Akap; Calcineurin; Calcium Channels; Cypher/ZASP; Phosphorylation; Protein Kinase A (PKA).

FIGURE 1.

Cypher interacted directly with RIIα regulatory subunit of PKA. A, an amphipathic helical wheel plot for amino acid residues AA200–217 of Cypher1c (AF114378_1) reveals a hydrophobic surface on one side (shaded). B–D, GST-tagged Cypher1c (B), Cypher2c (C), or Cypher cardiac-specific region (CCSR, AA108–227) (D) and FLAG-tagged PKA regulatory subunits (RIα, RIβ, RIIα, RIIβ) were co-expressed in HEK293 cells. FLAG-tagged proteins were immunoprecipitated with anti-FLAG antibody, and interacting proteins were verified with anti-GST antibody in immunoblot (IB). IP, immunoprecipitation. E, GST-tagged Cypher CCSR and its variants with an introduced proline (M1, T203P; M2, L204P; M3, L215P) were co-transfected with FLAG-tagged RIIα (RIα as a negative control) in HEK293 cells. Cell lysates were used for immunoprecipitation to verify the interaction. F, FLAG-tagged Cypher or its AA200–217 deletion mutant (ΔAA200–217) was co-expressed with GST-tagged RIIα. Cell lysates were used for immunoprecipitation to verify the interaction. G, FLAG-tagged Cypher was co-expressed with GST-tagged RIIα full-length (FL), its D/D (AA1–45), or its D/D deletion mutant (Δ45) in HEK293 cells. Cell lysates were used for immunoprecipitation to verify the interaction. H, FLAG-tagged Cypher, ENH, ALP, and CLP36 were co-expressed with GST-tagged RIIα (RIα as a negative control) in HEK293 cells. Protein interactions were determined by immunoprecipitation. I, Cypher protein was immunoprecipitated from neonatal mouse heart lysate by anti-Cypher antibody, and the interaction with PKA RII protein was verified by anti-PKARII antibody. Rabbit IgG and Cypher-null heart lysate were used as controls.

FIGURE 2.

Subcellular localization of Cypher (A and B) and PKARII (C and D) in heart tissues of adult mice. A and B, Cypher localized at Z lines (arrows) in mouse cardiomyocytes as judged by α-actinin co-staining. Z line localization of Cypher was not perturbed by treatment of mice with isoproterenol; however, Cypher showed additionally slight intercalated disk localization (arrowheads) in treated hearts when compared with controls (compare inset in A and B). C and D, PKARII was found to localize at intercalated discs (arrowheads) in control hearts, as judged by plakoglobin counterstaining. Upon isoproterenol treatment, PKARII was found to localize in a more pronounced way to Z lines (arrows), in addition to its intercalated disk localization (compare inset in C and D). Sarcomeric α-actinin was used to mark Z-lines (arrows); plakoglobin was use to mark cardiac intercalated disks (arrowheads). Scale bars: 20 μm.

FIGURE 3.

Cardiomyopathy-associated Cypher/ZASP mutation enhanced the PKA-Cypher interaction. A, GST-tagged human ZASP exon 4 encoded fragment and two mutants (S189L and T206I) were co-transfected with FLAG-tagged PKA RIIα (RIα as a control) in HEK293 cells. Protein interactions were assayed by immunoprecipitation (IP). IB, immunoblotting. B, stick models of RIIα D/D-Cypher Glu²⁰²–Leu²¹⁵ (WT, left; T203I mutant, right) complex (RII D/D dimer at the back in light brown and gray, Cypher Glu²⁰²–Leu²¹⁵ at the front in green) superimposed onto the RIIα D/D-AKAP-IS structure. C, surface representation of the Cypher helix (Glu²⁰²–Leu²¹⁵) (WT, left; T203I mutant, right) with the highlighted hydrophobic residues (yellow) involved in PKA RIIα binding.

FIGURE 4.

Cypher/ZASP tethered PKA to phosphorylate the LTCC cytosolic region at Ser¹⁹²⁸in vitro. A, the Cypher PDZ domain interacted with the LTCC C-terminal PDZ binding motif (VSSL). Myc-tagged Cypher or its PDZ domain deletion mutant (ΔPDZ) was co-expressed with FLAG-tagged LTCC cytosolic region (AA1510–2172) or its deletion mutant (ΔVSSL) in HEK293 cells. FLAG-tagged proteins were enriched by anti-FLAG antibody, and interacting proteins were verified by blotting with an anti-Myc antibody. IP, immunoprecipitation; IB, immunoblotting. B, FLAG-tagged LTCC cytosolic region with or without Myc-tagged Cypher was co-expressed in HEK293 cells. Forskolin (100 μm, 30 min) was used to activate PKA. LTCC protein was purified by anti-FLAG antibody, and the phosphorylation was detected by a phospho-(Ser/Thr) PKA substrate antibody. IBMX, 3-isobutyl-1-methylxanthine. C, LTCC and its mutants S1928A and C-terminal VSSL deletion (ΔVSSL) were expressed in HEK293 cells with Cypher. LTCC phosphorylation with or without the PKA agonist forskolin treatment was assessed.

FIGURE 5.

Cypher facilitated the phosphorylation of LTCC by PKA in vivo. A, neonatal rat cardiomyocytes were transfected with Lenti-FLAG-Cypher viruses (or empty vector viruses) and treated with forskolin (100 μm, 30 min). Cell lysates were analyzed for total and phosphorylated LTCC (phospho-Ser¹⁹²⁸-specific antibody (p-S1928)). IBMX, 3-isobutyl-1-methylxanthine. B, heart lysates from 1-day-old wild-type (WT) or Cypher-null mice (KO) were analyzed for LTCC, phosphorylated LTCC, PKA RII subunit, PKA catalytic subunit, and Cypher. C, neonatal mice were given isoproterenol (15 mg/kg in saline, 20 μl) at different time points before the hearts were dissected, and lysates were analyzed for the LTCC and its phosphorylation; the phosphorylation of Erk1/2 was used to show that the PKA signal was activated by isoproterenol. GAPDH was used to show equal protein loading.

FIGURE 6.

Cypher directly interacted with CaN and blocked its activity. A, CaN protein was precipitated from adult mouse heart lysates, and the interaction with Cypher was verified with an anti-Cypher antibody. Rabbit IgG was used for control. IP, immunoprecipitation; IB, immunoblotting. B, GST alone or GST-tagged Cypher1c, 2c, PDZ domain (AA1–80), and LIM domains (AA547–723) were co-transfected with FLAG-tagged CaN in HEK293 cells. Interacting proteins were verified by immunoprecipitation. C, GST alone or GST-tagged Cypher1c, 2c, 2c with PDZ domain deletion (2cΔPDZ), CCSR, and 2c^227–327 was co-transfected with FLAG-tagged CaN in HEK293 cells. Interactions were verified by immunoprecipitation. D, FLAG-tagged CaN and its mutants (Δcat: Δ45–345, ΔAI: Δ455–477) were co-transfected with GST-tagged Cypher1c in HEK293 cells. Protein interactions were verified by immunoprecipitation. E, WT and Cypher-null neonatal heart lysates were probed with an anti-CaN antibody. F, CaN proteins (or CaM) were precipitated from neonatal WT, and Cypher-null hearts and interacting CaM (or CaN) were determined by probing with an anti-CaM antibody (or CaN antibody). G, CaN enzymatic activity from WT and Cypher-null neonatal heart lysates was measured (n = 4). The values were normalized to the average WT value. H, the mRNA levels of modulatory calcineurin-interacting protein 1, the exon4 isoform, MCIP1.4, in 1-day-old WT and Cypher KO mouse hearts (n = 3) measured by real-time PCR. The data are presented as the ratios of MCIP1.4 to 18 S RNA, an internal standard, and were normalized to the WT values. *, p < 0.05. Error bars indicate mean ± S.E.

FIGURE 7.

Cypher was phosphorylated at Ser²⁶⁵ and Ser²⁹⁶ by PKA. A, Cypher Ser²⁶⁵ and Ser²⁹⁶ were potential PKA phosphorylation sites. B, heart lysates from adult mice treated with isoproterenol (Iso, 15 mg/kg, 20 μl) were immunoprecipitated for Cypher, and Cypher phosphorylation was detected by phospho-(Ser/Thr) PKA substrate antibody. C, FLAG-tagged Cypher and its variants Cypher S265A, S296A, and the double mutation S265A/S296A (indicated by S265/S296A) were expressed in HEK293 cells, and purified proteins were assayed for phosphorylation by phospho-(Ser/Thr) PKA substrate antibody.