Multi-tasking role of the mechanosensing protein Ankrd2 in the signaling network of striated muscle

Abstract

Background: Ankrd2 (also known as Arpp) together with Ankrd1/CARP and DARP are members of the MARP mechanosensing proteins that form a complex with titin (N2A)/calpain 3 protease/myopalladin. In muscle, Ankrd2 is located in the I-band of the sarcomere and moves to the nucleus of adjacent myofibers on muscle injury. In myoblasts it is predominantly in the nucleus and on differentiation shifts from the nucleus to the cytoplasm. In agreement with its role as a sensor it interacts both with sarcomeric proteins and transcription factors.

Methodology/principal findings: Expression profiling of endogenous Ankrd2 silenced in human myotubes was undertaken to elucidate its role as an intermediary in cell signaling pathways. Silencing Ankrd2 expression altered the expression of genes involved in both intercellular communication (cytokine-cytokine receptor interaction, endocytosis, focal adhesion, tight junction, gap junction and regulation of the actin cytoskeleton) and intracellular communication (calcium, insulin, MAPK, p53, TGF-β and Wnt signaling). The significance of Ankrd2 in cell signaling was strengthened by the fact that we were able to show for the first time that Nkx2.5 and p53 are upstream effectors of the Ankrd2 gene and that Ankrd1/CARP, another MARP member, can modulate the transcriptional ability of MyoD on the Ankrd2 promoter. Another novel finding was the interaction between Ankrd2 and proteins with PDZ and SH3 domains, further supporting its role in signaling. It is noteworthy that we demonstrated that transcription factors PAX6, LHX2, NFIL3 and MECP2, were able to bind both the Ankrd2 protein and its promoter indicating the presence of a regulatory feedback loop mechanism.

Conclusions/significance: In conclusion we demonstrate that Ankrd2 is a potent regulator in muscle cells affecting a multitude of pathways and processes.

Figure 1. Ankrd2 can bind proteins via their PDZ domain.

Panels (A), (B), (C) and (D) show respectively PDZ arrays I, II, III and IV (Panomics/Affymetrix, USA) probed with His-tagged Ankrd2 (15 µg/ml). On the left are diagrams showing of the positions of the GST-PDZ proteins on the membrane; PDZ proteins that interact with Ankrd2 are highlighted. On the right are the membranes after probing with His-Ankrd2: (A) on PDZ array I a very strong positive signal was detected for the Reversion-induced LIM protein (RIL); weak positive signals for the PDZ and LIM domain protein 1 (hCLIM1) and Discs large homolog 4 (Dlg4). (B) on PDZ array II positive signals were detected for domain 1 and 2 of Zonula occludens (ZO-1 and ZO-2). (C) on PDZ array III positive signals were detected for syntenin-2 beta, domain 2 (SDB2-D2); for partitioning-defective 3 homolog, domain 3 (PARD-3) and for Scribble domain 4 (SCRIB1-D4). (D) on PDZ array IV positive signals were detected for domains 6 and 13 of the MUPP1 protein (MPDZ, Multi-PDZ domain protein); for domain 1 of Discs large homolog 5 (DLG5-D1); for syntrophin 2 (SNTB1) and also for RIM2 (RIMS2). His-tagged ligand was spotted in duplicate along the bottom and right edge for alignment and as a positive control.

Figure 2. Ankrd2 interacts with tight junction protein ZO-1 (TJP1).

The left panel shows GST-Ankrd2 pull down of radiolabeled ZO-1: only the GST-Ankrd2 bound to ZO-1 and not GST protein alone. GST or GST-Ankrd2 bound to glutathione-Sepharose 4B and was incubated for 3 h at RT with IVTT ³⁵S ZO-1. Immobilized complexes were then washed and subjected to SDS-PAGE. The input was 10% of the total amount of IVTT ³⁵S-ZO-1 was used in each binding reaction. In the right panel a SDS-PAGE gel stained with Coomassie blue shows that equal amounts of GST-Ankrd2 and GST were used in this experiment.

Figure 3. Ankrd2 can interact with proteins containing SH3 domains.

A SH3 protein array (Panomics/Affymetrix, USA) was probed with His-tagged Ankrd2 protein (15 µg/ml). The upper panel is a schematic diagram of the array showing the positions of the spotted GST-proteins; proteins positive for interaction with Ankrd2 are highlighted. The lower panel shows the membrane after hybridization with His-Ankrd2 protein. Ankrd2 bound strongly to the following SH3 proteins: Cortactin, CRK-D2, Y124, PEXD, Stam, and PLCγ. The positive controls (in duplicate) intended for alignment are seen at the bottom and the right edge of the blot.

Figure 4. The Ankrd2 protein can interact with several transcription factors (TF).

The upper panel is a schematic diagram of the Transcription Factor Array II (Panomics/Affymetrix, USA) showing the positions of the spotted His tagged TF proteins. The lower panel shows the TF protein-protein array membrane after probing with GST-Ankrd2 protein (15 µg/ml). The Ankrd2 protein bound very strongly to MeCP2 and strongly to HAND2, HDAC1, HOXA5, HEY, JUN, JUNB, KLF12, LDB1, LHX2, NFIL3 and PAX6. Weaker binding was seen with HNFG4, MAFK, MAX, NR1H2 and p53. The positive controls are at the bottom and right edge of the membranes. The TF proteins that interact with the Ankrd2 protein are highlighted.

Figure 5. Mapping the interaction sites for YB-1, p53, PML and telethonin/Tcap on Ankrd2.

(A) Diagram of Ankrd2 modular structure and deletants used in GST pull-down experiments: A, almost full length Ankrd2 protein (aa 5–333); sA, Ankrd2 protein with a 97 aa N-terminal deletion (aa 98–333); N, N-terminal (aa 5–120); NA, N-terminal plus ankyrin repeats (aa 5–284); CA, C-terminal plus ankyrin repeats (aa 121–333); C, C-terminal (aa 280–333). (B) and (C) GST pull down assays, equal amounts of GST proteins, immobilized on glutathione Sepharose (Figure S2) were mixed with cell extracts containing telethonin/Tcap, YB-1, p53 and PML. The resins were washed, subjected to SDS-PAGE and immunoblotted. Negative control is GST, positive controls (INPUT): for telethonin, 1 µg of U2OS cell lysate; for endogenous p53, 500 ng of COS7 cell lysate; for YB-1 500 ng of lysate of COS7 cell overexpressing FLAG-YB-1; and for PML, 500 ng of lysate of COS7 cells overexpressing FLAG-PML.

Figure 6. Transcriptional regulation of the Ankrd2 promoter by Nkx2.5, p53 and NFkB.

Both Nkx2.5 (A) and p53 (B) are upstream effectors of Ankrd2 gene expression. C2C12 cells were transfected with Ankrd2 (−439/+7)-LUC and Renilla luciferase reporter plasmids along with increasing amounts of expression vectors for Nkx2.5 and p53 as indicated. (C) Canonical NFκB does not affect Ankrd2 promoter activity. C2C12 were co-transfected with Ankrd2 (−439/+7)-LUC and Renilla luciferase plasmids and 5 hrs after transfection cells were treated with increasing amounts of TNFα in order to activate NFκB. In all of these experiments the firefly luciferase activity was normalized against the Renilla luciferase. The histograms show the mean of at least three independent experiments; the bars indicate the standard deviation. *p<0.05 versus control sample. (D) C2C12 cells were grown in the presence of 0.1, 1and 20 ng/ml of TNFα for 20 h and nuclear (NE) and cytoplasmic (CE) extracts prepared. Activation of NFκB by TNFα was confirmed by Western blot detection of NFκB subunit p50 in the nuclear extract (upper two panels). Efficiency of protein separation was monitored by histone H3 subcellular localization (lower two panels).

Figure 7. Ankrd2 promoter DNA can interact with some transcription factors that also interact with the Ankrd2 protein.

The upper panel is a schematic diagram of the Transcription Factor Array II (Panomics/Affymetrix, USA) showing the positions of the spotted His tagged TF proteins. The bottom panel shows the TF protein array membrane after hybridization with the biotinylated DNA of the Ankrd2 promoter (−1,173 to −4 bp). The Ankrd2 promoter DNA bound strongly to MeCP2, LHX2, NFIL3 and PAX6 and more weakly to HAND2 and HOXA5. The positive controls are at the bottom and right edge of the membranes. The TF proteins that interact with the Ankrd2 promoter are highlighted.

Figure 8. Ankrd1/CARP enhances the transcriptional ability of MyoD, but has no effect on Nkx2.5 induced up-regulation of the Ankrd2 promoter.

C2C12 were co-transfected with both Ankrd2 (−439/+7)-LUC and Renilla luciferase reporter plasmids as well as a constant amount of MyoD-pCDNA3 (A) or Nkx2.5-pCDNA3 (B), along with increasing amounts of an expression vector for Ankrd1/CARP, as indicated. In each assay the amount of total DNA used in transfections was kept constant by the addition of pCDNA3 vector. The firefly luciferase activity was normalized against Renilla luciferase. The histograms show the mean of at least three independent experiments performed in triplicate; the bars indicate the standard deviation. *p<0.05 versus control sample.