NIMA-related kinase 9 regulates the phosphorylation of the essential myosin light chain in the heart

Figures

• 

  Fig. 1. Identification of ELC kinases in adult piscine heart protein. Immunoprecipitation (IP) was performed by using custom ELC antibodies covalently coupled to magnetic beads. Liquid chromatography followed by mass spectrometry (LC-MS) was used for identification. A Validation of ELC precipitation by immunoblot (IB) before performing LC-MS (see B and C). B Classification of ELC enriched proteins identified by LC-MS. From 3452 spectra 110 proteins were identified in total. 106 proteins were found to interact with ELC. 3 different kinases were found. (Peptide FDR: 5%, minimum of two identified peptides per protein) C Ranking of ELC enriched proteins by foldchange of unique peptide counts enriched by ELC compared to unique peptide counts of the same protein captured by the isotype control (IgG Ctrl). Data of the screening experiment are shown without statistical analysis. Candidates were validated using independent methods. The string intersection score was calculated for known ELC interacting proteins67. (NA: not available) D Two representative mass spectrometry spectra of unique peptides accepted to identify NIMA related kinase 9 (NEK9) (left panel) or calcium/calmodulin-dependent…

• 

  Fig. 2. Characterization of ELC interacting kinases. A NEK9 and CamK2G RNA expression in human total RNA tissue panel. Ribosomal protein large subunit P0 (RPLP0) was used as housekeeping gene. B NEK9 (left panel) and CamK2G (right panel) protein expression in piscine and human heart tissue to detect cross-species applicability. C mRNA expression profile of NEK9 and CamK2G in different human tissues from 4 weeks post-conception to adulthood68. D, E Relationship of NEK9-ELC binding efficiency on intracellular Ca2+-concentration. NEK9-ELC interaction was analyzed after overexpression of myc-tagged ELC protein in human cells followed by specific immunoprecipitation (IP) and immunoblot (IB) analysis (D). Ca2+-influx was obtained by ionophore stimulation. Quantification of NEK9-ELC interaction dependent on Ca2+-influx illustrated as the averaged intensity of precipitated NEK9 protein normalized to actin protein expression of input protein lysate (E). Data are mean ± SD (n = 3). **P < 0.01 by the analysis of ordinary one-way ANOVA followed by Bonferroni’s multiple comparisons test.

• 

  Fig. 3. NEK9 regulates ELC-phosphorylation pattern in-vitro. A Validation of siRNA mediated knockdown (KD) of NEK9 in HEK293 cells by quantitative real time PCR. mRNA expression shown as 2(−ΔCt) normalized to RPLP0 (ribosomal protein large subunit P0). Ca2+-influx was obtained by ionophore stimulation. Data are mean ± SD (n = 3). ****P < 0.0001 by analysis of ordinary one-way ANOVA followed by Bonferroni’s multiple comparisons test. B Representative immunoblot (IB) of NEK9 protein expression after specific knockdown (KD). C 2D immunoblot of ELC-phosphorylation pattern after myc-tagged ELC overexpression and Ca2+ -influx using myc-specific antibody. After protein isolation samples were split and half of the sample was treated with phosphatase inhibitor (Native) and the other half was incubated with phosphatase (Dephospho). D Representative 2D immunoblot of myc-tagged ELC-phosphorylation pattern using myc-specific antibody (above) and ELC-phosphorylation illustrated as averaged intensity normalized to total ELC protein (down). ELC-phosphorylation was analyzed after myc-tagged ELC overexpression and siRNA knockdown (KD) of NEK9 or after overexpression (Ox) of NEK9 in human cells.…

• 

  Fig. 4. Functional analysis of NEK9 in-vivo. A Lateral view of zebrafish embryo hearts 72 h post fertilization (hpf) injected with control oligonucleotides or anti-sense oligonucleotides blocking the translational start site of nek9 (KD NEK9) (left). Immunostaining of atrium-specific (A) and ventricle-specific (V) myosin heavy chains (green: antibody against atrial-specific myosin (S46), red: antibody against ventricular and atrial myosin (MF20)) (right). B Phenotype rescue of KD NEK9. Zebrafish eggs were injected with nek9 mRNA (Ox + KD NEK9) followed by KD NEK9 in one-cell stage. Analysis was performed at 72 hpf. Data are mean ± SD (Ctrl, n = 143; KD NEK9, n = 192; Ox + KD NEK9, n = 155 fish embryos out of 3 experiments). *P < 0.05; **P < 0.01; ***P < 0.001 by the analysis of two-tailed paired Student’s t-test. C Fractional shortening (FS) of zebrafish morphants at 72 hpf. Data are mean ± SD (Ctrl, n = 25; KD NEK9, n = 21; Ox + KD NEK9, n = 21 fish embryos out of 3 experiments). *P < 0.05; **P < 0.01 by the analysis of two-way ANOVA followed by Bonferroni’s multiple comparisons test. The respective heart rate is shown in Supplementary Fig. 4A. D–F Two heterozygous transgenic zebrafish…

• 

  Fig. 5. Analysis of the ELC-NEK9 interaction. Two heterozygous transgenic zebrafish lines were generated by CRISPR/Cas9. A NEK978del protein is lacking the ATP-binding domain shown in the 3D structure of NEK9 (A). NEK9500del is harboring a frame shift mutation leading to a premature stop codon. Sanger sequencing results of the affected allele are shown in Supplementary Fig. 5C. B, C Piscine ELC and mutated piscine myc-tagged NEK9 protein either lacking the ATP-binding domain (NEK978del) or harboring a premature stop codon (NEK9500del) were overexpressed in HEK293 cells. The ELC-NEK9 interaction was analyzed by specific immunoprecipitation (IP) and immunoblot (IB) (B). Quantification of ELC-NEK9 interaction illustrated as averaged intensity of precipitated ELC protein normalized to ELC expression of input protein lysate (C). Data are mean ± SD (n = 3). *P < 0.05; ***P < 0.001 by the analysis of ordinary one-way ANOVA followed by Bonferroni’s multiple comparisons test. D, E Validation of ELC-NEK9 interaction. Human myc-tagged ELC protein and different deletion variants of human flag-tagged NEK9 protein were overexpressed and interaction intensity was quantified after IP and IB. Data are…

• 

  Fig. 6. Functional coupling of ELC and NEK9 in-vivo. A Schematic illustration of genetic sensitizing by nek9 in an ELC phospho-deficient genetic background. The concept is based on the progressive amplification of a response by functionally coupled factors. B Penetrance of NEK9 KD in zebrafish embryos at 72 hpf. Different concentrations of antisense oligonucleotides against zebrafish nek9 were injected in a genetic wild-type background (KD NEK9: knockdown of NEK9 resulting in a significant heart failure phenotype; LD NEK9: low-dose knockdown showing no phenotypical effect). Data are mean ± SD (Ctrl, n = 517; LD NEK9, n = 162; KD NEK9, n = 461 fish embryos out of 4 (Ctrl), 3 (LD NEK9) and 5 (KD NEK9) experiments). For statistical analysis a mixed effect model followed by Bonferroni’s multiple comparisons test was used. **P < 0.01 shown for the corresponding embryos injected with Ctrl oligonucleotides. C Validation of NEK9 KD or LD NEK9 by immunoblot (IB). D Lateral view of heterozygous lazy susan (lazm647/+) embryo hearts 72 hpf injected with control oligonucleotides or low-dose antisense oligonucleotides against zebrafish ilk (LD ILK) or nek9 (LD NEK9). E Genotype-phenotype-association…

• 

  Fig. 7. ELC posttranslational modifications in human left ventricular tissue. A Representative 2D silver stain of human LV tissue. B Selected spot areas 1 to 6 were analyzed by liquid chromatography followed by mass spectrometry (LC-MS). Peptide counts uniquely assigned to ventricular (v) ELC or vRLC are shown for 6 independent samples. The mean percentage of the protein coverage of vELC and vRLC are given above (table inset). Data are mean ± SD. C Schematic illustration of identified ELC or RLC-phosphorylation sites and deamidated amino acid residues detected in human LV tissue (n = 6; DCM: n = 4; Ctrl: n = 2). D Validation of polyclonal rabbit antibody against human ELC (Biozol, GeneTex, ZF127578) by immunoblot (IB) of different amounts of human LV protein. No cross reactivity with vRLC was detected. E Quantification of relative ELC protein expression illustrated as averaged intensity normalized to total ELC protein. ELC expression intensities were plotted against the amount of human heart protein. Exposure time was varied from 0.25 to 5 min. For 2 min exposure R = 0.9383 and P < 0.0001 was calculated by two-tailed Spearman Rank Correlation (n = 5). F Validation of in-vitro…

• 

  Fig. 8. ELC-phosphorylation is regulated in human dilated cardiomyopathy. A Ratio of ventricular (v) MLC and atrial (a) MLC mRNA expression in human left ventricular tissue of DCM patients and HTX controls (HTX: n = 28; DCM: n = 44). Samples were analyzed by deep mRNA sequencing (mRNAseq)38. Data are mean ± SEM. Statistical significance was tested by two-tailed unpaired Student’s t-test. B Quantification of relative basal (Basal) and phosphorylated (+1P, + 2P) ELC protein illustrated as averaged intensity normalized to total ELC protein (Control: n = 9; DCM: n = 11). 6 HTX controls (Supplementary Table 1) and 3 healthy controls were included. Data are mean ± SD. **P < 0.01 by the analysis of two-tailed unpaired Student’s t-test. C Representative ELC 2D immunoblot (IB) of human LV heart tissue of a DCM patient and a healthy control. Native ELC protein forms (left panel) and ELC protein forms after in-vitro dephosphorylation (right panel). Exposure times were varied from 1 to 5 min. D Correlation analysis of phosphorylated ELC expression (+2P) and left ventricular ejection fraction (LVEF). R = −0.6193 and P = 0.0036 by the analysis of two-tailed Pearson Correlation (Control: n = 9; DCM: n…