Warscheid Lab - C-terminal FLNc in vivo kinase assays

In vivo kinase assay of mouse FLNc

  • Organism: C2C12 mouse myotubes
  • Instrument: Thermo Scientific Orbitrap Velos Elite
  • SpikeIn: No
Abstract
The Z-disc is a protein-rich structure critically important for myofibril development and integrity. Since a role of the Z-disc for signal integration and transduction was recently suggested, its precise phosphorylation landscape warranted in-depth analysis. We therefore established a site-resolved protein phosphorylation map of the Z-disc in skeletal myocytes and found that it is a phosphorylation hotspot in living cells, underscoring its functions in signalling and disease-related processes. In an exemplary fashion, we analysed the actin-binding multi-adaptor protein filamin C (FLNc), which is essential for Z-disc assembly and maintenance, and found that PKC phosphorylation at distinct serine residues in its hinge 2 region prevents its cleavage at an adjacent tyrosine residue by calpain 1. With this quantitative in vivo kinase assay, we show that the phosphorylation site S2625 in mouse FLNc is significantly down-regulated upon treatment of C2C12 myotubes with the PKCα inhibitor Gö6976.
Experiment Description
Light, medium and heavy SILAC labelling of C2C12 cells was performed with 13C6 L-arginine and 12C6 L-lysine, 12C6 L-arginine and D4 L-lysine and 13C615N4 L-arginine and 13C615N2 L-lysine, respectively. Labelled myoblasts were seeded into six well plates and differentiation was induced by reduction of the dialyzed FCS content to 2% in the absence of sodium pyruvate. Cells were differentiated for at least 4d. Prior to signalling experiments, cells were starved 16 h overnight and sarcomere formation was improved by electrical pulse stimulation. Tryptic digests were seperated via high-pH reversed phase chromatography and 2 fractions per biological replicate containing the FLNc peptide GASYSSIPK were futher enriched for phosphorylated isoform by titanium dioxide enrichment. Treatment of the biological replicates was performed as follows: experiment 1: light - control; medium - PMA; heavy - Gö6976; experiment 2: light - Gö6976; medium - control; heavy - PMA, experiment 3: light - PMA; medium - Gö6976; heavy - control
Created on 5/12/16, 2:41 PM
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InVivoKinaseAssay_2016-05-12_09-04-24.sky.zip (998 KB)2016-05-1211396

Figure 4 G-H, Experimental approach of quantitative in vivo kinase assay in contracting myotubes (G) and results for the regulated

peptide, phosphorylated at S2625 (H). Quantification of data shown was performed with skyline for n=3 independent experiments.

Shown is the mean log2 ratio of the area under the curve ± SEM.  **p ≤0.006.

Figure S3C, Extracted ion chromatogram of the phosphopeptide GApSYSSIPK within

fraction 1 in the biological replicate 3 of the in vivo kinase assay.