Warscheid Lab - FLNc d23-24 in vitro kinase assay

In vitro kinase assay of human and mouse FLNc domain 23-24

  • Organism: Human, Mouse
  • Instrument: Obritrap Velos Elite, QExactive Plus
  • 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 PKCα phosphorylation at distinct serine residues in its hinge 2 region prevents its cleavage at an adjacent tyrosine residue by calpain 1. In our in vitro kinase assay we demonstrate that mouse FLNc is predominantly phosphorylated at S2625 whereas human FLNc is phosphorylated at the neighbouring serines 2623/2624.
Experiment Description
Recombinantly expressed and purified mouse and human FLNc d23-24 WT and phosphosite mutants were dialyzed over night at 4°C in dialysis buffer (1 mM DTT, 100 mM KCl, 20 mM HEPES pH 7.4, 10 mM MgCl2). For MS-coupled kinase assays, a sample volume corresponding to 100 µg protein was added to a volume of 200 µl H2O and mixed with 5x PKC buffer [25 mM DTT, 100 mM KCl, 20 mM HEPES pH 7.4, 10 mM MgCl2, 0.5 mM CaCl2, 2.5 mM ATP, sodium fluoride, sodium pyrophosphate, sodium orthovanadate, β-glycerophosphate, 0.15% CHAPS, 1x PKC lipid activator (Merck Millipore, Darmstadt, Germany)]. The assay was started by adding 200 ng PKCα (Sigma-Aldrich) and the reaction was performed under shaking at 200 rpm for 20 min at 30°C for each FLNc construct. For further analysis, samples of three independent replicates were diluted 1:4 (v/v) with 50 mM ammonium bicarbonate and subjected to in-solution digestion using sequencing grade trypsin (1:50) (Promega) for 3.5 h at 200 rpm and 42°C. Single protein digests were acidified with TFA [final concentration 1% (v/v)], subjected to TiO2 enrichment, and the resulting phosphopeptide-enriched fractions analysed by LC-MS/MS using alternative fragmentation methods.
Created on 5/12/16 2:41 PM
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Figure 4 A-F

A, B, Radioactive in vitro kinase assays. Recombinant wildtype and phosphosite mutants of mouse (A) and human (B) FLNc Ig-like domains 23-24 (d23-24) were treated with PKCα in the presence of [γ-33P]ATP and analysed by SDS-PAGE followed by autoradiography or Coomassie staining. S2625 of mFLNC d23-24 was replaced by A or D; S2623/S2624 of hFLNC d23-24 by AA or DD. As a control, PKCα was incubated in [γ-33P]ATP-containing kinase buffer without hFLNc d23-24 (B). WT, wildtype; A, alanine; D, aspartate; +, 10 ng PKCα; ++, 20 ng PKCα

C, D, MS-based in vitro kinase assays. Reactions were performed as described in (A, B) using unlabelled ATP and PKCα. MS data from three independent kinase experiments for mFLNc d23-24 WT, AA and DD sites mutants (C) and mFLNc WT, A, and D site mutants (D) were quantified. Intensities of phosphopeptides distinctive for a specific phosphorylation site (red) were added up per experiment and represented as normalized mean ± SEM.

E, F, Fragmentation spectra of mono-phosphorylated peptides of mouse (E) and human (F) FLNc d23-24 WT forms. PKCα-dependent phosphorylation of mFLNc-S2625 and hFLNc-S2623 was determined by higher-collisional dissociation and electron transfer dissociation, respectively. Fragment ions exhibiting a neutral loss of phosphoric acid (H3PO4; 97.9768 u) are marked with an asterisk (*); loss of water (H2O) as indicated. Phosphorylated residues are depicted in red; b/c- and y/z-ion series in red and blue, respectively.

Figure S3. 

A, Radioactive in vitro kinase assays. Recombinant wildtype and phosphosite mutants of mouse and human FLNc Ig-like domains 23-24 (d23-24) were treated with [γ-33P]ATP in the absence of PKCα and analysed by SDS-PAGE followed by autoradiography or Coomassie staining. S2625 of mFLNC d23-24 was replaced by A or D; S2623/S2624 of hFLNC d23-24 by AA or DD. As a control, PKCα was incubated in [γ-33P]ATP-containing kinase buffer without hFLNc d23-24 (B). WT, wildtype; A, alanine; D, aspartate

B, Abundance of recombinant mouse and human FLNc d23-24 wildtype and phosphosite mutants in mass spectrometry-based in vitro kinase assays. Shown are the mean values of the total peptide intensity measured for each of the FLNc d23-24 constructs (see Figure S2B). Data derived from three independent experiments per construct. Error bars represent the SEM. WT, wildtype; A, alanine, D, aspartate; n.s., not significant