Figure 1: MLP is a substrate and inhibitor of PKCα kinase activity in vitro.

(a) In vitro kinase assay using PKCα and GST tagged phospholamban cytoplasmic N-terminus (GST-PLN(N)) as substrate with increasing GST-MLP or GST amounts. Shown are representative autoradiography images for phosphorylated proteins (upper panel) or Coomassie staining for total proteins (lower panel). Dashed line indicates that proteins were run on the same gel, but imaged using different exposure times. (b) Quantification of GST-PLN(N) phosphorylation or PKCα auto-phosphorylation with increasing GST-MLP or GST concentrations. Mean values with s.e., sample size (n) and P values are shown. (c) In vitro kinase assay using PKCα and GST tagged WT and mutant MLP as substrate. Representative autoradiography images for either Leu44Pro (L44P), Lys69Arg (K69R), Gly72Arg (G72R) mutant MLP (top panel), or Ser54Arg/Glu55Gly (S54R/E55G) or Cys58Gly (C58G; bottom panel) mutant MLP (bottom panel) in comparison with WT MLP are shown. Dashed line indicates proteins were run on the same gel, but non-consecutive. (d) Quantification of MLP phosphorylation by PKCα. Phosphorylation efficiency of WT MLP was arbitrarily set to 1. Mutants identified in human HCM patients show lower MLP phosphorylation, while MLP mutants associated with DCM display increased phosphorylation values. A representative Coomassie stained gel showing total GST-tagged MLP used for in vitro kinase assays is depicted (bottom panel; dashed line indicates that proteins were run on the same gel, but non consecutive.). Shown are mean values and s.e., as well as P values and sample sizes (n, in brackets below bar graphs). (e,f) Analysis of MLP phosphorylation in IDCM. (e) Quantification of MLP phosphorylation indicates significantly elevated phosphorylation levels in IDCM patients, compared to NF controls. Shown are mean values and s.e., as well as sample size (n, in brackets below bar graphs), and P values. (f) MLP phosphorylation levels for quantification in (e) were determined by Phostag analysis. SDS samples of protein extracts of NF and IDCM patient heart samples were run on conventional SDS PAGE (middle) and on 12% polyacrylamide gels with 50 μM Phostag reagent (top) and immunoblotted for MLP. A human control sample with uncharacterized disease status (ctl) showing non-phosphorylated (P0) and phosphorylated (P1) MLP was used to indicate Phostag gel migration pattern. Phosphorylated proteins (P1) migrate slower due to their interaction with the Phostag reagent compared to unphosphorylated protein (P0). GAPDH (bottom) was used to show equal loading.