The Cardioprotective PKA-Mediated Hsp20 Phosphorylation Modulates Protein Associations Regulating Cytoskeletal Dynamics

Figures

• 

  Figure 1. Identification of Hsp20/14-3-3 and 14-3-3/CFL2 protein complexes in cardiac muscle. (a) MBP-14-3-3 pull down assays determine its interactions with Hsp20 and CFL2 in cardiac homogenates. (b) Immunoprecipitation assays using 14-3-3 or IgG control antibodies confirm the association of 14-3-3/Hsp20 and 14-3-3/CFL2 endogenously in cardiac muscle extracts. (c,d) Coomassie staining showing purified GST-Hsp20 and GST-CFL2 recombinant proteins. Hsp20/14-3-3 and 14-3-3/CFL2 interactions are direct, as shown by blot overlay assays. Specifically, 14-3-3 interacts with (c) phosphorylated Hsp20 and (d) both phosphorylated and non-phosphorylated CFL2, as determined by two independent experiments. No unspecific binding to MBP has been observed in control experiments (Figure S1).

• 

  Figure 2. Minimal binding regions of interactions. (a) Diagrammatic representation of the various CFL2 deletion constructs. ADF, actin-depolymerizing factor. (b) Coomassie blue stained gel showing purified recombinant GST and GST-CFL2 deletion proteins. Blot overlay assays using GST-CFL2 deletion constructs and MBP-14-3-3 determine that 14-3-3 binds to N-terminal region of CFL2 that includes amino acids 1-55. (c) Coomassie blue stained gel showing purified MBP-14-3-3 deletion proteins as well as GST-CFL2, GST and GST-p-Hsp20 recombinant proteins. (d,e) Blot overlay assays using MBP-14-3-3 N- or C-terminal deletion constructs determine the regions of 14-3-3 that bind to Ser16 phosphorylated-Hsp20 and CFL2. No unspecific binding to MBP was observed in control experiments (Figure S1).

• 

  Figure 3. Phosphorylated Hsp20 displaces CFL2 from 14-3-3 protein complex. (a) Pull down assays using MBP-14-3-3 recombinant protein and lysates from HEK 293 cell transfected with myc-CFL2. Binding of CFL2 to 14-3-3 was examined in the presence of GST-Hsp20 or Ser16-phosphorylated GST-Hsp20. (b) Quantification of CFL2/14-3-3 binding in the presence of GST-Hsp20 or Ser16-phosphorylated GST-Hsp20 (* p < 0.05, t test, two-tailed; n = 3). Data are the mean ± SD. (c) Coomassie blue stained gel showing purified recombinant GST-Hsp20-S16A and GST-Hsp20-S16D recombinant proteins. (d) Pull down assays using MBP-14-3-3 recombinant protein and lysates from HEK 293 cell transfected with myc-CFL2. Binding of CFL2 to 14-3-3 was examined in the presence of GST-Hsp20-S16A, GST-Hsp20-S16D, or phosphorylated GST-Hsp20-S16A and phosphorylated GST-Hsp20-S16D. (e) Quantification of CFL2/14-3-3 binding in the presence of GST-Hsp20-S16A or GST-Hsp20-S16D (* p < 0.05 vs. Hsp20-S16A -PKA, t test, two-tailed; n = 3). Data are the mean ± SD. (f) Quantification of 14-3-3 binding to GST-Hsp20-S16A or GST-Hsp20-S16D (* p < 0.05 vs. Hsp20-S16A-PKA, t test, two-tailed; n = 3). Data are the mean ± SD. (g)…

• 

  Figure 4. Localization of Hsp20 protein in HEK 293 cells and redistribution in the presence of 14-3-3. (a) Immunofluorescence analysis of HEK 293 cells transfected with myc-tagged-Hsp20 and examination of its localization with the cytoskeletal marker, actin. Scale bar 5 μm. (b) Fractionation study of myc-Hsp20 and examination of its localization in cytosolic soluble (S) and cytoskeletal insoluble (I) cell fractions. (c) 1 μM ISO treatment results in efficient Ser16 phosphorylation of myc-Hsp20. (d) Immunofluorescence analysis of HEK 293 cells transfected with myc-tagged-Hsp20 and examination of its localization with the cytoskeletal actin following ISO treatment. Calculation of Pearson’s correlation coefficient determined no significant alteration in the subcellular distribution of myc-Hsp20 following ISO treatment. Data are the mean ± SD; n = 15 cells from each group. Scale bar 5 μm. (e) Fractionation of ISO treated HEK 293 cells transfected with myc-Hsp20 and GFP vector or (g) GFP-14-3-3 construct and determination of myc-Hsp20 subcellular location. (f,h) Quantification of myc-Hsp20 distribution in the presence of GFP or GFP-14-3-3 (* p < 0.05, t test, two-tailed; n = 3). Data are the…

• 

  Figure 5. The human P20L mutation in Hsp20 inhibits its association with 14-3-3. (a) Coomassie staining of GST-Hsp20 recombinant proteins. Blot overlay assays using GST-Hsp20-WT or P20L and MBP-14-3-3 determine diminished binding of PKA-treated P20L mutant protein to 14-3-3. No unspecific binding to MBP was observed in control experiments (Figure S1). (b) Immunofluorescence analysis of HEK 293 cells transfected with myc-Hsp20-P20L and examination of its localization with the cytoskeletal marker, actin. Calculation of Pearson’s correlation coefficient determined no significant alteration in the subcellular distribution of myc-Hsp20-P20L, when compared to myc-Hsp20. Data are the mean ± SD; n = 15 cells from each group. Scale bar 5 μm. (c) Fractionation of 1 μM ISO treated HEK 293 cells transfected with myc-Hsp20-P20L and GFP-14-3-3 determines no significant alteration of myc-Hsp20-P20L subcellular location. (d) Quantification of myc-Hsp20-P20L distribution in the presence GFP- 14-3-3. Data are the mean ± SD; n = 3.

• 

  Figure 6. Ser16- Hsp20-P20L fails to displace CFL2 from 14-3-3 protein complex. (a) Pull down assays using MBP-14-3-3 recombinant protein and lysates from HEK 293 cells transfected with myc-CFL2. Binding of CFL2 to 14-3-3 was examined in the presence of GST-Hsp20-P20L or Ser16-phosphorylated GST-Hsp20-P20L. (b) Quantification of CFL2/14-3-3 binding in the presence of GST-Hsp20-P20L or Ser16-phosphorylated GST-Hsp20-P20L. Data are the mean ± SD; n = 3. (c) Representative pull down assay showing the diminished effect of Hsp20-P20L in competing with CFL2 for 14-3-3 binding, when compared to WT Hsp20. (d) Quantification of CFL2/14-3-3 binding in the presence of GST-Hsp20 or GST-Hsp20-P20L (* p < 0.05 vs. Hsp20-PKA, t test, two-tailed; n = 3). Data are the mean ± SD; n = 3. (e) Reduced Ser16 phosphorylation of Hsp20-P20L mutant, in comparison to Hsp20-WT protein.

• 

  Figure 7. Hsp20 phosphorylation enhances CFL2 mediated actin depolymerization. (a) In vitro depolymerization assays were performed in the presence of GST-CFL2, MBP-14-3-3 and GST-Hsp20 recombinant proteins. Phosphorylated Hsp20 enhanced depolymerization of F-actin by CFL2, while this effect was reduced in the presence of phosphorylated Hsp20-P20L mutant protein. (b) Calculation of the slope of F-actin depolymerization of the different protein combinations. (* p < 0.05 vs. CFL2; # p < 0.05 vs. CFL2:14-3-3:phospho-Hsp20; † p < 0.05 vs. CFL2:14-3-3:Hsp20; ‡ p < 0.05 vs. CFL2:14-3-3: Hsp20, t-test, two-tailed; n = 3). Data are the mean ± SD.

• 

  Figure 8. Graphical representation of the proposed contribution of Hsp20 phosphorylation to the regulation of actin cytoskeleton dynamics. (a) Hsp20 phosphorylation by PKA during β-adrenergic stimulation leads to Hsp20 subcellular translocation to the cytoskeleton, where it interacts with 14-3-3. For this association to occur, phosphorylated Hsp20 competes with CFL2 for 14-3-3-binding that results in CFL2 dissociation from 14-3-3. This triggers CFL2 activation, leading to enhanced F-actin depolymerization activity and regulation of actin dynamics in order to maintain cytoskeletal integrity and function. (b) The human phosphorylation impairing Hsp20-P20L mutated protein exhibits diminished phosphorylation at Ser16 and significantly reduced binding to 14-3-3, with consequent failure to displace CFL2 from 14-3-3. As a result, aberrant regulation of CFL2-mediated F-actin depolymerization. This would lead to cytoskeletal disruption and myofibrillar damage during stress conditions, contributing towards cardiac dysfunction.