Protein kinase C zeta. A novel regulator of both phosphorylation and de-phosphorylation of cardiac sarcomeric proteins

Abstract

Our experiments investigated associations of specific isoforms of protein kinase C (PKC) with individual proteins in the cardiac troponin complex. Troponin I (cTnI) associated with PKCepsilon and zeta and troponin T (cTnT) associated with PKC alpha, delta, and epsilon. Based on its association with cTnI, we hypothesized that PKCzeta is a major regulator of myofilament protein phosphorylation. To test this, we infected adult cardiac myocytes with adenoviral constructs containing DsRed monomer-tagged wild type (WT) and the following constitutively active forms of PKCzeta: the pseudo-substrate region (A119E), 3'-phospho-inositide-dependent kinase-1 (T410E), and auto-phosphorylation (T560E). The A119E and T410E mutants displayed increased localization to the Z-discs compared with WT and T560E. Immunoprecipitations were performed in myocytes expressing PKCzeta using PKC phospho-motif antibodies to determine the phosphorylation of cTnI, cTnT, tropomyosin, myosin-binding protein C, and desmin. We did not find serine (Ser) phosphorylation of cTnI or cTnT. However, we observed a significant decrease in threonine (Thr) phosphorylation of cTnI and cTnT notably by PKCzeta T560E. Ser phosphorylation of tropomyosin was increased by all three active mutants of PKCzeta. Ser/Thr phosphorylation of myosin-binding protein C increased primarily by PKCzeta A119E. Both PKCzeta A119E and T410E mutants increased desmin Ser/Thr phosphorylation. To explain the apparent Thr dephosphorylation of cTnI and cTnT, we hypothesized that PKCzeta exists as a complex with p21-activated kinase-1 (Pak1) and protein phosphatase 2A (PP2A), and this was confirmed by immunoprecipitation Western blot. Our data demonstrate that PKCzeta is a novel regulator of myofilament protein phosphorylation.

FIGURE 1. GST-troponin pulldown assays

A, PKA_C pulldown by GST-cTnI (left panel) and PP2A pulldown by GST-cTnI and GST-cTnT (right panel). B, PKC pulldowns. G, GST only; I, GST-cTnI; T, GST-cTnT; C, GST-cTnC.

FIGURE 2. Localization of PKCζ in cultured adult rat ventricular myocytes

WT and mutants PKCζ cDNAs were tagged with DsRed monomer (DsRedM) and adenovirally expressed. Fixed cells were counter-stained with an α-actinin antibody (Upstate) and visualized by confocal microscopy. Data are representative of three independent experiments of >30 cells per experiment. Scale bar represents 10 microns.

FIGURE 3. Thin filament protein phosphorylation by PKCζ

A, cTnI:PKC phospho-motifs are highlighted (Ser) and underlined (Thr), representative IP-Westerns of cTnI Ser and Thr phosphorylation (left), quantification of Thr phosphorylation of cTnI (right). B, cTnT:PKC phospho-motifs are highlighted (Ser) and underlined (Thr), representative IP-Westerns of cTnT Ser and Thr phosphorylation (left), quantification of cTnT Thr phosphorylation (right). C, Tm:PKC phospho-motifs are highlighted (Ser) and underlined (Thr), representative IP-Westerns of Tm Ser and Thr phosphorylation (left panels), quantification of Ser phosphorylation (right panel). *, p < 0.05 versus Un, **, p < 0.01 versus Un.

FIGURE 4. MyBP-C phosphorylation by PKCζ

A, PKC phospho-motifs are highlighted (Ser) and underlined in MyBP-C. B, representative IP-Westerns of MyBP-C phosphorylation: PKC phospho-serine IP-Western (top panel); PKC phospho-threonine IP-Western (middle panel); lysate only (bottom panel). C, quantification of PKC Ser (top panel) and Thr phosphorylation (bottom panel). *, p < 0.05 versus Un; **, p < 0.01 versus Un.

FIGURE 5. Desmin phosphorylation by PKCζ

A, desmin amino acid sequence with PKC phospho-motifs highlighted (Ser) and underlined (Thr). B, representative IP-Westerns of desmin phosphorylation: PKC phospho-serine IP-Western (top panel), PKC phospho-threonine IP-Western (middle panel), lysate only (bottom panel). C, quantification of PKC Ser (top panel) and Thr (bottom panel) phosphorylation. *, p < 0.05 versus Un, **, p < 0.01 versus Un.

FIGURE 6. IP of PKCζ, Pak1, and PP2A in brain (B) and myocyte (M) lysates

A, IP with an antibody to PKCζ and blotted for Pak1, PP2A, and PKCζ. B, Pak1 immunoprecipitation followed by blotting for PKCζ, PP2A, and Pak1. C, immunoprecipitation with PP2A antibody followed by PKCζ, Pak1, and PP2A antibodies.