Protein kinase C zeta. A novel regulator of both phosphorylation and de-phosphorylation of cardiac sarcomeric proteins.
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.
Wu
Steven C
SC
Center for Cardiovascular Research, Department of Physiology and Biophysics, University of Illinois at Chicago College of Medicine, Chicago, Illinois 60612, USA.
Solaro
R John
RJ
D020816:Amino Acid Motifs / Q000235:genetics / Q000276:immunology; D019943:Amino Acid Substitution / Q000276:immunology; D000818:Animals; D000906:Antibodies / Q000276:immunology; D007527:Isoenzymes / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D008297:Male; D018832:Molecular Chaperones / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D009124:Muscle Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D020125:Mutation, Missense / Q000276:immunology; D009206:Myocardium / Q000166:cytology / Q000201:enzymology*; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D051381:Rats; D012518:Sarcomeres / Q000201:enzymology* / Q000235:genetics / Q000276:immunology; D054462:p21-Activated Kinases / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D000099221:Protein Kinase C zeta
D000906:Antibodies; D007527:Isoenzymes; D018832:Molecular Chaperones; D009124:Muscle Proteins; D010749:Phosphoprotein Phosphatases; D054462:p21-Activated Kinases; C490392:Prkcz protein, rat; D000099221:Protein Kinase C zeta; C515906:Pak1 protein, rat