Novel determinant of PKC-epsilon anchoring at cardiac Z-lines.
The Z-line represents a critical link between the transverse tubule network and cytoskeleton of cardiac cells with a role in anchoring structural proteins, ion channels, and signaling molecules. Protein kinase C-epsilon (PKC-epsilon) regulates cardiac excitability, cardioprotection, and growth, possibly as a consequence of translocation to the Z-line/T tubule region. To investigate the mechanism of PKC-epsilon translocation, fragments of its NH2-terminal 144-amino acid variable domain, epsilonV1, were fused with green fluorescent protein and evaluated by quantitative Fourier image analysis of decorated myocytes. Deletion of 23 amino acids from the NH2-terminus of epsilonV1, including an EAVSLKPT motif important for binding to a receptor for activated C kinase (RACK2), reduced but did not abolish Z-line binding. Further deletions of up to 84 amino acids from the NH2-terminus of epsilonV1 also did not prevent Z-line decoration. However, deletions of residues 85-144 from the COOH-terminus strongly reduced Z-line binding. COOH-terminal deletions caused 2.5-fold greater loss of binding energy (deltadeltaG) than did NH2-terminal deletions. Synthetic peptides derived from these regions modulated epsilonV1 binding and cardiac myocyte function, but also revealed considerable heterogeneity within populations of adult cardiac myocytes. The COOH-terminal subdomain important for Z-line anchoring maps to a surface in the epsilonV1 crystal structure that complements the eight-amino acid RACK2 binding site and two previously identified membrane docking motifs. PKC-epsilon anchoring at the cardiac Z-line/T tubule appears to rely on multiple points of contact probably involving protein-lipid and protein-protein interactions.
Robia
Seth L
SL
Dept. of Physiology, University of Wisconsin, Madison, WI 53706, USA.
Kang
Misuk
M
Walker
Jeffery W
JW
D000818:Animals; D004734:Energy Metabolism / Q000502:physiology; D005456:Fluorescent Dyes; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D049452:Green Fluorescent Proteins; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D008958:Models, Molecular; D009200:Myocardial Contraction / Q000502:physiology; D032383:Myocytes, Cardiac / Q000201:enzymology* / Q000648:ultrastructure*; D015388:Organelles / Q000201:enzymology*; D021381:Protein Transport / Q000502:physiology; D051381:Rats
D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins