NMR Structure/Function Relationships of Peptides Corresponding to the C1B1 Region Of PKC

Abstract

Purpose: : PKCγ is a serine/threonine kinase that inhibits the activity of membrane gap junctions in response to oxidative stress. In the absence of oxidative induction, PKCγ is held in an inactive state in the cytoplasm by docking to 14-3-3 proteins at the C1B regulatory domain of PKCγ. The purpose of this study is to prove the existence of a structure /function relationship in the binding of PKCγ to 14-3-3 through its C1B domain.

Methods: : Synthetic peptides of 12 amino acids in length corresponding to the C1B1 region (101-112) of PKCγ were tested for our studies. The peptides were wild type and three mutants M1 (Y108F), M2 (S109A) and M3 (H106F) C1B1 peptides. The gap junction activity scrape loading /dye transfer assay was done on 90% confluent N/N 1003A cells pre-treated with 100uM of C1B1 or each of the three mutants for 2 hours. Dye was allowed to permeate the cells for 2 min. Cell viability analysis was performed by a fluorometric method on 30 *10³ N/N cells pre-treated with 50uM, 100uM and 200uM concentration of each of the four C1B peptides followed by the treatment with 100uM H2O2 for 12 hours. For in vivo competition assay, N/N cells were treated for four hour with 100 uM peptides and PKCγ was immunoprecipitated thereafter. The samples were then blotted with antibody against 14-3-3ε. The images were scanned and total average pixel intensity was recorded to determine the amount of 14-3-3 competed off of PKCγ by C1B1 peptides. For structural determination of C1B1 peptides 1D and 2D 1H-1H NMR experiments were performed on a 500MHz UNITY plus spectrometer at 10 degree C in H2O :TFE-d3 (1:1V/V).

Results: : Wild type C1B1 and mutant M1, but not M2 and M3, decreased the amount of dye transfer in N/N 1003A cells by 40% which indicated PKCγ activation and subsequent gap junction inhibition. Cells treated with C1B1 and M1 peptides were protected from death by H2O2 but cells treated with M2 and M3 were vulnerable to H2O2 .In vivo competition assay demonstrated C1B1 and mutant M1 to be more effective than M2 and M3 at competing 14-3-3 off of PKCγ .NMR studies showed a flexible structure for all the peptides with the structure of M3 more organized than C1B1, M1 or M2.

Keywords: gap junctions/coupling • stress response