Dietary fish oil blocks carcinogen-induced down-regulation of colonic protein kinase C isozymes

1. The visual system of Hermissenda has been studied extensively as a site of cellular plasticity produced by classical conditioning. Previous research has shown that one-trial conditioning, consisting of light paired with serotonin (5-HT) results in short- and long-term enhancement of light-elicited generator potentials in identified type B-photoreceptors. Recent evidence suggests that 5-HT exerts its effects on the induction of short-term enhancement by activation of protein kinase C (PKC), a Ca(2+)-activated and phospholipid-dependent protein kinase. However, the contribution of protein kinases in general, and specifically PKC in long-term enhancement has not been established. 2. The protein kinase inhibitors H-7 and sphingosine blocked the induction of short-term enhancement when applied before one-trial conditioning. However, the conditions that are sufficient to block the induction of short-term enhancement do not block long-term enhancement. Sphingosine and H-7 do not block the induction and expression of long-term enhancement when applied before one-trial conditioning. 3. Pretreatment before conditioning with 12-O-tetradecanoyl-phorbol-13-acetate (TPA), which leads to down-regulation of PKC, also did not block long-term enhancement. Down-regulation by itself did not produce enhancement, although the transient peak of light-elicited generator potentials was reduced by pretreatment with TPA. 4. The results suggest that the induction of short- and long-term enhancement involve parallel processes, and thus the expression of long-term cellular plasticity produced by one-trial conditioning does not depend on the induction or expression of short-term enhancement.

Download Full-text

Invariant Leu Preceding Turn Motif Phosphorylation Site Controls the Interaction of Protein Kinase C with Hsp70

Journal of Biological Chemistry ◽

10.1074/jbc.m604076200 ◽

2006 ◽

Vol 281 (43) ◽

pp. 32461-32468 ◽

Cited By ~ 26

Author(s):

Tianyan Gao ◽

Alexandra C. Newton

Keyword(s):

Protein Kinase C ◽

Heat Shock ◽

Protein Kinase ◽

Phosphorylation Site ◽

Insoluble Fraction ◽

Down Regulation ◽

Kinase C ◽

Shock Proteins ◽

Invariant Residue ◽

Autophosphorylation Site

Heat shock proteins play important roles in regulating signal transduction in cells by associating with, and stabilizing, diverse signaling molecules, including protein kinases. Previously, we have shown that heat shock protein Hsp70 associates with protein kinase C (PKC) via an interaction that is triggered by dephosphorylation at the turn phosphorylation motif. Here we have identified an invariant residue in the carboxyl terminus of PKC that mediates the binding to Hsp70. Specifically, we show that Hsp70 binds to Leu (Leu-640) immediately preceding the conserved turn motif autophosphorylation site (Thr-641) in PKC βII. Co-immunoprecipitation experiments reveal that mutation of Leu-640 to Gly decreases the interaction of Hsp70 with PKC βII. This weakened interaction between Hsp70 and the mutant PKCs results in accumulation of dephosphorylated PKC in the detergent-insoluble fraction of cells. In addition, the Hsp70-binding mutant is considerably more sensitive to down-regulation compared with WT PKC: disruption of Hsp70 binding leads to accelerated dephosphorylation and enhanced ubiquitination of mutant PKC upon phorbol ester treatment. Last, pulse-chase experiments demonstrate that Hsp70 preferentially binds the species of mature PKC that has become dephosphorylated compared with the newly synthesized protein that has yet to be phosphorylated. Thus, Hsp70 binds a hydrophobic residue preceding the turn motif, protecting PKC from down-regulation and sustaining the signaling lifetime of the kinase.

Download Full-text

Abnormal protein kinase C down regulation and reduced substrate levels in non-phorbol ester-responsive 3T3-TNR9 cells.

Molecular and Cellular Biology ◽

10.1128/mcb.10.5.2122 ◽

1990 ◽

Vol 10 (5) ◽

pp. 2122-2132 ◽

Cited By ~ 7

Author(s):

H P Biemann ◽

R L Erikson

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Phorbol Ester ◽

Kinase Activity ◽

Subcellular Location ◽

Parental Cell ◽

Down Regulation ◽

Kinase C ◽

Cell Variant ◽

Swiss 3T3 Cell

The cell line TNR9 (E. Butler-Gralla and H. R. Herschman, J. Cell. Physiol. 107:59-67, 1981) in a Swiss 3T3 cell variant that expresses protein kinase C (PKC) but is mitogenically nonresponsive to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). We have found that PKCs purified from variant and parental cells are identical as judged by kinase activity, protease mapping, and column chromatography. We analyzed cellular levels and subcellular location of PKC in TPA-treated 3T3 and TNR9 cells via immunoprecipitation of [35S]methionine-labeled protein and assay of immune-complex PKC kinase activity. TNR9 cells grew to higher densities than parental 3T3 cells. TNR9 cells at maximal density did not down regulate PKC in response to long-term TPA treatment. We compared the 80-kilodalton (kDa) PKC substrate phosphorylation in 3T3 and TNR9 cells by using two-dimensional gels and found that TNR9 cells treated with TPA for 30 min contained only 10 to 15% as much 32Pi associated with the 80-kDa as did parental cells. The TNR9 80-kDa substrate was present at reduced levels compared with the parental-cell 80-kDa substrate as judged by immunoblot and silver staining. Thus, the loss of mitogenic responsiveness to TPA in TNR9 cells is accompanied by resistance to TPA-mediated down regulation of PKC and reduced phosphosubstrate levels.

Download Full-text