Intracellular Zinc as a Phosphorylation Modulator of Protein Kinase C Delta

Abstract

Protein kinase C delta (PKC delta) is a Ser/Thr-specific kinase that acts upstream of many important cellular processes including apoptosis, cell survival and proliferation. The activity and specificity of PKC delta is determined by its subcellular localization and phosphorylation state. Increase in the concentration of intracellular free zinc has been shown to promote pro-apoptotic events during hypoxia and other cellular stress. Recently, zinc has emerged as an important modulator of phosphorylation. We hypothesized that zinc modulates activity of PKC delta by inhibiting the phosphorylation of this kinase. To test the hypothesis, cultured HeLa cells were treated with a combination of zinc and zinc ionophore pyrithione, after the treatment, the phosphorylation state of PKC delta was evaluated. The changes in intracellular zinc was monitored with the cell-permeable zinc-specific fluorescent probe FluoZin-3, AM. The application of zinc and pyrithione resulted in rapid increases in intracellular zinc within 60 seconds of zinc application. Levels of intracellular zinc plateaued within 4 minutes of application and remained elevated for 10 minutes during observation. Intracellular zinc increase was not detected in the cells treated with pyrithione alone. Following confirmation of intracellular zinc increase, HeLa cells were exposed to 1, 10 and 50 μM zinc and 10 μM pyrithione for 30 minutes. Treated cells were then lysed and immunoblotted for phosphorylated PKC delta. We selected to investigate phosphorylation site in the activation loop of PKC delta at threonine 505 (T505) due to previous suggestions that this site influences substrate specificity of PKC delta. Our results indicate that zinc significantly inhibits phosphorylation of PKC delta at T505 in a concentration dependent manner, with higher zinc concentrations resulting in greater inhibition of phosphorylation. We attribute these results to three possible scenarios: 1) zinc inhibits kinases that phosphorylate PKC delta at T505; 2) zinc stimulates phosphatases that remove the phosphate from PKC delta at T505 or 3) zinc directly prevents phosphorylation at T505 by interacting with this phosphorylation site. We used molecular modeling software to further investigate potential interaction zinc has with the T505 phosphorylation site. Notably, we found a probable His-Cys3 zinc-binding site directly adjacent to T505 composed of residues H197, C200, C393 and C509. Energy-minimized models of this potential zinc-binding site indicated that, in the presence of zinc, H197 is oriented such that it blocks access to T505. This model suggests a structural mechanism by which zinc could act to inhibit phosphorylation of T505 in PKC and possibly other similar proteins. In summary, we have shown that zinc inhibits phosphorylation of T505 in the activation loop of PKC delta and we further propose a novel mechanism by which zinc directly interferes with phosphorylation by binding to a His-Cys3 zinc binding site that is proximal to T505. Support or Funding Information This research was supported in part by a NIH grant NS081629 to YVL. Solvent exposed surface of energy-minimized computer model of PKC delta with and without zinc. Zoom: sticks rendering of open phosphorylation site at Thr505 (magenta) without zinc and closed phosphorylation site when zinc binds to His197 (blue) and Cys200, Cys393 and Cys509 (yellow). Distances to zinc atom are in Angstroms. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.