The Role of Tyrosine Phosphorylation of Protein Kinase C Delta in Infection and Inflammation

Abstract

Protein Kinase C (PKC) is a family composed of phospholipid-dependent serine/threonine kinases that are master regulators of inflammatory signaling. The activity of different PKCs is context-sensitive and these kinases can be positive or negative regulators of signaling pathways. The delta isoform (PKCδ) is a critical regulator of the inflammatory response in cancer, diabetes, ischemic heart disease, and neurodegenerative diseases. Recent studies implicate PKCδ as an important regulator of the inflammatory response in sepsis. PKCδ, unlike other members of the PKC family, is unique in its regulation by tyrosine phosphorylation, activation mechanisms, and multiple subcellular targets. Inhibition of PKCδ may offer a unique therapeutic approach in sepsis by targeting neutrophil-endothelial cell interactions. In this review, we will describe the overall structure and function of PKCs, with a focus on the specific phosphorylation sites of PKCδ that determine its critical role in cell signaling in inflammatory diseases such as sepsis. Current genetic and pharmacological tools, as well as in vivo models, that are used to examine the role of PKCδ in inflammation and sepsis are presented and the current state of emerging tools such as microfluidic assays in these studies is described.

Keywords: PKC; PKCδ; inflammation; microfluidics; phosphorylation; sepsis.

Figure 1

Structure of the three main classes of Protein Kinase C (PKC)s along with their respective activators. The hinge domain separates the regulatory domain and the catalytic domain. The regulatory domain contains: the pseudosubstrate (binds to C4 when not activated) for keeping the enzyme inactive; the C1 domain (including C1A and C1B) for DAG/PS/phorbol ester binding for cPKCs and nPKCs; the C2 domain for Ca²⁺ binding; the C2-like domain for nPKC spatial distribution; and the C1 domain (in aPKCs) for PS binding. The catalytic domain contains the C3 domain for ATP binding and C4 domain for substrate/pseudosubstrate binding.

Figure 2

Schematic drawing of the activation steps of cPKCs. Following the three distinct phosphorylations at the activation loop, the turn motif, and the hydrophobic motif (for example, in human PKC β-II, corresponding to threonine 500, serine 641, and threonine 6601, respectively), PKCs are released into the cytosol, but with the pseudosubstrate occupying the substrate-binding site. Binding to Ca²⁺, PS, and DAG results in membrane translocation and subsequent conformational change, which releases the pseudosubstrate from the substrate-binding site.

Figure 3

Important amino acid sequences (activators, inhibitors, regulatory signals) and tyrosine phosphorylation sites on PKCδ. Adapted from Malavez et al., 2009 [27].

Figure 4

Microfluidic-based in vitro assay for studying the role of PKCδ in regulating neutrophil-endothelial cell interaction. (A) The assay is manufactured by soft lithography on polydimethylsiloxane (PDMS) and assembled on a microscope glass slide with plastic tubes (dark blue) allowing access to individual vascular channels and the tissue compartment. (B) 3D reconstruction of confocal images of human brain microvascular endothelial cells (HBMVEC) stained for F-actin with fluorescently labelled phalloidin (green) and for cell nuclei with Draq 5 (red) after 72 hrs of flow culture (0.1 μL/min). (C) PKCδ inhibition with a PKCδ-TAT peptide inhibitor (PKCδ-i) reduces neutrophil migration across activated HBMVEC. Data are presented as mean ± SEM (n = 3). ** p < 0.01, * p < 0.05 compared to the other two groups by ANOVA with Tukey-Kramer post-hoc. Reprinted with permission from Tang et al., 2018 [25].

Figure 5

Immunohistochemical analysis of PKCδ phosphorylation at tyrosine 155 (pPKCδ₁₅₅; red) in lung tissue sections at 24 h post-surgery of sham-operated animals (Sham) (A,B) and CLP-operated animals that received 200 μg/kg PKCδ-TAT (CLP + PKCδ-TAT) (E,F) or a similar volume of PBS vehicle only (CLP + PBS) (C,D) immediately following surgery. (A,C,E) Tissue sections were also stained for CD68 (green), a marker for the cells of the macrophage lineage. Yellow/orange indicates co-localization of pPKCδ₁₅₅ and CD68. (B,D,F) Tissue sections were also stained for rat endothelial cell antigen-1 (RECA-1; green), a marker for rat endothelial cells. Yellow/orange indicates co-localization of pPKCδ₁₅₅ and RECA-1. All scale bars = 100 microns. Reprinted with permission from Mondrinos et al., 2015 [24].