PKC β pseudosubstrate

We showed previously that enteropathogenic Escherichia coli (EPEC) infection of intestinal epithelial cells induces inflammation by activating NF-kappa B and upregulating IL-8 expression. We also reported that extracellular signal-regulated kinases (ERKs) participate in EPEC-induced NF-kappa B activation but that other signaling molecules such as PKC zeta may be involved. The aim of this study was to determine whether PKC zeta is activated by EPEC and to investigate whether it also plays a role in EPEC-associated inflammation. EPEC infection induced the translocation of PKC zeta from the cytosol to the membrane and its activation as determined by kinase activity assays. Inhibition of PKC zeta by the pharmacological inhibitor rottlerin, the inhibitory myristoylated PKC zeta pseudosubstrate (MYR-PKC zeta-PS), or transient expression of a nonfunctional PKC zeta significantly suppressed EPEC-induced I kappa B alpha phosphorylation. Although PKC zeta can activate ERK, MYR-PKC zeta-PS had no effect on EPEC-induced stimulation of this pathway, suggesting that they are independent events. PKC zeta can regulate NF-kappa B activation by interacting with and activating I kappa B kinase (IKK). Coimmunoprecipitation studies showed that the association of PKC zeta and IKK increased threefold 60 min after infection. Kinase activity assays using immunoprecipitated PKC zeta-IKK complexes from infected intestinal epithelial cells and recombinant I kappa B alpha as a substrate showed a 2.5-fold increase in I kappa B alpha phosphorylation. PKC zeta can also regulate NF-kappa B by serine phosphorylation of the p65 subunit. Serine phosphorylation of p65 was increased after EPEC infection but could not be consistently attenuated by MYR-PKC zeta-PS, suggesting that other signaling events may be involved in this particular arm of NF-kappa B regulation. We speculate that EPEC infection of intestinal epithelial cells activates several signaling pathways including PKC zeta and ERK that lead to NF-kappa B activation, thus ensuring the proinflammatory response.

Vascular smooth muscle (VSM) plays an important role in maintaining vascular tone. In addition to Ca2+-dependent myosin light chain (MLC) phosphorylation, protein kinase C (PKC) is a major regulator of VSM function. PKC is a family of conventional Ca2+-dependent α, β, and γ, novel Ca2+-independent δ, ɛ, θ, and η, and atypical ξ, and ι/λ isoforms. Inactive PKC is mainly cytosolic, and upon activation it undergoes phosphorylation, maturation, and translocation to the surface membrane, the nucleus, endoplasmic reticulum, and other cell organelles; a process facilitated by scaffold proteins such as RACKs. Activated PKC phosphorylates different substrates including ion channels, pumps, and nuclear proteins. PKC also phosphorylates CPI-17 leading to inhibition of MLC phosphatase, increased MLC phosphorylation, and enhanced VSM contraction. PKC could also initiate a cascade of protein kinases leading to phosphorylation of the actin-binding proteins calponin and caldesmon, increased actin-myosin interaction, and VSM contraction. Increased PKC activity has been associated with vascular disorders including ischemia-reperfusion injury, coronary artery disease, hypertension, and diabetic vasculopathy. PKC inhibitors could test the role of PKC in different systems and could reduce PKC hyperactivity in vascular disorders. First-generation PKC inhibitors such as staurosporine and chelerythrine are not very specific. Isoform-specific PKC inhibitors such as ruboxistaurin have been tested in clinical trials. Target delivery of PKC pseudosubstrate inhibitory peptides and PKC siRNA may be useful in localized vascular disease. Further studies of PKC and its role in VSM should help design isoform-specific PKC modulators that are experimentally potent and clinically safe to target PKC in vascular disease.

Since the identification of protein kinase C (PKC) in the late 1970s, there have been many attempts to define its involvement in pancreatic beta-cell responses to physiological secretagogues. Early studies made use of PKC inhibitors such as polymyxin B and staurosporine, but their lack of selectivity made results difficult to interpret. Phorbol ester-induced PKC downregulation and measurements of PKC translocation within beta-cells provided useful information, but these studies were further complicated by the identification of novel PKC isoforms which do not possess diacylglycerol-binding sites or do not translocate upon stimulation. Second-generation PKC inhibitors, such as Ro 31-8220 and Go 6976, show improved selectivity and have helped clarify the situation. In addition, the use of antisense oligonucleotides or pseudosubstrate peptide inhibitors to selectively deplete or inhibit particular PKC isoforms has provided valuable information. The application of these varied methodologies has allowed us to develop a fuller understanding of the role played by PKC in beta-cell stimulus-response coupling.