Chromatinized Protein Kinase C-θ: Can It Escape the Clutches of NF-κB?

Abstract

We recently provided the first description of a nuclear mechanism used by Protein Kinase C-theta (PKC-θ) to mediate T cell gene expression. In this mode, PKC-θ tethers to chromatin to form an active nuclear complex by interacting with proteins including RNA polymerase II, the histone kinase MSK-1, the demethylase LSD1, and the adaptor molecule 14-3-3ζ at regulatory regions of inducible immune response genes. Moreover, our genome-wide analysis identified many novel PKC-θ target genes and microRNAs implicated in T cell development, differentiation, apoptosis, and proliferation. We have expanded our ChIP-on-chip analysis and have now identified a transcription factor motif containing NF-κB binding sites that may facilitate recruitment of PKC-θ to chromatin at coding genes. Furthermore, NF-κB association with chromatin appears to be a prerequisite for the assembly of the PKC-θ active complex. In contrast, a distinct NF-κB-containing module appears to operate at PKC-θ targeted microRNA genes, and here NF-κB negatively regulates microRNA gene transcription. Our efforts are also focusing on distinguishing between the nuclear and cytoplasmic functions of PKCs to ascertain how these kinases may synergize their roles as both cytoplasmic signaling proteins and their functions on the chromatin template, together enabling rapid induction of eukaryotic genes. We have identified an alternative sequence within PKC-θ that appears to be important for nuclear translocation of this kinase. Understanding the molecular mechanisms used by signal transduction kinases to elicit specific and distinct transcriptional programs in T cells will enable scientists to refine current therapeutic strategies for autoimmune diseases and cancer.

Chromatinized Protein Kinase C-θ Directly Regulates Inducible Genes in Epithelial to Mesenchymal Transition and Breast Cancer Stem Cells

“…This is consistent with active PKC-acting within the nucleus as part of an active transcription complex on inducible genes enriched in the mesenchymal state and elevated in CSCs. The data are also consistent with our recent report showing that PKC-exists as a chromatin-tethered adapter protein on immune responsive genes and is important for inflammatory responses (38). An accessible chromatin configuration prevails in the mesenchymal state across the proximal promoter of key inducible genes such as CD44 and is enriched in an active chromatin complex consisting of NF-B, PKC-, and Pol II-associated complex, which is conducive to enhanced CD44 transcription (see Fig.…”

“…To ascertain the contribution of nuclear PKC-in CSC-inducible-gene regulation, we utilized previously validated PKC-wildtype (WT) and nuclear localization sequence (NLS) constructs (38). We observed reduced nuclear localization of PKC-in NLS mutant-transfected MCF-7 cells ( high /CD24 low CSCs compared to PKC-WT constructs in MCF-IM cells (Fig.…”

Protein kinase C in the immune system: from signalling to chromatin regulation

“…The SPT‐like motif appears to reside close to where the regulatory and kinase/catalytic domains are and deletions in the regulatory or kinase domains of PKC are shown to affect nuclear translocation . Mutation studies substituting the SPT motif in PKC θ with an alternative motif that cannot be phosphorylated impaired the localization of PKC θ to the nucleus, suggesting that phosphorylation of SPT is required for nuclear translocation . Interestingly, mutating the putative NLS motif has no effect on the distribution of PKC in the cell, suggesting that the SPT‐like motif may be an alternative NLS motif used by signalling kinases that do not have the canonical NLS for nuclear transport .…”

“…In addition to phosphorylating histones, PKC can also interact with and phosphorylate histone modifiers (Table ). Specifically in the immune system, PKC θ not only has a cytoplasmic signalling role during TCR activation, it is also present in the nucleus of T cells to regulate transcription of inducible immune genes . In stimulated human Jurkat T cells, PKC θ associates with the N‐terminal transcriptional activation domain of p300 and phosphorylates S384 on p300 leading to its transcriptional activation .…”

“…). The assembly of this complex is dependent on NF‐ κ B as inhibiting NF‐ κ B impaired PKC θ , RNA polymerase II and lysine‐specific demethylase 1 recruitment to immune gene promoters upon TCR activation . This formation of an active transcriptional complex on key inducible genes can also be seen in breast cancer stem cells, where PKC θ acts as a molecular switch that regulates epithelial to mesenchymal transition .…”