Research Directions - Kolettas group

Research Directions
NF-κB Signalling: The NF-κB transcription factors are pivotal regulators of gene expression programs culminating in stress-like responses. They bind to DNA as hetero- or homodimers that are selectively derived from five possible subunits (RelA/p65, c-Rel, RelB, p50 and p52). All NF-κB family members contain an N-terminal Rel homology domain that mediates DNA binding and dimerization and a nuclear localization domain. The Rel subfamily members RelA/p65, c-Rel and RelB also contain a C-terminal transactivation domain, which is absent in the p50 and p52 subunits. In addition the p50 and p52 subunits are processed from precursor proteins p105 (NF-κB1) and p100 (NF-κB2), respectively. The p50/p65 heterodimers are bound to IκBs (inhibitors of NF-κB) thereby sequestering them in the cytoplasm of most cells in the absence of a stress-like response. Kinases that directly activate NF-κB mediate the site-specific phosphorylation of two amino terminal serines on each IκB (Ser32/36 of IκBα), which makes adjacent lysines targets for ubiquitination thereby resulting in proteasome mediated IκB degradation. NF-κB is then free to translocate to the nucleus and bind DNA leading to the activation of a host of target genes. IκB phosphorylation is mediated by a high molecular weight signalsome complex comprising two direct IκB kinases, IKKα and IKKβ, and two molecules of a regulatory, docking/adapter protein, NEMO. IKKα and IKKβ are serine/threonine kinases possessing an amino-terminal catalytic domain and two carboxyl-proximal interaction motifs resembling leucine zipper and helix-loop-helix domains. Activation of IKKβ depends upon signal induced phosphorylation of Ser177/181 in its T-activation loop. NIK (NF-κB-inducing kinase) physically interacts with and specifically activates IKKα by phosphorylating IKKα at Ser 176/180 in vitro and in vivo. NF-κB can be activated through three Signalling pathways: the classical IKK-dependent pathway leading to nuclear translocation of p50/p65 heterodimers, the non-classical or alternative pathway involving activation of IKKα homodimers leading to nuclear translocation of RelB/p52 and a subset of p50/p65 heterodimers and an atypical IKK-independent pathway activating p50/p65 heterodimers. In response to DNA damage, ATM activation induces the inhibitor of NF-κB kinase (IKK) complex and NEMO/IKKγ was shown to associate with ATM after the induction of DSBs leading to IKKβ activation suggesting that the DNA damage-induced NF-κB response is essential for cell survival.

NF-κB Signalling Patways.
The canonical pathway is induced by TNFα and IL-1 and results in the IKKβ-dependent activation of p50/RelA(p65) heterodimers.

The non-canonical pathway is induced by LPS resulting in the activation of IKKα leading to the activation and nuclear translocation of p52/RelB heterodimers that target distinct κB elements.

Research is focused on: (a) oncogenic stress and (b) Nuclear factor-kappa B (NF-κB) implicated in: (i) replicative (RS) and stress-induced senescence (SIPS) such as ROS- and oncogene-induced senescence (OIS), and (ii) DNA damage leading to growth arrest or apoptosis. Specifically, our research is focused on the regulation and mechanisms of action of the inhibitor of NF-κB kinase (IKK) complex, which is essential for the activation of the NF-κB transcription factor family that are pivotal regulators of stress-like responses and the survival and growth of normal and malignant cells

1. Role NF-κB on cellular senescence and apoptosis
We study, in collaboration with Prof. Kenneth B. Marcu (Biochemistry & Cell Biology Dept, Stony Brook University, NY, USA) the molecular and biochemical mechanisms of cellular senescence of human diploid fibroblasts (HDFs). We investigate the role of NF-κB and its activating IKK kinases, IKKα and IKKβ, on both replicative and stress-induced senescence of HDFs, but also on oxidative stress-induced apoptosis of tumour cells.

2. Role NF-κB on the regulation of genes implicated in DNA damage response
We study, in collaboration with Prof. K. B. Marcu and Prof. Vassilis Gorgoulis (Histology & Embryology Dept, University of Athens Medical School, Greece) the role of NF-κB and its activating IKK kinases, IKKα and IKKβ, in DNA damage in response to oxidative stress induced by hydrogen peroxide and chemotherapeutic agents.

3. Role NF-κB on cellular transformation and tumourigenicity
We also study, in collaboration with Prof. K. B. Marcu and Prof. Vassilis Gorgoulis, the role of the NF-κB and its activating IKK kinases, IKKα and IKKβ, in oncogene-induced transformation and tumourigenicity, and in epithelial-mesenchymal transition (EMT) and metastasis.

4. Interaction of NF-κB signalling pathways with p53

We also study, in collaboration with Prof. K. B. Marcu, the interaction between NF-κB Signalling pathways with the tumor suppressor protein p53. We have generated normal and cancer cells carrying wild-type and different p53 mutants in normal and NF-κB-compromised background and investigate their responses to chemotherapeutic agents. In addition, we are attempting to investigate the role of p53 in cellular senescence of human diploid fibroblasts.