No abstract

The IκB kinase (IKK) complex is an enzyme that regulates the activation of the transcription factor NF‐κB in response to a diverse set of extracellular signals to control expression of many genes including those that mediate the cell survival program involving immune and inflammatory responses. IKK is composed of two catalytic subunits: IKK2/β and IKK1/α in addition to a non‐catalytic subunit known as the NF‐κB essential modulator (NEMO)/IKKγ. In this study, we focus on IKKβ which is the subunit primarily responsible for signal‐dependent activity in pro‐inflammatory signaling. Like many protein kinases, IKKβ activation is achieved through phosphorylation of two serine residues (S177 and S181 in IKKβ) located within its activation loop. However, the molecular details of how the activation loop serine residues undergo signal‐induced phosphorylation is still unknown. The x‐ray crystal structure and mutational analyses from our laboratory identified specific crystallographic interfaces (V‐shaped and KD‐KD interfaces) that are critical for IKKβ activation. Disruption of these interfaces reduced the ability of IKKβ to be activated in cells, suggesting the significance of protein‐protein contacts in activation. It is known that an N‐terminal segment of NEMO comprising the IKKβ binding domain (IKKBD) is responsible for a stable interaction with the C‐terminal NEMO binding domain (NBD) of IKKβ. Here, we propose a mechanistic model of IKKβ activation which invokes a transient second site interaction between the NEMO Ubiquitin binding in ABIN and NEMO (UBAN) domain and the IKKβ V‐shaped interface via a ubiquitin (Ub)‐chain. Engagement of poly‐Ub chain‐bound NEMO to IKKβ at this second site leads to conformational changes of the activation loop in an allosteric manner, resulting in the phosphorylation of S177 and S181. Cell transfection studies using V‐shaped interface mutants indicates that the second site of interaction critical for IKKβ activation coincides with the V‐shaped interface in a NEMO‐dependent manner. In vitro binding assays show that the NEMO UBAN (which lacks the IKKBD) binds to IKKβ (devoid of the NBD) in the presence of poly‐Ub. In vitro activation assays reveal that the activation loop is phosphorylated in an ATP, NEMO UBAN, and poly‐Ub‐dependent manner. We conclude that the molecular interactions between the V‐shaped interface of IKKβ and the poly‐Ub‐bound NEMO UBAN is responsible for signal‐dependent IKKβ activation loop phosphorylation. Our biochemical and cell‐based results give a detailed molecular understanding behind IKKβ activation in pro‐inflammatory signaling.Support or Funding InformationThis project was funded by an NIH R01 grant to Gourisankar Ghosh and supported by the California Metabolic Research Foundation at San Diego State University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cell division is a tightly regulated process, and if not controlled, may lead to the formation of tumors. Epidermal growth factor receptor (EGFR) is a transmembrane receptor that causes cell division in a wide range of cell types in response to several different growth factors. Mutations that activate EGFR in the absence of growth factors can contribute to cancer. The Dalton School SMART Team (Students Modeling A Research Topic) modeled EGFR using 3D printing technology. EGFR is a kinase that phosphorylates other proteins to turn them on or off. EGFR consists of an extracellular ligand‐binding domain, an anchoring intramembrane segment, a juxtamembrane connector segment, and a phosphorylating intracellular kinase domain. Generally, EGFR remains in an inactive form and becomes active when a ligand growth factor binds to the extracellular domain. Ligand binding changes the shape of the intracellular kinase domain, including the active site, turning it on. Active EGFR initiates a signaling pathway that causes cell growth and division. However, mutations in EGFR, including deletion of the extracellular domain or kinase domain point mutations, can activate EGFR in the absence of a growth factor, which may cause the formation of tumors because cells grow and divide uncontrollably. Therefore, EGFR is a potential target for cancer therapy. Supported by a grant from the Camille and Henry Dreyfus Foundation.

In Drosophila, the IMD pathway is indispensable for proper innate immune responses. Infection by gram‐negative bacteria elicits a signaling cascade culminating in the rapid induction of antimicrobial peptide gene expression by the NF‐κB transcription factor Relish. Signal‐dependent activation of Relish is an essential component in the IMD pathway and is regulated by the Drosophila melanogaster IκB Kinase (DmIKK) complex. DmIKK is composed of two subunits: the catalytic subunit IKKβ and non‐catalytic subunit IKKγ. Like many protein kinases, DmIKK must be activated for proper catalysis. Although there has been extensive research into the signaling pathways of the Drosophila innate immune response, little is known about the molecular mechanisms that lead to DmIKK activation. Here we report expression, purification, and in vitro characterization of a catalytically active multi‐subunit DmIKK complex. Support or Funding Information This work is supported in part by the California Metabolic Research Foundation. S. Cohen is a recipient of the Arne N. Wick predoctoral fellowship.