Elsevier

Methods in Enzymology

Volume 322, 2000, Pages 393-400
Methods in Enzymology

[36] - Assaying for IκB Kinase Activity

https://doi.org/10.1016/S0076-6879(00)22038-7Get rights and content

Introduction

The NF-κ-B/Rel transcription factors serve as key regulators of a wide variety of target genes that includes immune regulatory genes and growth control, cell survival, and cell death genes.1, 2, 3 NF-κ-B/Rel exists in various combinations of homo- and heterodimeric DNA-binding subunits derived from the expression of a family of five related genes.4 NF-κB is held in the cytoplasm in an inactive state by a group of inhibitor molecules collectively termed IκBs.5 IκBα, IκBβ, and IκBε are the three most widely studied of the IκBs. Cellular stimulation by a variety of inducing agents including proinflammatory cytokines, ultraviolet light (UV), phorbol esters, mitogens, and infection by some viruses and bacteria results in the proteolysis of the IκBs by the 26S proteasome and release of NF-κB.1,6, 7, 8 Other extracellular stimuli such as reoxygenation of cells or treatment of cells with pervanadate (a tyrosine phosphatase inhibitor) result in tyrosine phosphorylation of IκBα and cause its dissociation from NF-κB, thereby allowing activation of the transcription factor.9 The newly liberated NF-κB is rapidly translocated to the nucleus, where it activates its target genes.

The key event in NF-κB activation is its release from the IκBs, which in most instances occurs by proteolysis. Signal-induced IκB proteolysis is triggered only after the inducible phosphorylation of the IκBs at conserved amino-terminal serine residues.4,10 It has been unclear whether phosphorylation at these conserved serines resulted from the action of an induced IκB kinase(s) or the inactivation of a phosphatase. We now know that for a number of extracellular stimuli, especially tumor necrosis factor α (TNF-α) and interleukin 1 (IL-1), there is an inducible IκB kinase activity that exists as a high molecular weight (∼900,000) complex (IKK). The IKK complex was found to contain the two related kinases IKKα and IKKβ (also known as IKK1 and IKK2, respectively), and these subunits are believed to be the catalytic polypeptides.11, 12, 13, 14 Both IKKα and IKKβ are strongly activated by the proinflammatory cytokines TNF-α and IL-1, with kinetics that are consistent with the physiologically relevant phosphorylation of IκBα prior to its degradation.11, 12, 13 IKK complexes containing IKKα and IKKβ are activated to a lesser degree by the phorbol ester tetradecanoyl phorbol acetate (TPA) or phorbol myristate acetate (PMA) and UV.11, 12, 13 Biochemically, the identity of additional IKK components and the exact stoichiometry of IKKα and IKKβ in the IKK complex remain to be determined. In addition, it will be important to determine if homodimeric IKKα or IKKβ-IKK complexes exist and, if so, what their function(s) are.

Section snippets

Rationale

To ascertain if there is IκB kinase activity induced by a specific stimulus in a particular cell type under examination, the first step should be to examine the cellular extract for NF-κB DNA-binding activity. Assuming that there is NF-κB DNA-binding activity in response to the stimulus, IκB abundance levels should then be examined by immunoblot analysis. Immunoblot analysis using specific IκB antibodies will reveal whether the IκBs are degraded. If IκB is being degraded in response to

Immunoprecipitations

Cells are left untreated or stimulated for 10 min with TNF-α (20 ng/ ml) and then harvested by decanting the tissue culture medium and quickly washing the cells three times (5 ml each) with ice-cold phosphate-buffered saline (PBS). The cells are then scraped into 1 ml of ice-cold PBS and transferred to a 1.5-ml microcentrifuge tube and pelleted for 30 sec at 2000g, and the PBS is completely removed by vacuum. The cell pellet is then resuspended in solution I supplemented to 0.42 M NaCl and 10%

Chromatographic Purification of Inducible IκB Kinase

IKK activity can be purified approximately 25,000-fold by using the threestep purification scheme described below. For most applications this amount of enrichment is not necessary and a simple anion-exchange (Mono Q) chromatographic step coupled with a gel-filtration (Superose-6) step will yield ∼500-fold enrichment of the IKK activity. Tissue culture cells (3–5 × 107 cells), e.g., HeLa cells, are left either nontreated or stimulated with TNF-α (20 ng/ml) for 10 min and then washed with

Potential Advantages / Disadvantages and Problems

Potential problems that may arise in using the immunoprecipitation assay for IKK activity are relatively few. In fact, immunoprecipitation of IKK activity containing IKKα and IKKβ is the most rapid and efficient way to determine if a specific stimulus can activate IKK activity in the cell type of interest. The main problem that one may encounter in detecting IKK activity could arise from the stringency of the wash buffer containing urea. Some antibodies to IKKα, IKKβ, or other IKK components

First page preview

First page preview
Click to open first page preview

References (0)

Cited by (0)

View full text