A cytokine-responsive IkappaB kinase that activates the transcription factor NF-kappaB.

Laboratory of Gene Regulation and Signal Transduction, Department of Pharmacology, University of California at San Diego, La Jolla 92093-0636, USA.
Nature (Impact Factor: 42.35). 09/1997; 388(6642):548-54. DOI: 10.1038/41493
Source: PubMed

ABSTRACT Nuclear transcription factors of the NF-kappaB/Rel family are inhibited by IkappaB proteins, which inactivate NF-kappaB by trapping it in the cell cytoplasm. Phosphorylation of IkappaBs marks them out for destruction, thereby relieving their inhibitory effect on NF-kappaB. A cytokine-activated protein kinase complex, IKK (for IkappaB kinase), has now been purified that phosphorylates IkappaBs on the sites that trigger their degradation. A component of IKK was molecularly cloned and identified as a serine kinase. IKK turns out to be the long-sought-after protein kinase that mediates the critical regulatory step in NF-kappaB activation.

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To determine the biological relevance of DNA-PK phosphorylation of I␬B␣, murine severe combined immunodeficiency (SCID) cell lines which lack the DNA-PKcs gene were analyzed. Gel retardation analysis using extract prepared from these cells demonstrated constitutive nuclear NF-␬B DNA binding activity, which was not detected in extracts prepared from SCID cells complemented with the human DNA-PKcs gene. Furthermore, I␬B␣ that was phosphorylated by DNA-PK was a more potent inhibitor of NF-␬B binding than nonphosphorylated I␬B␣. These results suggest that DNA-PK phosphorylation of I␬B␣ increases its interaction with NF-␬B to reduce NF-␬B DNA binding properties. NF-␬B comprises a family of proteins including p50, p52, p65 or RelA, p100, p105, and c-Rel which regulate the expression of a variety of cellular and viral genes (reviewed in references 7, 75, and 79). Each of these proteins contains a region known as the Rel homology domain which is critical for the DNA binding and dimerization properties of these proteins. One of the major regulatory mechanisms which control NF-␬B activity is the unique cellular localization of different members of this family. In unstimulated cells, p65 or RelA is nearly exclusively localized in the cytoplasm (4–6, 13, 34), but it trans-locates to the nucleus upon treatment of the cells with a variety of inducers such as phorbol esters, interleukin 1, and tumor necrosis factor alpha (TNF-␣) (43, 73). RelA dimerizes with other NF-␬B family members (7, 75, 79) and activates gene expression via its potent transactivation domain (8, 67, 70). Thus, cellular proteins which regulate the nuclear transloca-tion of NF-␬B are critical for the control of NF-␬B activation of viral and cellular genes. The I␬B proteins constitute a group of cytoplasmic proteins that bind to NF-␬B and sequester these proteins in the cyto-plasm by preventing their nuclear localization. A number of different I␬B proteins have been identified including I␬B␣, I␬B␤, I␬B␥ (reviewed in reference 79), and I␬Bε (80). I␬B␣ (41) and I␬B␤ (76) are the best studied of these regulatory proteins. Treatment of cells with a variety of agents such as phorbol esters, TNF-␣, and UV irradiation results in the degradation of I␬B␣ and I␬B␤ and the nuclear translocation of NF-␬B (12, 17, 43, 73). I␬B present in the nucleus terminates the induction process in response to TNF-␣ and other activa-tors (2, 3, 60). I␬B␣ and I␬B␤ have distinct functional domains. For example , the N terminus and the ankyrin repeats of I␬B␣ are required for the cytoplasmic regulation of NF-␬B while C-terminal sequences are required to regulate NF-␬B function in the nucleus (60). The activity of I␬B is regulated by its phosphor-ylation state. The C termini of the I␬B␣ and I␬B␤ proteins contain PEST domains with serine and threonine residues that are phosphorylated by cellular kinases which regulate the intrinsic stability of these proteins (10, 11, 25, 57, 61, 66, 81). In addition, the amino termini of these proteins each contain two closely spaced serine residues that are also capable of being phosphorylated by cellular kinases (16, 17, 28, 32, 77). Serine residues at positions 32 and 36 of I␬B␣ (16, 17, 28, 32, 77) and 19 and 23 of I␬B␤ (62) are phosphorylated when cells are treated with various agents such as TNF-␣ and phorbol esters. Phosphorylation of these residues leads to their ubiquitination and proteasome-mediated degradation (1, 23, 24, 28, 32, 58, 69, 77). Mutations of these amino-terminal serine residues in I␬B␣ and I␬B␤ prevent the degradation of these proteins upon treatment of cells with TNF-␣ or phorbol esters and inhibit the nuclear translocation of NF-␬B (16, 28, 62, 77). Biochemical fractionation has been performed to identify cellular kinases that are capable of phosphorylating I␬B␣. A protein complex migrating at approximately 700 kDa is capable of phosphorylating I␬B␣ on serine residues 32 and 36, resulting in I␬B␣ degradation by the proteasome (24, 51). Two related kinases isolated from a similar-size complex, IKK␣ and IKK␤, phosphorylate serine residues 32 and 36 in I␬B␣ (27, 63, 65, 83, 85). Another kinase, RSK1, also phosphorylates the amino terminus of I␬B␣ (71). In contrast to IKK␣ and IKK␤, RSK1 phosphorylates I␬B␣ exclusively on serine residue 32. Cellular kinases are also capable of phosphorylating the carboxy terminus of I␬B␣. For example, casein kinase II phosphorylates se
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