A cytokine-responsive IkappaB kinase that activates the transcription factor NF-kappaB.
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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ABSTRACT: This study presents the anti-inflammatory and antioxidative properties of dried plum (Prunus domestica L.) polyphenols in macrophage RAW 264.7 cells. We hypothesized that dried plum polyphenols have strong anti-inflammatory and antioxidant properties against lipopolysaccharide (LPS)-induced production of the pro-inflammatory markers, nitric oxide (NO) and cyclooxygenase-2 (COX-2), and the lipid peroxidation product, malondialdehyde, in activated macrophage RAW 264.7 cells. To test this hypothesis, macrophage RAW 264.7 cells were stimulated with either 1 µg/ml (for measurement of NO production) or 1 ng/ml (for measurement of COX-2 expression) of LPS to induce inflammation and were treated with different doses of dried plum polyphenols (0.0, 0.1,1,10,100 and 1000 µg/ml). Dried plum polyphenols at a dose of 1000 µg/ml was able to significantly (P < 0.05) reduce NO production by 43%. Additionally, LPS-induced expression of COX-2 was significantly (P < 0.05) reduced by 100 and 1000 µg/ml dried plum polyphenols. To investigate the antioxidant activity of dried plum polyphenols, macrophage RAW 264.7 cells were stimulated with 100 µg/ml of FeSO4 + 1mM/ml of H2O2 to induce lipid peroxidation. Dried plum polyphenols at a dose of 1000 µg/ml showed a 32% reduction in malondialdehyde production. These findings indicate that dried plum polyphenols are potent anti-inflammatory and antioxidative agents in vitro.04/2015; DOI:10.1039/C5FO00173K
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ABSTRACT: Regulation of the IB␣ and IB␤ proteins is critical for modulating NF-B-directed gene expression. Both IB␣ and IB␤ are substrates for cellular kinases that phosphorylate the amino and carboxy termini of these proteins and regulate their function. In this study, we utilized a biochemical fractionation scheme to purify a kinase activity which phosphorylates residues in the amino and carboxy termini of both IB␣ and IB␤. Pep-tide microsequence analysis by capillary high-performance liquid chromatography ion trap mass spectroscopy revealed that this kinase was the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). DNA-PK phosphorylates serine residue 36 but not serine residue 32 in the amino terminus of IB␣ and also phosphor-ylates threonine residue 273 in the carboxy terminus of this protein. To determine the biological relevance of DNA-PK phosphorylation of IB␣, 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, IB␣ that was phosphorylated by DNA-PK was a more potent inhibitor of NF-B binding than nonphosphorylated IB␣. These results suggest that DNA-PK phosphorylation of IB␣ 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 IB 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 IB proteins have been identified including IB␣, IB␤, IB␥ (reviewed in reference 79), and IBε (80). IB␣ (41) and IB␤ (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 IB␣ and IB␤ and the nuclear translocation of NF-B (12, 17, 43, 73). IB present in the nucleus terminates the induction process in response to TNF-␣ and other activa-tors (2, 3, 60). IB␣ and IB␤ have distinct functional domains. For example , the N terminus and the ankyrin repeats of IB␣ 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 IB is regulated by its phosphor-ylation state. The C termini of the IB␣ and IB␤ 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 IB␣ (16, 17, 28, 32, 77) and 19 and 23 of IB␤ (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 IB␣ and IB␤ 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 IB␣. A protein complex migrating at approximately 700 kDa is capable of phosphorylating IB␣ on serine residues 32 and 36, resulting in IB␣ 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 IB␣ (27, 63, 65, 83, 85). Another kinase, RSK1, also phosphorylates the amino terminus of IB␣ (71). In contrast to IKK␣ and IKK␤, RSK1 phosphorylates IB␣ exclusively on serine residue 32. Cellular kinases are also capable of phosphorylating the carboxy terminus of IB␣. For example, casein kinase II phosphorylates seMolecular and Cellular Biology 08/1998; 18(7):270-7306. · 5.04 Impact Factor
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ABSTRACT: Focal adhesion kinase (FAK) is a major signaling molecule which functions downstream of integrins or in conjunction with mitogenic signaling pathways. FAK is overexpressed and/or activated in many types of human tumors, in which it promotes cell adhesion, survival, migration and invasion. In addition to FAK's ability to regulate signaling through its scaffolding activities, FAK encodes an intrinsic kinase activity. Although some FAK substrates have been identified, a more comprehensive analysis of substrates is lacking. In this study, we use a protein microarray to screen the human proteome for FAK substrates. We confirm that several of the proteins identified are bona fide in vitro FAK substrates, including several factors which are known to regulate the NFκB pathway. Finally, we identify a role for FAK's kinase activity in both canonical and non-canonical NFκB signaling. Our screen therefore represents the first high throughput screen for FAK substrates and provides the basis for future in-depth analysis of the role of FAK's kinase activity in the processes of tumorigenesis.International journal of biological sciences 01/2015; 11(4):404-10. DOI:10.7150/ijbs.10273 · 4.37 Impact Factor