Control of IκB-α proteolysis by site-specific, signal-induced phosphorylation

Science

Volumn 267, Issue 5203, 1995, Pages 1485-1488

  Brown, Keith ^a   Gerstberger, Susan ^a   Carlson, Louise ^a   Franzoso, Guido ^a   Siebenlist, Ulrich ^a  

^a NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

IMMUNOGLOBULIN ENHANCER BINDING PROTEIN; SERINE; TRANSCRIPTION FACTOR;

ANIMAL CELL; ARTICLE; CONTROLLED STUDY; HUMAN; HUMAN CELL; MOUSE; NONHUMAN; PATHOGENESIS; PRIORITY JOURNAL; PROTEIN DEGRADATION; PROTEIN PHOSPHORYLATION; PROTEIN TRANSPORT; SIGNAL TRANSDUCTION; STRESS; TRANSCRIPTION INITIATION;

ANIMALIA;

EID: 0028986075     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.7878466     Document Type: Article

References (24)

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10. r transfectants were selected by incubation of the etectroporated cells for 3 to 4 weeks in medium supplemented with geneticin G418 [(Gibco-BRL, Garthersburg, MD), at 400 μg/ml for 1 week; 200 μg/ml. 2 to 3 weeks] and then screened for human IκB-α by immunoblotting. The results with each transfected IκB-α species were confirmed with independently transfected cell clones.
11. V. Bours et al., Mol. Cell. Biol. 12, 685 (1992); G. Franzoso et al., Nature 369, 339 (1992); V. Bours et al., Cell 72, 729 (1993).
12. V. Bours et al., Mol. Cell. Biol. 12, 685 (1992); G. Franzoso et al., Nature 369, 339 (1992); V. Bours et al., Cell 72, 729 (1993).
13. V. Bours et al., Mol. Cell. Biol. 12, 685 (1992); G. Franzoso et al., Nature 369, 339 (1992); V. Bours et al., Cell 72, 729 (1993).
14. We constructed the ΔN and ΔC IκB-α mutants by cloning into PMT2T (9) the 1296-base pair (bp) Xho I-Eco RI fragment and the 921-bp Eco RI-Xmn I fragment, respectively, of the human gene (15). Point mutants of IκB-α were obtained in a Bluescript plasmid (Stratagene, La Jolla, CA) containing nucleotides 1 to 1550 of IκB-α complementary DNA (15) by site-directed mutagenesis [P. Bressler et al., J. Virol. 67, 288 (1993)]; the mutants were then cloned into PMT2T as 1550-bp Eco RI fragments encoding fulllength IκB-α.
15. To permit detection of the newly phosphorylated IκB-α in this experiment, we included phosphatase inhibitors during cell extraction (2, 5). To enhance detection, we trapped the phosphorylated intermediate by blocking proteolysis with calpain inhibitor I (2).
16. K. Brown, S. Gerstberger, L. Carlson, G. Franzoso, U. Siebenlist, unpublished data.
17. 36 mutations act directly at one or both of these sites to block phosphorylation.
18. S. Haskill et al., Cell 65, 1281 (1991).
19. S. Rogers, R. Wells, M. Rechsteiner, Science 234, 364 (1986).
20. A similar AC mutant has previously been shown to inhibit DNA binding of NF-κB in vitro [E. N. Hatada, M. Naumann, C. Scheidereit, EMBO J. 12, 2781 (1993)].
21. 32 (12). Phosphatase treatment resulted in a single spot (12). Endogenous IκB-α from human cells was indistinguishable from exogenously expressed human IκB-α on these gels.
22. R. de Martin et al., EMBO J. 12, 2773 (1994).
23. It remains to be determined if one or both serines are phosphorylated. Mutation of one of these closely spaced serines may block phosphorylation of the other; even if both serines are phosphorylated, their phosphorylation may be linked.
24. We thank K. Kelly and A. S. Fauci for review of the manuscript, A. S. Fauci for continued support, and Y. Ward for expert help and advice.