Direct phosphorylation of IκB by IKKα and IKKβ: Discrimination between free and NF-κB-bound substrate

Science

Volumn 281, Issue 5381, 1998, Pages 1360-1363

  Zandi, Ebrahim ^a   Chen, Yi ^a   Karin, Michael ^a  

^a NONE

Author keywords

[No Author keywords available]

Indexed keywords

IMMUNOGLOBULIN ENHANCER BINDING PROTEIN; PROTEIN KINASE; CHUK PROTEIN, HUMAN; HELIX LOOP HELIX PROTEIN; HYBRID PROTEIN; I KAPPA B KINASE; IKBKB PROTEIN, HUMAN; IKBKE PROTEIN, HUMAN; LEUCINE ZIPPER PROTEIN; ONCOPROTEIN; PROTEIN SERINE THREONINE KINASE; RECOMBINANT PROTEIN; RELB PROTEIN, HUMAN; TRANSCRIPTION FACTOR; TRANSCRIPTION FACTOR RELB;

ANIMAL CELL; ARTICLE; ENZYME SPECIFICITY; HUMAN; HUMAN CELL; INFLAMMATION; INSECT; NONHUMAN; PRIORITY JOURNAL; PROTEIN DOMAIN; PROTEIN PHOSPHORYLATION; PROTEIN PROTEIN INTERACTION; PROTEIN PURIFICATION; ANIMAL; ARMYWORM; CELL LINE; CHEMISTRY; DIMERIZATION; DRUG ANTAGONISM; ENZYME ACTIVATION; GENETICS; HELA CELL; METABOLISM; MUTATION; PHOSPHORYLATION;

ANIMALS; CELL LINE; DIMERIZATION; ENZYME ACTIVATION; HELA CELLS; HELIX-LOOP-HELIX MOTIFS; HUMANS; I-KAPPA B KINASE; LEUCINE ZIPPERS; MUTATION; NF-KAPPA B; PHOSPHORYLATION; PROTEIN-SERINE-THREONINE KINASES; PROTO-ONCOGENE PROTEINS; RECOMBINANT FUSION PROTEINS; RECOMBINANT PROTEINS; SPODOPTERA; TRANSCRIPTION FACTOR RELB; TRANSCRIPTION FACTORS;

EID: 0032575379     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: None     Document Type: Article

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16. 6-FLAG-IKKβ. Primers for FLAG-tagged and HA-tagged IKKβ were 5′-AGCTATCGCGGCCGCGACTACAAGGACGACGATGACAAAAGCTGGTCACCTTCC-3′, 5′-AGCTAGCGCGGCCGCTACCCTTACGATGTTCCTGATTACGCTAGCTGGTCACCTTCC-3′, and 5′-TGACTCCCGGGTCATGAGGCCTGCTCC-3′. For the generation of recombinant viruses, we followed the manufacturer's (Pharmingen) recommendations for growth and maintenance of Sf 9 cells, preparation of recombinant baculoviruses, and infection of the cells. For in vivo recombination, we transfected the transfer vectors and BaculoGold DNA into Sf9 cells using SuperFect (Qiagen). To prepare a control viral stock, we cotransfected Sf 9 cells with unmodified pAcSGHis NT-B vector and BaculoGold DNA. After 5 days, culture supernatants were collected and amplified by two rounds of infection. For expression of both IKKα and IKKβ, the corresponding recombinant viruses were mixed in various ratios before infection of Sf 9 cells. The efficiency of infection was determined by indirect double immunofluorescence using antibodies to HA and FLAG.
17. 6-FLAG-IKKβ proteins were produced in Sf9 cells after infection with high-titer recombinant viruses. The proteins were purified as described (1). Briefly, cell extracts were fractionated on Q-Sepharose and the fractions containing IKK were applied to an adenosine triphosphate (ATP) affinity column. Bound proteins were eluted with 20 mM ATP and applied to a Superose 6 gel filtration column. The fractions containing IKK (200 to 250 kD range) were then bound to Ni-Sepharose and washed extensively with AP buffer (500 mM NaCl, 1% Triton X-100, and 2 M urea) (1) and eluted with 200 mM imidazole. The eluted IKKs were either separated by SDS-polyacrylamide gel electrophoresis (PAGE) (8% gel) and visualized by silver staining or further purified by immunoaffinity chromatography with anti-FLAG (M2)-coupled Sepharose for IKKβ or anti-HA-coupled Sepharose for IKKα.
18. 36, IKKα and to a lesser extent IKKβ also phosphorylate sites close to the COOH-terminus of IκBα (1).
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22. Purified IKK proteins were cross-linked in the presence of bovine serum albumin (BSA, 1 mg/ml) in AP buffer containing 300 mM NaCl, 1% Triton X-100, and 2 mM ethylene glycol-bis(succinimidylsuccinate) (EGS; Pierce) for 20 min at room temperature. The cross-linker was inactivated by addition of 500 mM tris-HCl (pH 8.0). Cross-linked proteins were either immunoprecipitated (as described below) before separation by SDS-PAGE (4.5% gel) or directly subjected to SDS-PAGE. Proteins were visualized by immunoblotting with specific antibodies. For immunoprecipitation, the cross-linked IKK preparations were divided in two; one portion was immunoprecipitated with anti-IKKα and the other with anti-IKKβ. The immune complexes were separated by SDS-PAGE (4.5% gel) and proteins were transferred to a membrane and immunoblotted with an antibody to the other subunit than the one used for immunoprecipitation.
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30. We thank D. Rothwarf and F. Mercurio for insightful discussions and reagents; Z. Radic for help with kinetic analysis; G. Ghosh for purified NF-κB proteins (18); G. Cadwell for excellent technical assistance; and B. Thompson for help with manuscript preparation. E.Z. was supported by a junior postdoctoral fellowship from the American Cancer Society. Supported by NIH grant Al 43477 (M.K.).