PMID- 10527519
OWN - NLM
STAT- MEDLINE
DCOM- 19991213
LR  - 20161124
IS  - 0003-2697 (Print)
IS  - 0003-2697 (Linking)
VI  - 274
IP  - 2
DP  - 1999 Oct 15
TI  - Assay for IkappaB kinases using an in vivo biotinylated IkappaB protein
      substrate.
PG  - 220-8
AB  - IkappaB kinases (IKK)-1 and -2 are related kinases that are induced by stimuli
      such as TNF or IL-1 to phosphorylate serines 32 and 36 of IkappaBalpha, the
      regulatory subunit of the transcription factor NF-kappaB. A procedure for an IKK 
      protein kinase assay is described that uses an in vivo biotinylated IkappaB
      protein substrate, [gamma-(33)P]ATP, and capture onto a streptavidin membrane.
      Residues 1-54 of the IkappaBalpha substrate were expressed as a fusion with
      glutathione S-transferase (GST) and a short (22 amino acid) biotinylation
      sequence that allowed modification during bacterial expression. Using the
      streptavidin capture assay the phosphorylation activities of recombinant IKK-1
      and -2 were characterized. The assay provided a convenient way to compare IKK
      protein and peptide substrate preferences; biotinylated GST-IkappaBalpha(1-54)
      was more readily phosphorylated by both IKK-1 and IKK-2 compared to biotinylated 
      myelin basic protein or a 20-mer biotinylated peptide containing serines 32 and
      36 of IkappaBalpha. IKK-1 had 83-fold less activity than IKK-2, and the IKK-1+2
      complex had approximately 2-fold more activity than IKK-2. IKK-1+2 and IKK-2 had 
      similar K(m) values for ATP and GST-biotin-IkappaB(1-54) and were similarly
      inhibited by staurosporine and two of its analogues K252a and K252b, suggesting
      that most of the IkappaBalpha kinase activity in the IKK-1+2 complex may be
      attributed to IKK-2. Several features of the assay including the broad linear
      binding range of the streptavidin membranes for the protein substrate
      GST-biotin-IkappaB(1-54) (1-4000 pmol of protein/cm(2)), the low background, and 
      its capacity for both biotinylated peptides and proteins make it a useful tool
      for quantitating IKK activity. These factors and the ease of expressing in vivo
      biotinylated GST fusions will make this assay approach suitable for a wide
      variety of protein kinases.
CI  - Copyright 1999 Academic Press.
FAU - Wisniewski, D
AU  - Wisniewski D
AD  - Department of Molecular Design and Diversity, Merck Research Laboratories,
      Rahway, New Jersey, 07065, USA.
FAU - LoGrasso, P
AU  - LoGrasso P
FAU - Calaycay, J
AU  - Calaycay J
FAU - Marcy, A
AU  - Marcy A
LA  - eng
SI  - GENBANK/AF012890
SI  - GENBANK/AF029684
PT  - Journal Article
PL  - United States
TA  - Anal Biochem
JT  - Analytical biochemistry
JID - 0370535
RN  - 0 (I-kappa B Proteins)
RN  - 6SO6U10H04 (Biotin)
RN  - EC 2.7.11.1 (Protein-Serine-Threonine Kinases)
RN  - EC 2.7.11.10 (CHUK protein, human)
RN  - EC 2.7.11.10 (I-kappa B Kinase)
RN  - EC 2.7.11.10 (IKBKB protein, human)
RN  - EC 2.7.11.10 (IKBKE protein, human)
SB  - IM
MH  - Biotin/metabolism
MH  - Biotinylation
MH  - Cell Line
MH  - Cloning, Molecular
MH  - Electrophoresis, Polyacrylamide Gel
MH  - Humans
MH  - I-kappa B Kinase
MH  - I-kappa B Proteins/*metabolism
MH  - Mass Spectrometry
MH  - Molecular Sequence Data
MH  - Protein-Serine-Threonine Kinases/*analysis
EDAT- 1999/10/21 00:00
MHDA- 1999/10/21 00:01
CRDT- 1999/10/21 00:00
PHST- 1999/10/21 00:00 [pubmed]
PHST- 1999/10/21 00:01 [medline]
PHST- 1999/10/21 00:00 [entrez]
AID - 10.1006/abio.1999.4287 [doi]
AID - S0003-2697(99)94287-2 [pii]
PST - ppublish
SO  - Anal Biochem. 1999 Oct 15;274(2):220-8. doi: 10.1006/abio.1999.4287.