PMID- 10593898
OWN - NLM
STAT- MEDLINE
DCOM- 20000127
LR  - 20190508
IS  - 0021-9258 (Print)
IS  - 0021-9258 (Linking)
VI  - 274
IP  - 51
DP  - 1999 Dec 17
TI  - Peptides corresponding to the N and C termini of IkappaB-alpha, -beta, and
      -epsilon as probes of the two catalytic subunits of IkappaB kinase, IKK-1 and
      IKK-2.
PG  - 36146-52
AB  - The signal-inducible phosphorylation of serines 32 and 36 of IkappaB-alpha is the
      key step in regulating the subsequent ubiquitination and proteolysis of
      IkappaB-alpha, which then releases NF-kappaB to promote gene transcription. The
      multisubunit IkappaB kinase (msIKK) responsible for this phosphorylation contains
      two catalytic subunits, termed IKK-1 and IKK-2. Using recombinant IKK-2, a
      kinetic pattern consistent with a random, sequential binding mechanism was
      observed with the use of a peptide corresponding to amino acids 26-42 of
      IkappaB-alpha. Values of 313 microM, 15.5 microM, and 1.7 min(-1) were obtained
      for K(peptide), K(ATP), and k(cat), respectively. The value of alpha, a factor by
      which binding of one substrate changes the dissociation constant for the other
      substrate, was determined to be 0.2. Interestingly, the recombinant IKK-1 subunit
      gave similar values for alpha and K(ATP), but values of 1950 microM and 0.016
      min(-1) were calculated for K(peptide) and k(cat), respectively. This suggests
      that the IKK-2 catalytic subunit provides nearly all of the catalytic activity of
      the msIKK complex with the IKK-1 subunit providing little contribution to
      catalysis. Using peptides corresponding to different regions of IkappaB-alpha
      within amino acids 21-47, it was shown that amino acids 31-37 provide most
      binding interactions (-4.7 kcal/mol of binding free energy) of the full-length
      IkappaB-alpha (-7.9 kcal/mol) with the IKK-2. This is consistent with the
      observation that IKK-2 is able to phosphorylate the IkappaB-beta and
      IkappaB-epsilon proteins, which have consensus phosphorylation sites nearly
      identical to that of amino acids 31-37 of IkappaB-alpha. A peptide corresponding 
      to amino acids 279-303 in the C-terminal domain of IkappaB-alpha was unable to
      activate IKK-2 to phosphorylate an N-terminal peptide, which is in contrast to
      the results observed with the msIKK. Moreover, the IKK-2 catalyzes the
      phosphorylation of the full-length IkappaB-alpha and the amino acid 26-42 peptide
      with nearly equal efficiency, while the msIKK catalyzes the phosphorylation of
      the full-length IkappaB-alpha 25,000 times more efficiently than the 26-42
      peptide. Therefore, the C terminus of IkappaB-alpha is important in activating
      the msIKK through interactions with subunits other than the IKK-2.
FAU - Burke, J R
AU  - Burke JR
AD  - Drug Discovery Research, Bristol-Myers Squibb Pharmaceutical Research Institute, 
      Princeton, New Jersey 08543, USA. james.burke@bms.com
FAU - Wood, M K
AU  - Wood MK
FAU - Ryseck, R P
AU  - Ryseck RP
FAU - Walther, S
AU  - Walther S
FAU - Meyers, C A
AU  - Meyers CA
LA  - eng
PT  - Journal Article
PL  - United States
TA  - J Biol Chem
JT  - The Journal of biological chemistry
JID - 2985121R
RN  - 0 (I-kappa B Proteins)
RN  - 0 (Peptide Fragments)
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  - Amino Acid Sequence
MH  - Binding Sites
MH  - Enzyme Activation
MH  - Humans
MH  - I-kappa B Kinase
MH  - I-kappa B Proteins/*chemistry/*metabolism
MH  - Kinetics
MH  - Molecular Sequence Data
MH  - Peptide Fragments/chemistry/metabolism
MH  - Protein Binding
MH  - Protein-Serine-Threonine Kinases/*metabolism
MH  - Substrate Specificity
EDAT- 1999/12/14 00:00
MHDA- 1999/12/14 00:01
CRDT- 1999/12/14 00:00
PHST- 1999/12/14 00:00 [pubmed]
PHST- 1999/12/14 00:01 [medline]
PHST- 1999/12/14 00:00 [entrez]
AID - 10.1074/jbc.274.51.36146 [doi]
PST - ppublish
SO  - J Biol Chem. 1999 Dec 17;274(51):36146-52. doi: 10.1074/jbc.274.51.36146.