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.
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.
Burke
J R
JR
Drug Discovery Research, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543, USA. james.burke@bms.com
Wood
M K
MK
Ryseck
R P
RP
Walther
S
S
Meyers
C A
CA
D000595:Amino Acid Sequence; D001665:Binding Sites; D004789:Enzyme Activation; D006801:Humans; D051550:I-kappa B Kinase; D020672:I-kappa B Proteins / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D013379:Substrate Specificity
D020672:I-kappa B Proteins; D010446:Peptide Fragments; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human