Peptides corresponding to the N and C termini of I kappa B-alpha, -beta, and -epsilon as probes of the two catalytic subunits of I kappa B kinase, IKK-1 and IKK-2

The signal-inducible phosphorylation of serines 32 and 36 of I kappa B-alph a is the key step in regulating the subsequent ubiquitination and proteolys is of I kappa B-alpha, which then releases NF-KB to promote gene transcript ion, The multisubunit I kappa B kinase (msIKK) responsible for this phospho rylation contains two catalytic subunits, termed IKK-1 and IKK-2. Using rec ombinant IKK-2, a kinetic pattern consistent with a random, sequential bind ing mechanism was observed with the use of a peptide corresponding to amino acids 26-42 of I kappa B-alpha Values of 313 mu M 15.5 mu M, 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 dissociati on constant for the other substrate, was determined to be 0.2. Interestingl y, the recombinant IKK-1 subunit gave similar values for alpha and K-ATP, b ut values of 1950 mu M and 0.016 min(-1) were calculated for K-peptide and k(cat), respectively. This suggests that the IKK-2 catalytic subunit provid es nearly all of the catalytic activity of the msIKK complex with the IKK-1 subunit providing Little contribution to catalysis. Using peptides corresp onding to different regions of I kappa B-alpha within amino acids 21-47, it was shown that amino acids 31-37 provide most binding interactions (-4.7 k cal/mol of binding free energy) of the full-length I kappa B-alpha (-7.9 kc al/mol) with the IKK-2, This is consistent with the observation that IKK-2 is able to phosphorylate the I kappa B-beta and I kappa B-epsilon proteins, which have consensus phosphorylation sites nearly identical to that of ami no acids 31-37 of I kappa B-alpha A peptide corresponding to amino acids 27 9-303 in the C-terminal domain of I kappa B-alpha was unable to activate IK K-2 to phosphorylate an N-terminal peptide, which is in contrast to the res ults observed with the msIKK. Moreover, the IKK-2 catalyzes the phosphoryla tion of the full-length I kappa B-alpha and the amino acid 26-42 peptide wi th nearly equal efficiency, while the msIKK catalyzes the phosphorylation o f the full-length I kappa B-alpha 25,000 times more efficiently than the 26 -42 peptide, Therefore, the C terminus of I kappa B-alpha is important in a ctivating the msIKK through interactions with subunits other than the IKK-2 .