SummaryWe have defined the regions of the cytoplasmic domain of the leukocyte integrin lymphocyte function-associated antigen 1 (LFA-1) that are required for active binding of its extracellular domain to intercellular adhesion molecule 1 (ICAM-1). The NH2-terminal 28 amino acids in the cytoplasmic domain are dispensable, but a segment of 5 amino acids including three contiguous threonines (758-760) and Phe 766 in the COOH-terminal third of the cytoplasmic domain are required for binding to ICAM-1. Mutation and phosphoamino acid analysis show that Set 756 is the major residue phosphorylated in response to phorbol ester. Furthermore, multiple mutations demonstrate that serine phosphorylation can be dissociated from phorbol ester-stimulated binding of LFA-1 to ICAM-1. The sites we have defined are previously unremarked, are well conserved in the ~31, 133, and B7 integrin subunits, and may be of broad importance in regulating adhesiveness of integrins.

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Activation of the transcription factor NF-B is regulated by the phosphorylation and subsequent degradation of its inhibitory subunit, IB. A large multiprotein complex, the IB kinase (IKK), catalyzes the phosphorylation of IB. The two kinase components of the IKK complex, IKK␣ and IKK␤, were overexpressed in insect cells and purified to homogeneity. Both purified IKK␣ and IKK␤ specifically catalyzed the phosphorylation of the regulatory serine residues of IB␣. Hence, IKK␣ and IKK␤ were functional catalytic subunits of the IKK complex. Purified IKK␣ and IKK␤ also preferentially phosphorylated serine as opposed to threonine residues of IB␣, consistent with the substrate preference of the IKK complex. Kinetic analysis of purified IKK␣ and IKK␤ revealed that the kinase activity of IKK␤ on IB␣ is 50 -60-fold higher than that of IKK␣. The primary difference between the two activities is the K m for IB␣. The kinetics of both IKK␣ and IKK␤ followed a sequential Bi Bi mechanism. No synergistic effects on IB␣ phosphorylation were detected between IKK␣ and IKK␤. Thus, in vitro, IKK␣ and IKK␤ are two independent kinases of IB␣.The transcription factor NF-B plays a critical role in immune and inflammatory responses. It is regulated by the signaling of receptors for inflammatory cytokines such as TNF-␣, 1 IL-1, or other external stimuli (1). In resting cells, NF-B is sequestered in the cytoplasm through its association with inhibitory proteins termed IB. Two IB proteins, IB␣ and IB␤, are rapidly phosphorylated at Ser residues in the Nterminal region upon stimulation by TNF-␣ and IL-1. The regulated phosphorylation is at Ser-32 and Ser-36 of IB␣ and, correspondingly, Ser-19 and Ser-23 of IB␤ (2, 3). The more recently cloned IB isoform, IB⑀, also contains the two conserved Ser residues at the N terminus for signal-induced degradation (4). Phosphorylated IB␣ and IB␤ are subsequently ubiquitinated and undergo ubiquitin-dependent degradation by the 26 S proteasome (3, 5). Degradation of IB results in the release of NF-B, which then translocates to the nucleus, where it up-regulates the transcription of its target genes (1).A 500 -900-kDa protein complex that contains the TNF-␣-induced IB kinase (IKK) has been purified and characterized independently by two groups (6, 7). The IKK complex phosphorylates IB␣ at the specific Ser residues that target the protein for ubiquitination and degradation. Two kinase subunits of the IKK complex, denoted IKK␣ and IKK␤, have been cloned (6 -10). IKK␣ or IKK␤ overexpressed in mammalian cells specifically phosphorylates IB␣ and IB␤ after immunoprecipitation, and their kinase activities can be induced by TNF-␣ or IL-1 (6 -10). In HeLa cells, expression of antisense IKK␣ inhibited NF-B activation by TNF-␣ or IL-1 (6). Furthermore, overexpression of dominant-negative mutants of either IKK␣ or IKK␤ blocked TNF-␣/IL-1-induced NF-B activation (8, 10). Thus, both IKK␣ and IKK␤ contribute to the activity of the IKK complex and are involved in NF-B activation. An additional protein kinase, NF-B-inducing kina...