Here we report the identification of a novel PMA-inducible IkappaB kinase complex, distinct from the well-characterized high-molecular weight IkappaB kinase complex containing IKKalpha, IKKbeta, and IKKgamma. We have characterized one kinase from this complex, which we designate IKKepsilon. Although recombinant IKKepsilon directly phosphorylates only serine 36 of IKBalpha, the PMA-activated endogenous IKKepsilon complex phosphorylates both critical serine residues. Remarkably, this activity is due to the presence of a distinct kinase in this complex. A dominant-negative mutant of IKKepsilon blocks induction of NF-kappaB by both PMA and activation of the T cell receptor but has no effect on the activation of NF-KB by TNFalpha or IL-1. These observations indicate that the activation of NF-kappaB requires multiple distinct IkappaB kinase complexes, which respond to both overlapping and discrete signaling pathways.

A critical step in the signal-induced activation of the transcription factor NF-B is the site-specific phosphorylation of its inhibitor, IB, that targets the latter for degradation by the ubiquitin-proteasome pathway. We have previously shown that mitogen-activated protein kinase͞ERK kinase kinase 1 (MEKK1) can induce both this site-specific phosphorylation of IB␣ at Ser-32 and Ser-36 in vivo and the activity of a high molecular weight IB kinase complex in vitro. Subsequently, others have identified two proteins, IB kinase ␣ (IKK-␣) and IB kinase ␤ (IKK-␤), that are present in a tumor necrosis factor ␣-inducible, high molecular weight IB kinase complex. These kinases are believed to directly phosphorylate IB based on the examination of the kinase activities of IKK immunoprecipitates, but more rigorous proof of this has yet to be demonstrated. We show herein that recombinant IKK-␣ and IKK-␤ can, in fact, directly phosphorylate IB␣ at Ser-32 and Ser-36, as well as homologous residues in IB␤ in vitro, and thus are bona fide IB kinases. We also show that MEKK1 can induce the activation of both IKK-␣ and IKK-␤ in vivo. Finally, we show that IKK-␣ is present in the MEKK1-inducible, high molecular weight IB kinase complex and treatment of this complex with MEKK1 induces phosphorylation of IKK-␣ in vitro. We conclude that IKK-␣ and IKK-␤ can mediate the NF-B-inducing activity of MEKK1.The transcriptional activator protein NF-B plays a critical role in immune and inflammatory responses (for recent reviews, see refs. 1 and 2). NF-B is sequestered in the cytoplasm of most cell types by virtue of its association with the IB family of inhibitor proteins, which include IB␣, IB␤, and IB. Upon exposure to a wide variety of stimuli, including the proinflammatory cytokines tumor necrosis factor ␣ (TNF-␣) and interleukin 1, UV-irradiation, virus infection, bacterial lipopolysaccharide, and oxidative stress, the IB protein is phosphorylated at its N terminus. In the case of IB␣, this occurs at Ser-32 and Ser-36; the corresponding residues in IB␤ are Ser-19 and Ser-23. This phosphorylation then targets IB for degradation by the ubiquitin-proteasome pathway, thereby liberating NF-B, which is then free to translocate to the nucleus and bind to DNA (3).The mechanism by which the phosphorylation of the N terminus of IB occurs has recently been the subject of intense investigation (4, 5). A large, approximately 700-kDa multiprotein complex isolated from uninduced HeLa cells was originally demonstrated to phosphorylate IB␣ at . This complex could be activated in vitro by the mitogenactivated protein kinase kinase kinase (MAP3K) MEKK1, and the latter was shown to induce the site-specific phosphorylation of IB␣ in vivo (7). A model was proposed in which MEKK1 activates IB␣ kinase activity, which in turn leads to the phosphorylation of IB␣. Inherent in this model was the possibility that other MAP3Ks might also activate IB␣ kinase activity (7).Subsequently, the MAP3K NF-B inducing kinase (NIK) was identified in a yeast two-hybrid scr...

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Nearly 350 IgG-based therapeutics are approved for clinical use or are under development for many diseases lacking adequate treatment options. These include molecularly engineered biologicals comprising the IgG Fc-domain fused to various effector molecules (so-called Fc-fusion proteins) that confer the advantages of IgG, including binding to the neonatal Fc receptor (FcRn) to facilitate in vivo stability, and the therapeutic benefit of the specific effector functions. Advances in IgG structure-function relationships and an understanding of FcRn biology have provided therapeutic opportunities for previously unapproachable diseases. This article discusses approved Fc-fusion therapeutics, novel Fc-fusion proteins and FcRn-dependent delivery approaches in development, and how engineering of the FcRn–Fc interaction can generate longer-lasting and more effective therapeutics.