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J. Biol. Chem., Vol. 283, Issue 1, 76-86, January 4, 2008

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Phosphorylation of Serine 68 in the IB Kinase (IKK)-binding Domain of NEMO Interferes with the Structure of the IKK Complex and Tumor Necrosis Factor--induced NF-B Activity^*

Lysann Palkowitsch, Julia Leidner, Sankar Ghosh, and Ralf B. Marienfeld¹

From the Institute of Physiological Chemistry, Ulm University, Albert-Einstein-Allee 11, Ulm 89081, Germany and the Department of Immunobiology and Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06520-8011

The IB-Kinase (IKK) complex is a multisubunit protein complex crucial for signal-induced phosphorylation of the IB proteins and thus controls the activity of the transcription factor NF-B. Besides the two kinases IKK and IKKβ, the IKK complex contains NEMO/IKK, an additional subunit with regulatory and adaptor functions. NEMO not only confers structural stability to the IKK complex but also participates in the activation process of the IKK complex by linking the IKK subunits to upstream activators. In this study we analyze the IKKβ-mediated phosphorylation of the IKK-binding domain of NEMO. In vitro, IKKβ phosphorylates three serine residues in the domain of NEMO at positions 43, 68, and 85. However, mutational analysis revealed that only the phosphorylation of serine 68 in the center of the IKK-binding domain plays an essential role for the formation and the function of the IKK complex. Thus, Ser⁶⁸ phosphorylation attenuates the amino-terminal dimerization of NEMO as well as the IKKβ-NEMO interaction. In contrast, the NEMO-IKK interaction was only mildly affected by the phosphorylation of Ser⁶⁸. However, functional analysis revealed that Ser⁶⁸ phosphorylation primarily affects the activity of IKK. Furthermore, in complementation experiments of NEMO-deficient murine embryonic fibroblasts, a S68A-NEMO mutant enhanced, whereas a S68E mutant decreased, TNF--induced NF-B activity, thus emphasizing the inhibitory role of the Ser⁶⁸ phosphorylation on the signal-induced NF-B activity. Finally, we provide evidence that the protein phosphatase PP2A is involved in the regulation of the Ser⁶⁸-based mechanism. In summary, we provide evidence for a signal-induced phosphorylation-dependent alteration of the IKK complex emphasizing the dynamic nature of this multisubunit kinase complex.

Received for publication, October 26, 2007

^* This work was supported by an Emmy-Noether-Fellowship of the Deutsche Forschungsgemeinschaft (Grant MA-2367/1-1). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 49-0731-502-2897; Fax: 49-0731-502-2892; E-mail: ralf.marienfeld{at}uni-ulm.de.

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