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J. Biol. Chem., Vol. 282, Issue 41, 30303-30310, October 12, 2007

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Structure-Function Analysis of the WIP Role in T Cell Receptor-stimulated NFAT Activation

EVIDENCE THAT WIP-WASP DISSOCIATION IS NOT REQUIRED AND THAT THE WIP NH₂ TERMINUS IS INHIBITORY^*

Xiaoyun Dong, Genaro Patino-Lopez, Fabio Candotti, and Stephen Shaw¹

From the Experimental Immunology Branch, NCI, National Institutes of Health, Bethesda, Maryland 20892-1360 and the Genetics and Molecular Biology Branch, National Human Genome Research Institute, Bethesda, Maryland 20892

WASP and its binding partner WIP play important roles in T cells both in actin polymerization and in interleukin-2 transcription. Aberrations thereof contribute to the pathology of Wiskott-Aldrich syndrome (WAS). To directly evaluate the cooperativity of WIP and WASP in interleukin-2 transcription, we investigated how the WIP-WASP complex regulates NF-AT-mediated gene transcription. We developed an improved model system for analysis, using WIP and WASP cotransfection into Jurkat cells, in which strong induction of NFAT reporter activation is observed with anti-T cell receptor (TCR) antibody without the phorbol 12-myristate 13-acetate usually used previously. Using this system, our findings contradict a prevailing conceptual model of TCR-induced WIP-WASP dissociation by showing in three ways that the WIP-WASP complex mediates TCR-induced NFAT activation without dissociation. First, phosphorylation of WIP Ser⁴⁸⁸ does not cause dissociation of the WIP-WASP complex. Second, WIP-WASP complexes do not dissociate demonstrably after TCR stimulation. Third, a fusion protein of WIP to WASP efficiently mediates NFAT activation. Next, our studies clarify that WIP stabilization of WASP explains otherwise unexpected results in TCR-induced NFAT activation. Finally, we find that the NH₂ terminus of WIP is a highly inhibitory region for TCR-mediated transcriptional activation in which at least two elements contribute: the NH₂-terminal polyproline and the NH₂-terminal actin-binding WH2 domain. This suggests that WIP, like WASP, is subject to autoinhibition. Our data indicate that the WIP-WASP complex plays an important role in WASP stabilization and NFAT activation.

Received for publication, June 15, 2007 , and in revised form, July 31, 2007.

^* This research was supported in part by the Intramural Research Program of NCI, National Institutes of Health (NIH), and the National Human Genome Research Institute, NIH. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1-3.

¹ To whom correspondence should be addressed: National Institutes of Health, Bldg. 10, Rm. 4B36, 10 Center Dr., MSC 1360, Bethesda, MD 20892-1360. Tel.: 301-435-6499; Fax: 301-496-0887; E-mail: sshaw{at}nih.gov.

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