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J. Biol. Chem., Vol. 283, Issue 17, 11772-11784, April 25, 2008

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HIV-1 Nef Binds PACS-2 to Assemble a Multikinase Cascade That Triggers Major Histocompatibility Complex Class I (MHC-I) Down-regulation

ANALYSIS USING SHORT INTERFERING RNA AND KNOCK-OUT MICE^*

From the Vollum Institute, Portland, Oregon 97239

Human immunodeficiency virus, type 1, negative factor (Nef) initiates down-regulation of cell-surface major histocompatibility complex-I (MHC-I) by assembling an Src family kinase (SFK)-ZAP70/Syk-phosphoinositide 3-kinase (PI3K) cascade through the sequential actions of two sites, Nef EEEE⁶⁵ and PXXP⁷⁵. The internalized MHC-I molecules are then sequestered in endosomal compartments by a process requiring Nef Met²⁰. How Nef assembles the multikinase cascade to trigger the MHC-I down-regulation pathway is unknown. Here we report that EEEE⁶⁵-dependent binding to the sorting protein PACS-2 targets Nef to the paranuclear region, enabling PXXP⁷⁵ to bind and activate a trans-Golgi network (TGN)-localized SFK. This SFK then phosphorylates ZAP-70 to recruit class I PI3K by interaction with the p85 C-terminal Src homology 2 domain. Using splenocytes and embryonic fibroblasts from PACS-2^-/- mice, we confirm genetically that Nef requires PACS-2 to localize to the paranuclear region and assemble the multikinase cascade. Moreover, genetic loss of PACS-2 or inhibition of class I PI3K prevents Nef-mediated MHC-I down-regulation, demonstrating that short interfering RNA knockdown of PACS-2 phenocopies the gene knock-out. This PACS-2-dependent targeting pathway is not restricted to Nef, because PACS-2 is also required for trafficking of an endocytosed cation-independent mannose 6-phosphate receptor reporter from early endosomes to the TGN. Together, these results demonstrate PACS-2 is required for Nef action and sorting of itinerant membrane cargo in the TGN/endosomal system.

Received for publication, September 10, 2007 , and in revised form, January 29, 2008.

^* This work was supported by National Institutes of Health National Research Service Awards DK076343 [GenBank] (to R. T. Y.), T32 NS007381 (to M. J. H.), T32 GM71338 (to F. P.), R01 AI49793, and R01 DK37274. 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. S1 and S2.

¹ To whom correspondence should be addressed: Vollum Institute, 3181 Sam Jackson Park Rd., Portland, OR 97239. Tel.: 503-494-6955; Fax: 503-494-1218; E-mail: thomasg{at}ohsu.edu.

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