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

Katelyn M. Atkins, Laurel Thomas, Robert T. Youker, Melanie J. Harriff, Franco Pissani, Huihong You, Gary Thomas

Research output: Contribution to journalArticle

61 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)11772-11784
Number of pages13
JournalJournal of Biological Chemistry
Volume283
Issue number17
DOIs
StatePublished - Apr 25 2008

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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