Functional Analysis of AP-2 α and μ2 Subunits

Motley, Alison M.; Berg, Nicola; Taylor, Marcus J.; Sahlender, Daniela A.; Hirst, Jennifer; Owen, David J.; Robinson, Margaret S.

doi:10.1091/mbc.e06-05-0452

Cited by 87 publications

(109 citation statements)

References 29 publications

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“…First, depletion of AP2M1 or NUMB reduced HCV entry. Second, consistent with the previously reported dominant negative effects of AP2M1 and NUMB (67,76,110), overexpression of either an AP2M1 or NUMB phosphorylation site mutant reduced HCV entry, albeit the NUMB mutant exhibited such a phenotype in HCVpp but not in HCVcc infection. Third, AP-2 overexpression rescued the effect of AAK1 and GAK depletion on HCV entry.…”

Section: Discussionsupporting

confidence: 88%

AP-2-Associated Protein Kinase 1 and Cyclin G-Associated Kinase Regulate Hepatitis C Virus Entry and Are Potential Drug Targets

Neveu

Ziv-Av

Barouch-Bentov

et al. 2015

J Virol

115

155

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Hepatitis C virus (HCV) enters its target cell via clathrin-mediated endocytosis. AP-2-associated protein kinase 1 (AAK1) and cyclin G-associated kinase (GAK) are host kinases that regulate clathrin adaptor protein (AP)-mediated trafficking in the endocytic and secretory pathways. We previously reported that AAK1 and GAK regulate HCV assembly by stimulating binding of the subunit of AP-2, AP2M1, to HCV core protein. We also discovered that AAK1 and GAK inhibitors, including the approved anticancer drugs sunitinib and erlotinib, could block HCV assembly. Here, we hypothesized that AAK1 and GAK regulate HCV entry independently of their effect on HCV assembly. Indeed, silencing AAK1 and GAK expression inhibited entry of pseudoparticles and cell culture grown-HCV and internalization of Dil-labeled HCV particles with no effect on HCV attachment or RNA replication. AAK1 or GAK depletion impaired epidermal growth factor (EGF)-mediated enhanced HCV entry and endocytosis of EGF receptor (EGFR), an HCV entry cofactor and erlotinib's cancer target. Moreover, either RNA interference-mediated depletion of AP2M1 or NUMB, each a substrate of AAK1 and/or GAK, or overexpression of either an AP2M1 or NUMB phosphorylation site mutant inhibited HCV entry. Last, in addition to affecting assembly, sunitinib and erlotinib inhibited HCV entry at a postbinding step, their combination was synergistic, and their antiviral effect was reversed by either AAK1 or GAK overexpression. Together, these results validate AAK1 and GAK as critical regulators of HCV entry that function in part by activating EGFR, AP2M1, and NUMB and as the molecular targets underlying the antiviral effect of sunitinib and erlotinib (in addition to EGFR), respectively. IMPORTANCEUnderstanding the host pathways hijacked by HCV is critical for developing host-centered anti-HCV approaches. Entry represents a potential target for antiviral strategies; however, no FDA-approved HCV entry inhibitors are currently available. We reported that two host kinases, AAK1 and GAK, regulate HCV assembly. Here, we provide evidence that AAK1 and GAK regulate HCV entry independently of their role in HCV assembly and define the mechanisms underlying AAK1-and GAK-mediated HCV entry. By regulating temporally distinct steps in the HCV life cycle, AAK1 and GAK represent "master regulators" of HCV infection and potential targets for antiviral strategies. Indeed, approved anticancer drugs that potently inhibit AAK1 or GAK inhibit HCV entry in addition to assembly. These results contribute to an understanding of the mechanisms of HCV entry and reveal attractive host targets for antiviral strategies as well as approved candidate inhibitors of these targets, with potential implications for other viruses that hijack clathrin-mediated pathways. H epatitis C virus (HCV) is a major global health problem, estimated to infect 170 million people worldwide (1, 2). HCV persistence results in severe liver disease, including cirrhosis, liver failure, and hepatocellular carcinoma (reviewed in reference...

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Section: Discussionsupporting

confidence: 88%

AP-2-Associated Protein Kinase 1 and Cyclin G-Associated Kinase Regulate Hepatitis C Virus Entry and Are Potential Drug Targets

Neveu

Ziv-Av

Barouch-Bentov

et al. 2015

J Virol

115

155

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“…Based on previous work it was predicted that AP2 fluorescence (marked by mu2-mCherry) should markedly decrease before scission, indicating the polarized segregation of AP2 in the nascent bud [31] and/or loss from developing buds before clathrin [41] (though see [42]). This was indeed the case, and, in addition, the adaptor protein Eps15, TfR7 (i.e., the receptor cargo), and the F-BAR domain proteins FCHo1 and FCHo2 showed similar signatures, suggesting that these proteins were also polarized and/or lost from the developing bud before clathrin ( Figure 4A, 4B,and 4F).…”

Section: Clathrin Adaptor Proteins and Receptor Cargomentioning

confidence: 98%

A High Precision Survey of the Molecular Dynamics of Mammalian Clathrin-Mediated Endocytosis

Perrais²,

2011

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Dual colour total internal reflection fluorescence microscopy is a powerful tool for decoding the molecular dynamics of clathrin-mediated endocytosis (CME). Typically, the recruitment of a fluorescent protein-tagged endocytic protein was referenced to the disappearance of spot-like clathrin-coated structure (CCS), but the precision of spot-like CCS disappearance as a marker for canonical CME remained unknown. Here we have used an imaging assay based on total internal reflection fluorescence microscopy to detect scission events with a resolution of ,2 s. We found that scission events engulfed comparable amounts of transferrin receptor cargo at CCSs of different sizes and CCS did not always disappear following scission. We measured the recruitment dynamics of 34 types of endocytic protein to scission events: Abp1, ACK1, amphiphysin1, APPL1, Arp3, BIN1, CALM, CIP4, clathrin light chain (Clc), cofilin, coronin1B, cortactin, dynamin1/ 2, endophilin2, Eps15, Eps8, epsin2, FBP17, FCHo1/2, GAK, Hip1R, lifeAct, mu2 subunit of the AP2 complex, myosin1E, myosin6, NECAP, N-WASP, OCRL1, Rab5, SNX9, synaptojanin2b1, and syndapin2. For each protein we aligned ,1,000 recruitment profiles to their respective scission events and constructed characteristic ''recruitment signatures'' that were grouped, as for yeast, to reveal the modular organization of mammalian CME. A detailed analysis revealed the unanticipated recruitment dynamics of SNX9, FBP17, and CIP4 and showed that the same set of proteins was recruited, in the same order, to scission events at CCSs of different sizes and lifetimes. Collectively these data reveal the fine-grained temporal structure of CME and suggest a simplified canonical model of mammalian CME in which the same core mechanism of CME, involving actin, operates at CCSs of diverse sizes and lifetimes.

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“…Two mutant HeLa cell lines, expressing CD8-furin AKGL and CD8-furin AKGL-AAAA , were generated for this study. A QuikChange mutagenesis kit (Stratagene, La Jolla, CA) was used to introduce the mutations into the CD8-furin plasmid (Seaman, 2004), and stable cell lines were selected as described previously (Motley et al, 2006). For the comparison between HeLa and T-cells, a viral-specific CD8 T cell line was used (MacAry et al, 2004).…”

Section: Cell Linesmentioning

confidence: 99%

HIV-1 Nef-induced Down-Regulation of MHC Class I Requires AP-1 and Clathrin but Not PACS-1 and Is Impeded by AP-2

Lubben

Sahlender

Motley

et al. 2007

MBoC

Self Cite

106

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Major histocompatibility complex class I is down-regulated from the surface of human immunodeficiency virus (HIV)-1-infected cells by Nef, a virally encoded protein that is thought to reroute MHC-I to the trans-Golgi network (TGN) in a phosphofurin acidic cluster sorting protein (PACS) 1, adaptor protein (AP)-1, and clathrin-dependent manner. More recently, an alternative model has been proposed, in which Nef uses AP-1 to direct MHC-I to endosomes and lysosomes. Here, we show that knocking down either AP-1 or clathrin with small interfering RNA inhibits the down-regulation of HLA-A2 (an MHC-I isotype) by Nef in HeLa cells. However, knocking down PACS-1 has no effect, not only on Nef-induced down-regulation of HLA-A2 but also on the localization of other proteins containing acidic cluster motifs. Surprisingly, knocking down AP-2 actually enhances Nef activity. Immuno-electron microscopy labeling of Nef-expressing cells indicates that HLA-A2 is rerouted not to the TGN, but to endosomes. In AP-2-depleted cells, more of the HLA-A2 localizes to the inner vesicles of multivesicular bodies. We propose that depleting AP-2 potentiates Nef activity by altering the membrane composition and dynamics of endosomes and causing increased delivery of HLA-A2 to a prelysosomal compartment. INTRODUCTIONLike many viruses, human immunodeficiency virus (HIV)-1 has evolved strategies to avoid detection and destruction by the host. One such strategy is the down-regulation of major histocompatability complex (MHC) class I from the plasma membrane of HIV-1-infected cells, which prevents the cells from being attacked by cytotoxic T lymphocytes. MHC-I down-regulation has been shown to be a function of a virally encoded protein called Nef (Schwartz et al., 1996;Collins et al., 1998). Nef is one of the so-called "accessory proteins" of both human and simian immunodeficiency viruses: although not required for viral replication in vitro, Nef plays a key role in the development of acquired immunodeficiency syndrome. Nef is a small protein, only ϳ200 amino acids, but it has been reported to interact with a large number of host cell proteins, including several proteins that are involved in membrane traffic. These interactions are thought to be responsible for the ability of Nef to modulate the surface expression of MHC-I and other molecules (for review, see Collins and Baltimore, 1999;Piguet et al., 1999;Doms and Trono, 2000;Roeth and Collins, 2006).Among the binding partners that have been identified for Nef are the adaptor protein (AP) complexes. There are four AP complexes in mammalian cells, two of which, AP-1 and AP-2, are highly enriched in clathrin-coated vesicles (CCVs). AP-1 facilitates clathrin-mediated trafficking between the trans-Golgi network (TGN) and endosomes (although there is still some question about directionality), and AP-2 facilitates clathrin-mediated endocytosis (Robinson, 2004). AP-3, which seems to be able to act both in a clathrin-dependent and in a clathrin-independent manner (Dell'Angelica et al., 1998;Peden et al., 2...

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Functional Analysis of AP-2 α and μ2 Subunits

Cited by 87 publications

References 29 publications

AP-2-Associated Protein Kinase 1 and Cyclin G-Associated Kinase Regulate Hepatitis C Virus Entry and Are Potential Drug Targets

AP-2-Associated Protein Kinase 1 and Cyclin G-Associated Kinase Regulate Hepatitis C Virus Entry and Are Potential Drug Targets

A High Precision Survey of the Molecular Dynamics of Mammalian Clathrin-Mediated Endocytosis

HIV-1 Nef-induced Down-Regulation of MHC Class I Requires AP-1 and Clathrin but Not PACS-1 and Is Impeded by AP-2

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