High expression of Notch-1 and Jagged-1 mRNA correlates with poor prognosis in breast cancer. Elucidating the cross-talk between Notch and other major breast cancer pathways is necessary to determine which patients may benefit from Notch inhibitors, which agents should be combined with them, and which biomarkers indicate Notch activity in vivo. We explored expression of Notch receptors and ligands in clinical specimens, as well as activity, regulation, and effectors of Notch signaling using cell lines and xenografts. Ductal and lobular carcinomas commonly expressed Notch-1, Notch-4, and Jagged-1 at variable levels. However, in breast cancer cell lines, Notch-induced transcriptional activity did not correlate with Notch receptor levels and was highest in estrogen receptor α–negative (ERα–), Her2/Neu nonoverexpressing cells. In ERα+ cells, estradiol inhibited Notch activity and Notch-1IC nuclear levels and affected Notch-1 cellular distribution. Tamoxifen and raloxifene blocked this effect, reactivating Notch. Notch-1 induced Notch-4. Notch-4 expression in clinical specimens correlated with proliferation (Ki67). In MDA-MB231 (ERα–) cells, Notch-1 knockdown or γ-secretase inhibition decreased cyclins A and B1, causing G2 arrest, p53-independent induction of NOXA, and death. In T47D:A18 (ERα+) cells, the same targets were affected, and Notch inhibition potentiated the effects of tamoxifen. In vivo, γ;-secretase inhibitor treatment arrested the growth of MDA-MB231 tumors and, in combination with tamoxifen, caused regression of T47D:A18 tumors. Our data indicate that combinations of antiestrogens and Notch inhibitors may be effective in ERα+ breast cancers and that Notch signaling is a potential therapeutic target in ERα– breast cancers.
Macroautophagy (hereafter autophagy) is a ubiquitous process in eukaryotic cells that is integrally involved in various aspects of cellular and organismal physiology. The morphological hallmark of autophagy is the formation of double-membrane cytosolic vesicles, autophagosomes, which sequester cytoplasmic cargo and deliver it to the lysosome or vacuole. Thus, autophagy involves dynamic membrane mobilization, yet the source of the lipid that forms the autophagosomes and the mechanism of membrane delivery are poorly characterized. The TRAPP complexes are multimeric guanine nucleotide exchange factors (GEFs) that activate the Rab GTPase Ypt1, which is required for secretion. Here we describe another form of this complex (TRAPPIII) that acts as an autophagy-specific GEF for Ypt1. The Trs85 subunit of the TRAPPIII complex directs this Ypt1 GEF to the phagophore assembly site (PAS) that is involved in autophagosome formation. Consistent with the observation that a Ypt1 GEF is directed to the PAS, we find that Ypt1 is essential for autophagy. This is an example of a Rab GEF that is specifically targeted for canonical autophagosome formation.utophagy is a catabolic process in which damaged or superfluous cytoplasmic components are degraded in response to stress conditions; it is evolutionarily conserved in eukaryotes and is integrally involved in development and physiology (1, 2). The morphological hallmark of autophagy is the formation of doublemembrane cytosolic vesicles, autophagosomes, which sequester cytoplasm. The autophagosomes then fuse with the lysosome, resulting in the degradation of the cargo. The mechanism of autophagosome formation is distinct from that used for vesicle formation in the secretory or endocytic pathways and is said to be de novo in that it does not occur by direct budding from a preexisting organelle. Instead, a nucleating structure, the phagophore, appears to expand by the addition of membrane possibly through vesicular fusion. One consequence of this mechanism is that it allows the sequestration of essentially any sized cargo, including intact organelles or invasive microbes, and this capability is critical to autophagic function. When autophagy is induced there is a substantial demand for membrane, and a major question in the field concerns the membrane origin; nearly every organelle has been implicated in this role (3). The early secretory pathway is likely one such membrane source for autophagy (4, 5).Rab GTPases are key regulators of membrane traffic that mediate multiple events including vesicle tethering and membrane fusion. These molecular switches cycle between an inactive (GDP-bound) and active (GTP-bound) conformation. The yeast Rab Ypt1, which is essential for ER-Golgi and Golgi traffic (6), is activated by the multimeric guanine nucleotide exchange factor (GEF) called TRAPP (7,8). Two forms of the TRAPP complexes have been identified (9). These two complexes share several subunits, including four (Bet3, Bet5, Trs23, and Trs31) that are essential to activate Ypt1. How each of th...
How the directionality of vesicle traffic is achieved remains an important unanswered question in cell biology. The Sec23p/Sec24p coat complex sorts the fusion machinery (SNAREs) into vesicles as they bud from the endoplasmic reticulum. Vesicle tethering to the Golgi begins when the tethering factor TRAPPI binds to Sec23p. Where the coat is released and how this event relates to membrane fusion is unknown. Here we use a yeast transport assay to demonstrate that an ER-derived vesicle retains its coat until it reaches the Golgi. A Golgi-associated kinase, Hrr25p (CK1δ ortholog), then phosphorylates the Sec23p/Sec24p complex. Coat phosphorylation and dephosphorylation are needed for vesicle fusion and budding, respectively. Additionally, we show that Sec23p interacts in a sequential manner with different binding partners, including TRAPPI and Hrr25p, to ensure the directionality of ER-Golgi traffic and prevent the back-fusion of a COPII vesicle with the ER. These events are conserved in mammalian cells.
The chemokine receptor CXCR4 is rapidly targeted for lysosomal degradation by the E3 ubiquitin ligase atrophin-interacting protein 4 (AIP4). Although it is known that AIP4 mediates ubiquitination and degradation of CXCR4 and that perturbations in these events contribute to disease, the mechanisms mediating AIP4-dependent regulation of CXCR4 degradation remain poorly understood. Here we show that AIP4 directly interacts with the amino-terminal half of nonvisual arrestin-2 via its WW domains. We show that depletion of arrestin-2 by small interfering RNA blocks agonist-promoted degradation of CXCR4 by preventing CXCR4 trafficking from early endosomes to lysosomes. Surprisingly, CXCR4 internalization and ubiquitination remain intact, suggesting that the interaction between arrestin-2 and AIP4 is not required for ubiquitination of the receptor at the plasma membrane but perhaps for a later post-internalization event. Accordingly, we show that activation of CXCR4 promotes the interaction between AIP4 and arrestin-2 that is consistent with a time when AIP4 co-localizes with arrestin-2 on endocytic vesicles. Taken together, our data suggest that the AIP4⅐arrestin-2 complex functions on endosomes to regulate sorting of CXCR4 into the degradative pathway.The chemokine receptor CXCR4, a G protein-coupled receptor (GPCR), 3 together with its cognate ligand, stromal cell-derived factor-1␣, also termed CXCL12, play an important role in several biological processes such as development of the heart and brain, leukocyte chemotaxis, and stem cell homing (1-3). Although CXCR4 dysregulation has been linked to several pathologies, especially cancer, the molecular mechanisms regulating CXCR4 remain poorly understood (4, 5). Activated CXCR4 is targeted for lysosomal degradation through a pathway involving ubiquitination of carboxyl-terminal tail lysine residues mediated by the E3 ubiquitin ligase atrophininteracting protein 4 (AIP4) (6, 7). AIP4 belongs to the neural precursor cell-expressed developmentally down-regulated gene 4-like family of homologous to E6-AP carboxyl-terminal domain E3 ubiquitin ligases, which interact with their target proteins either directly or indirectly via their WW domains or possibly other domains (8, 9).In addition, AIP4 regulates endosomal sorting of activated CXCR4 by targeting the receptor to the endosomal sorting complex required for transport pathway (7), which is a complex network of proteins that recognize and sort ubiquitinated cargo into the multivesicular body (10). Entry into this pathway requires the action of HRS, a ubiquitin-binding protein localized to flat clathrin lattices on endosomes adjacent to invaginating domains, where it sequesters ubiquitinated cargo destined for entry into the multivesicular body for subsequent degradation (11, 12). CXCR4 and AIP4 localize to HRS-positive microdomains on endosomes (7). In addition, AIP4 mediates CXCR4-dependent ubiquitination of HRS, an action that likely plays a role in the sorting function of HRS (7). Whether additional proteins play a role in ...
Transport protein particle (TRAPP; also known as trafficking protein particle), a multimeric guanine nucleotide-exchange factor for the yeast GTPase Ypt1 and its mammalian homologue, RAB1, regulates multiple membrane trafficking pathways. TRAPP complexes exist in three forms, each of which activates Ypt1 or RAB1 through a common core of subunits and regulates complex localization through distinct subunits. Whereas TRAPPI and TRAPPII tether coated vesicles during endoplasmic reticulum to Golgi and intra-Golgi traffic, respectively, TRAPPIII has recently been shown to be required for autophagy. These advances illustrate how the TRAPP complexes link Ypt1 and RAB1 activation to distinct membrane-tethering events.
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