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14-3-3 Protein Targets Misfolded Chaperone-Associated Proteins To Research Article 4173 14-3-3 protein targets misfolded chaperone-associated proteins to aggresomes Zhe Xu, Kourtney Graham, Molly Foote, Fengshan Liang, Raed Rizkallah, Myra Hurt, Yanchang Wang, Yuying Wu and Yi Zhou* Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, FL 32306, USA *Author for correspondence ([email protected]) Accepted 19 June 2013 Journal of Cell Science 126, 4173–4186 ß 2013. Published by The Company of Biologists Ltd doi: 10.1242/jcs.126102 Summary The aggresome is a key cytoplasmic organelle for sequestration and clearance of toxic protein aggregates. Although loading misfolded proteins cargos to dynein motors has been recognized as an important step in the aggresome formation process, the molecular machinery that mediates the association of cargos with the dynein motor is poorly understood. Here, we report a new aggresome-targeting pathway that involves isoforms of 14-3-3, a family of conserved regulatory proteins. 14-3-3 interacts with both the dynein-intermediate chain (DIC) and an Hsp70 co-chaperone Bcl-2-associated athanogene 3 (BAG3), thereby recruiting chaperone-associated protein cargos to dynein motors for their transport to aggresomes. This molecular cascade entails functional dimerization of 14-3-3, which we show to be crucial for the formation of aggresomes in both yeast and mammalian cells. These results suggest that 14-3-3 functions as a molecular adaptor to promote aggresomal targeting of misfolded protein aggregates and may link such complexes to inclusion bodies observed in various neurodegenerative diseases. Key words: Aggresome, 14-3-3, Dynein, BAG3, Adaptor Introduction structures of human 14-3-3 proteins reveal that they exist as Misfolded proteins are prone to forming aggregates that perturb homo- and heterodimers, with each monomer comprising nine a- normal cellular functions and lead to cytotoxicity (Ross and helices organized in an anti-parallel array (Liu et al., 1995; Xiao Poirier, 2005). In addition to chaperone-assisted refolding and et al., 1995). Co-crystallization of several ligand-bound 14-3-3 Journal of Cell Science ubiquitin-proteasome mediated degradation, the aggresome- complexes further demonstrate that the 14-3-3 dimer is arranged autophagy dependent clearance has recently been identified as in such a way that the ligand binding groove runs in opposite an important cellular response to toxic misfolded proteins directions for each monomer, thereby allowing simultaneous (Tyedmers et al., 2010). When proteasome-dependent binding of two ligands for one 14-3-3 dimer (Rittinger et al., degradation fails, misfolded proteins can be sequestered into 1999). large juxtanuclear inclusion bodies known as aggresomes, which 14-3-3 proteins are known to interact with over 200 proteins not only help alleviate the toxicity of small aggregates, but also that contain specific phosphoserine/phosphothreonine motifs facilitate their clearance by macroautophagy (Garcia-Mata et al., (Furukawa et al., 1993; Muslin et al., 1996). Through binding 2002; Kopito, 2000). Aggresome formation is an active process to their target proteins, 14-3-3 participates in a wide variety of in which misfolded proteins are loaded onto the dynein–dynactin biological processes, ranging from transcription to neuronal motor complex by protein adaptors for transport along development (Datta et al., 2000; Fantl et al., 1994; Fu et al., microtubules to the microtubule organizing center (MTOC) 2000; Peng et al., 1997; Skoulakis and Davis, 1998; Tzivion and (Chin et al., 2010; Ravikumar et al., 2005). While the underlying Avruch, 2002; Zha et al., 1996). In addition, 14-3-3 proteins have molecular details are not fully understood, previous studies have been implicated in a number of neurodegenerative disorders, identified the dynein motor and molecular adaptors [e.g. histone largely based on the observation that 14-3-3 proteins co-localize deacetylase 6 (HDAC6)] as critical components in the aggresome with the pathological inclusion bodies associated with these formation process (Kawaguchi et al., 2003; Ravikumar et al., diseases, including Lewy bodies in Parkinson’s disease, 2005). More recently, heat-shock protein 70 (Hsp70) and its co- neurofibrillary tangles in Alzheimer’s disease, mutant chaperone Bcl-2-associated athanogene 3 (BAG3) were shown to huntingtin aggregates in Huntington’s disease, etc. (Chen et al., be critical for targeting misfolded proteins to aggresomes 2003; Kawamoto et al., 2002; Umahara et al., 2004). However, it (Gamerdinger et al., 2011; Zhang and Qian, 2011). is not known how and why 14-3-3 assembles in these inclusion We report here that aggresome formation is profoundly bodies (Foote and Zhou, 2012). regulated by 14-3-3, a family of ubiquitous proteins that are It has been hypothesized that the presence of 14-3-3 proteins in abundantly expressed in the brain (Boston et al., 1982; Moore, various inclusion bodies may be a consequence of their 1967). 14-3-3 proteins are highly conserved from yeast to human associations with disease-related proteins such as a-synuclein and consist of seven mammalian isoforms (b, e, g, c, t/h, f and s) (a-Syn), parkin and tau, which are major components of (Rosenquist et al., 2000; Wang and Shakes, 1996). The crystal inclusions in certain neurodegenerative diseases (Hashiguchi 4174 Journal of Cell Science 126 (18) et al., 2000; Ostrerova et al., 1999; Sato et al., 2006). On the other (supplementary material Fig. S1). To further characterize the hand, 14-3-3 has been proposed to actively promote the function of 14-3-3 in a-Syn inclusion body formation, we co- formation of aggresome-like inclusions, thus acting as a transfected a-Syn-EGFPD155 with either exogenous 14-3-3 or sweeper to facilitate the sequestration and deposition of pSCM138 in cells and examined inclusion body formation disease-associated toxic proteins (Kaneko and Hachiya, 2006). microscopically. In line with our biochemical data, a majority of In support of this hypothesis, previous studies have shown that the cells with overexpressed 14-3-3 bore large a-Syn inclusion 14-3-3 plays an important role in aggresome formation. For bodies after a treatment of the proteasome inhibitor (ALLN), example, 14-3-3f is required for aggresome formation induced by while few ALLN-induced inclusion bodies were observed in cells the expression of a polyglutamine-expanded huntingtin protein co-transfected with pSCM138 (Fig. 1C). Further quantification (Htt86Q) in mammalian cells (Omi et al., 2008). Likewise, showed that overexpressing 14-3-3 in cells gave rise to deletion of bmh1, which encodes one of two yeast 14-3-3 significantly more frequent inclusion body formation than that homologs Bmh1, inhibits aggresomal targeting of another in control cells. In contrast, co-transfection of pSCM138 led to a disease-related huntingtin protein (Htt103QP) ectopically drastic decrease in the percentage of cells containing large expressed in yeast cells (Wang et al., 2009). Current studies, inclusion bodies (Fig. 1B). however, have not yet addressed whether the effect of 14-3-3 on It is known that overexpression of a-Syn leads to formation of aggresome formation is specific for the huntingtin proteins, and a specific type of inclusions termed aggresome, which is uniquely more importantly, which molecular step 14-3-3 might participate located in the vicinity of the centrosome and assembled by in the aggresome formation pathway. retrograde transport of dynein motors along the microtubule In this study, we have determined that 14-3-3 functions as a network (Opazo et al., 2008; Tanaka et al., 2004). Consistently, molecular adaptor to couple chaperone-associated misfolded these perinuclear structures were enriched with vimentin, a proteins with dynein motors through its dimeric binding to both known aggresome marker that displayed homogeneous BAG3 and the dynein intermediate chain (DIC). 14-3-3 thus distribution in cells without aggresome formation (Fig. 1C). plays a critical role in recruiting misfolded protein cargos onto Thus, the a-Syn-enriched inclusion bodies facilitated by 14-3-3 the cytoplasmic motor complex, thereby promoting aggresome were aggresomes. formation in response to accumulation of various misfolded proteins. Our findings set a stage for further determination of the 14-3-3 plays an important role in aggresome formation molecular detail and functional significance of the 14-3-3- To test whether 14-3-3 is able to promote aggresome formation mediated aggresomal targeting complex and provide a foundation of other aggregation-prone proteins, we utilized two other well- to investigate the protective role of 14-3-3 in neurodegenerative established aggresome formation models: GFP-250, which diseases. spontaneously forms aggresomes when expressed in cells; and a deletion mutant of cystic fibrosis transmembrane conductance Results regulator (CFTR), CFTRDF508, which concentrates in 14-3-3 facilitates the formation of a-Syn aggresomes aggresomes when proteasome activity is inhibited (Garcı´a-Mata 14-3-3 proteins have previously been reported to interact with a- et al., 1999; Johnston et al., 1998). As shown in Fig. 2A, 14-3-3 Journal of Cell Science Syn, one of the major components of Lewy bodies (Ostrerova et al., 1999; Sato et al., 2006). To examine the potential role of overexpression increased the percentage of cells bearing large 14-3-3 in a-Syn aggregation, we adopted an established method perinuclear GFP-250 aggresomes. In cells co-transfected with 14- to induce a-Syn aggregation by inhibition of proteasome 3-3-binding antagonist pSCM138, however, aggresome functions in cells expressing a-Syn-EGFP fusion proteins formation was suppressed and GFP-250 appeared to exist in (McLean et al., 2001). Consistent with the previous report, a- small aggregates in the cytoplasm. Similarly, GFP-CFTRDF508 Syn-EGFP fusion protein underwent C-terminal cleavage, and aggresome formation was enhanced in 14-3-3-overexpressed treatment of cells with the proteasome inhibitor ALLN resulted in cells, but significantly suppressed in cells co-transfected with a marked accumulation of C-terminal truncated fragments in the pSCM138 (Fig.
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