One-by-One Sample Preparation Method for Protein Network Analysis

Iemura, Shun‐ichiro; Natsume, Tohru

doi:10.5772/37931

Cited by 5 publications

(12 citation statements)

References 27 publications

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“…Previously, WDR26 is identified as an Axin1‐binding protein . In the present study, we found that WDR26, with Axin1, controlled β‐catenin levels to negatively regulate the expression of Wnt target genes.…”

supporting

confidence: 59%

WDR26 is a new partner of Axin1 in the canonical Wnt signaling pathway

et al. 2016

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The stability of β‐catenin is very important for canonical Wnt signaling. A protein complex including Axin/APC/GSK3β phosphorylates β‐catenin to be degraded by ubiquitination with β‐TrCP. In the recent study, we isolated WDR26, a protein that binds to Axin. Here, we found that WDR26 is a negative regulator of the canonical Wnt signaling pathway, and that WDR26 affected β‐catenin levels. In addition, WDR26/Axin binding is involved in the ubiquitination of β‐catenin. These results suggest that WDR26 plays a negative role in β‐catenin degradation in the Wnt signaling pathway.

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supporting

confidence: 59%

WDR26 is a new partner of Axin1 in the canonical Wnt signaling pathway

et al. 2016

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“…We over‐expressed Flag‐LRRK2(G1341V) in HEK293T cells and used anti‐Flag antibody to immunoprecipitate LRRK2(G1341V) and its associated proteins. We analyzed digests of these complexes using liquid chromatography‐coupled tandem mass spectrometry (Iemura & Natsume ). We identified BAG2 and HSC70 as LRRK2(G1341V)‐binding proteins.…”

Section: Resultsmentioning

confidence: 99%

“…Identification of associated proteins using LC‐MS/MS analysis has been previously described (Hanafusa et al . ; Iemura & Natsume ). In brief, Flag‐tagged human LRRK1 and LRRK2(G1341V) were expressed in HEK293T cells, and associated proteins were recovered from cell extracts by immunoprecipitation with anti‐Flag antibody.…”

Section: Methodsmentioning

confidence: 98%

Chaperone complex BAG2–HSC70 regulates localization of Caenorhabditis elegans leucine‐rich repeat kinase LRK‐1 to the Golgi

et al. 2016

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Mutations in LRRK2 are linked to autosomal dominant forms of Parkinson's disease. We identified two human proteins that bind to LRRK2: BAG2 and HSC70, which are known to form a chaperone complex. We characterized the role of their Caenorhabditis elegans homologues, UNC-23 and HSP-1, in the regulation of LRK-1, the sole homologue of human LRRK2. In C. elegans, LRK-1 determines the polarized sorting of synaptic vesicle (SV) proteins to the axons by excluding SV proteins from the dendrite-specific transport machinery in the Golgi. In unc-23 mutants, SV proteins are localized to both presynaptic and dendritic endings in neurons, a phenotype also observed in lrk-1 deletion mutants. Furthermore, we isolated mutations in the hsp-1 gene that can suppress the unc-23, but not the lrk-1 defect. We show that UNC-23 determines LRK-1 localization to the Golgi apparatus in cooperation with HSP-1. These results describe a chaperone-dependent mechanism through which LRK-1 localization is regulated.

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“…HEK293T cells transfected with plasmids expressing Lrp4‐Flag were solubilized in TNE buffer [13]. The lysates were incubated with mouse monoclonal anti‐Flag antibody‐conjugated agarose (M2, Sigma) at 4 °C for 1h and subjected to immunoprecipitation.…”

Section: Methodsmentioning

confidence: 99%

“…The lysates were incubated with mouse monoclonal anti‐Flag antibody‐conjugated agarose (M2, Sigma) at 4 °C for 1h and subjected to immunoprecipitation. Lrp4‐Flag and co‐precipitated proteins were eluted from the slurry with an excess of Flag peptides, digested with lysyl endopeptidase C (Wako), and analyzed with a highly sensitive direct nanoflow LC–MS/MS system as previously described [13,14].…”

Section: Methodsmentioning

confidence: 99%

Mesdc2 plays a key role in cell‐surface expression of Lrp4 and postsynaptic specialization in myotubes

et al. 2013

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a b s t r a c tLow-density lipoprotein receptor-related protein 4 (Lrp4) is essential for pre-and post-synaptic specialization at the neuromuscular junction (NMJ), an indispensable synapse between a motor nerve and skeletal muscle. Muscle-specific receptor tyrosine kinase MuSK must form a complex with Lrp4 to organize postsynaptic specialization at NMJs. Here, we show that the chaperon Mesdc2 binds to the intracellular form of Lrp4 and promotes its glycosylation and cell-surface expression. Furthermore, knockdown of Mesdc2 suppresses cell-surface expression of Lrp4, activation of MuSK, and postsynaptic specialization in muscle cells. These results suggest that Mesdc2 plays an essential role in NMJ formation by promoting Lrp4 maturation. Structured summary of protein interactions:Lrp4 physically interacts with CANX, LRPAP1, CCAR2, MESDC2, PDIA4, RPN1 and SDF2L1 by anti tag coimmunoprecipitation (View interaction) Mesdc2 physically interacts with Lrp4 by anti tag coimmunoprecipitation (View interaction) Mesdc2 physically interacts with Lrp4 by anti bait coimmunoprecipitation (View interaction)

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One-by-One Sample Preparation Method for Protein Network Analysis

Cited by 5 publications

References 27 publications

WDR26 is a new partner of Axin1 in the canonical Wnt signaling pathway

WDR26 is a new partner of Axin1 in the canonical Wnt signaling pathway

Chaperone complex BAG2–HSC70 regulates localization of Caenorhabditis elegans leucine‐rich repeat kinase LRK‐1 to the Golgi

Mesdc2 plays a key role in cell‐surface expression of Lrp4 and postsynaptic specialization in myotubes

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