Mass spectrometry approaches to study mammalian kinase and phosphatase associated proteins

Kean, Michelle J.; Couzens, Amber L.; Gingras, Anne‐Claude

doi:10.1016/j.ymeth.2012.06.002

Cited by 69 publications

(58 citation statements)

References 73 publications

(87 reference statements)

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“…Clone accession numbers are shown in supplemental Table S4. Stable Flp-In HeLa T-REx cell line pools were generated as described in (32). The PATL1, DCP1A, EIF4A1, PABPC1 and G3BP1 genes were cloned from the following clone accession number templates: AM392959, DQ893260, DQ893044, DQ893508, and DQ893058.…”

Section: Methodsmentioning

confidence: 99%

BioID-based Identification of Skp Cullin F-box (SCF)β-TrCP1/2 E3 Ligase Substrates*

Coyaud

Mis

Laurent

et al. 2015

Molecular & Cellular Proteomics

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The identification of ubiquitin E3 ligase substrates has been challenging, due in part to low-affinity, transient interactions, the rapid degradation of targets and the inability to identify proteins from poorly soluble cellular compartments. SCF ␤-TrCP1 and SCF ␤-TrCP2 are well-studied ubiquitin E3 ligases that target substrates for proteasomal degradation, and play important roles in Wnt, Hippo, and NFB signaling. Combining 26S proteasome inhibitor (MG132) treatment with proximity-dependent biotin labeling (BioID) and semiquantitative mass spectrometry, here we identify SCF ␤-TrCP1/2 interacting partners. Based on their enrichment in the presence of MG132, our data identify over 50 new putative SCF ␤-TrCP1/2 substrates. We validate 12 of these new substrates and reveal previously unsuspected roles for ␤-TrCP in the maintenance of nuclear membrane integrity, processing (P)-body turnover and translational control. Together, our data suggest that ␤-TrCP is an important hub in the cellular stress response. The technique presented here represents a complementary approach to more standard IP-MS methods and should be broadly applicable for the identification of substrates for many ubiquitin E3 ligases. Molecular & Cellular

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Section: Methodsmentioning

confidence: 99%

BioID-based Identification of Skp Cullin F-box (SCF)β-TrCP1/2 E3 Ligase Substrates*

Coyaud

Mis

Laurent

et al. 2015

Molecular & Cellular Proteomics

Self Cite

157

137

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“…Stable, tetracycline-inducible HEK293 T-REx and U-2 OS T-Rex cell lines were generated as previously described (22). For biochemical assays and MTMR3/4 overexpression analysis, protein expression was induced 24 h before harvest by addition of 1 g/ml tetracycline (Tet).…”

Section: Methodsmentioning

confidence: 99%

“…Affinity Purification and Mass Spectrometry-For each myotubularin and FLAG-GFP control, cell pellets from two 150-mm plates were lysed in 50 mM HEPES-KOH (pH 8.0), 100 mM KCl, 2 mM EDTA, 0.1% Nonidet P-40, and 10% glycerol and affinity-purified with M2-FLAG magnetic beads (Sigma-Aldrich), followed by on-bead trypsin digest as described (22). A spray tip was formed on fused silica capillary column (0.75 m ID, 350 m OD) using a laser puller (program ϭ 4; heat ϭ 280, FIL ϭ 0, VEL ϭ 18, DEL ϭ 200).…”

Section: Methodsmentioning

confidence: 99%

Myotubularin-related Proteins 3 and 4 Interact with Polo-like Kinase 1 and Centrosomal Protein of 55 kDa to Ensure Proper Abscission

St‐Denis

Gupta

Lin

et al. 2015

Molecular & Cellular Proteomics

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The myotubularins are a family of phosphatases that dephosphorylate the phosphatidylinositols phosphatidylinositol-3-phosphate and phosphatidylinositol-3,5-phosphate. Several family members are mutated in disease, yet the biological functions of the majority of myotubularins remain unknown. To gain insight into the roles of the individual enzymes, we have used affinity purification coupled to mass spectrometry to identify protein-protein interactions for the myotubularins. The myotubularin interactome comprises 66 high confidence (false discovery rate The myotubularins are a subfamily of protein tyrosine phosphatases (PTPs) 1 , consisting of sixteen conserved proteins. Despite containing the conserved C(X) 5 R catalytic motif found in all protein tyrosine phosphatases, myotubularins harbor active sites that do not dephosphorylate tyrosine, but instead catalyze the conversion of the phosphatidylinositol-type lipids From the ‡Lunenfeld

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“…Flp-In T-Rex 293 cells (Invitrogen) were stably transfected with N-terminally-FLAG-tagged PP2A constructs and protein expression was induced by tetracycline addition. FLAG-tagged proteins and their putative interaction partners were recovered by immunoprecipitation on anti-FLAG magnetic beads 40 .…”

Section: Methodsmentioning

confidence: 99%

Mechanistic insight into GPCR-mediated activation of the microtubule-associated RhoA exchange factor GEF-H1

et al. 2014

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Mass spectrometry approaches to study mammalian kinase and phosphatase associated proteins

Cited by 69 publications

References 73 publications

BioID-based Identification of Skp Cullin F-box (SCF)β-TrCP1/2 E3 Ligase Substrates*

BioID-based Identification of Skp Cullin F-box (SCF)β-TrCP1/2 E3 Ligase Substrates*

Myotubularin-related Proteins 3 and 4 Interact with Polo-like Kinase 1 and Centrosomal Protein of 55 kDa to Ensure Proper Abscission

Mechanistic insight into GPCR-mediated activation of the microtubule-associated RhoA exchange factor GEF-H1

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