Within the resting time ensure proper mixing. Cell breakage is performed by sonication with 3 × 60 s and 3 × 45 s cycles with always 60 s resting time. Add 5 mL of a 10% Triton X-100 solution and gently mix the solution. Pellets can be stored at − 20☌ for few months or used directly for purification. Harvest the cells by centrifugation (6500 × g, 10 min, 4☌) and transfer the pellets to a falcon tube. Induce MSP expression with 1 m M IPTG with additional incubation of 1 h at 37☌ and 4 h at 28☌ at 200 rpm shaking. Inoculate LB medium (8 × 600 mL) supplemented with kanamycin (30 μg/mL) and glucose (0.5% (v/v)) with 50 mL of the grown overnight culture and incubate at 37☌ and 200 rpm shaking until OD 600 reaches 1–1.2. Inoculate a 600-mL preculture of LB medium supplemented with kanamycin (30 μg/mL) and incubate overnight (37☌, 200 rpm shaking). Thereby the same protocol is used for all different variants of MSP: 1.įreshly transform the selected MSP expression plasmid (Addgene) in BL21 (DE3) star cells, streak them out on an agar plate containing kanamycin and incubate the plate overnight at 37☌. coli cells and purified via the N-terminal His 6-tag ( Denisov et al., 2004). The MSP can be produced by conventional expression in E. However, the mechanistic details whereby many of the scaffold proteins promote pathway activation remain to be elucidated. Additional scaffold proteins have been identified that participate in the activation of p38 and ERKs. Additionally, WDR62 (WD repeat domain 62) has been implicated in noncanonical activation of JNK via association with JNK and MKK7. In response to stress resulting from X-rays and genotoxic drugs, RACK1 (Receptor for Activated C Kinase 1) has been shown to bind and activate JNK1 and MTK1. Following cleavage by caspase-3, the C-terminal domain of GRASP1 is released to promote activation of JNK. In neurons, GRASP-1 (GRIP1-associated protein 1) binds both JNK1 and MEKK1. In 2016, another MKP, DUSP22, was reported to interact with ASK1, MKK7, and JNK1 to promote JNK activation independent of its phosphatase activity. DUSP19 physically interacts with MKK7 and indirectly regulates JNK. ĭUSP19 (dual-specificity phosphatase 19) is the first MAPK phosphatase (MKP) identified as a scaffold protein. Crk II activates HPK1 (hematopoietic progenitor kinase 1) and SEK1-dependent activation of the JNK pathway, by recruiting JNK1 to a p130Cas multiprotein complex. β-Arrestin-2 contains a MAP kinase docking site and mainly functions as a scaffold protein in activation of JNK3, by tethering ASK1, SEK1, and JNK3. JIP1 recruits DLK, MKK7, and JNK as a complex, whereas JIP3 binds SEK1 and MEKK1 to stimulate SEK1, leading to JNK3 activation. Scaffolds JIP1 (JNK-interacting protein 1) and JIP3 recruit several different activators and JNKs. POSH complexes with and sequentially stimulates RAC1, MLKs, SEK1, MKK7, and JNKs. The scaffold proteins POSH (plenty of SH3s) promote JNK pathway activation by directly interacting with GTP-bound RAC1. In melanoma cells, filamin was found to interact with SEK1 as a scaffold protein, in response to TNF-α stimulation. Several scaffold proteins have been identified that bind to JNKs and upstream activators. Scaffold proteins play key roles in providing a platform for signaling molecules to assemble, promoting the localization of signaling molecules at specific sites and coordinating positive and negative feedback signals for pathway regulation. Johnson, in Targeting Cell Survival Pathways to Enhance Response to Chemotherapy, 2019 4.2.3 The Role of Scaffold Proteins
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