Journal: Scientific reports

Article Title: Up-regulation of ST18 in pemphigus vulgaris drives a self-amplifying p53-dependent pathomechanism resulting in decreased desmoglein 3 expression.

doi: 10.1038/s41598-022-09951-x

Figure Lengend Snippet: Figure 2. Antibody-mediated DSG3 down-regulation affects p53 expression and activity in a p38MAPK- dependent manner. (a) NHEKs exposed to either AK23 or negative control antibody (NC) were stained for p53 (green) and DAPI (blue) (scale bar = 20um); (b) p53 protein expression in NHEKs exposed to either AK23 or negative control antibody (NC) was assessed using immunoblotting with anti-p53 antibody. β-actin served as a loading control (left panel). Protein levels were quantified and data was normalized to levels observed in negative control antibody-treated cells (right panel). Results represent the mean ± SE of four independent experiments (*p < 0.05 by 2-tailed t test); (c) NHEKs were transfected with a luciferase reporter construct under the regulation of a p53 binding site or with a control reporter, and were then treated either with AK23 antibody or negative control antibody (NC). Results represent the mean ± SE of three independent experiments (***p < 0.001 by 2-tailed t test); (d) NHEKs were transfected with a luciferase reporter construct under the regulation of a p53 binding site or a control reporter. Cells were additionally transfected with control (si-control) or p38MAPK-specific (si-p38) siRNAs. Twenty four hours post-transfection, cells were treated either with AK23 antibody (AK23 Ab) or negative control antibody (NC Ab). Results represent the mean ± SE of four independent experiments (*p < 0.05 by one way ANOVA test). Original blots are presented in Supplementary Fig. 4.

Article Snippet: Briefly, primary keratinocytes were co-transfected with the same pGL2 vectors, Renilla expression vector and control siRNA (Life Technologies, Carlsbad, CA, 452002) or a specific p38 alpha MAPK14-specific siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, SC-29433).

Techniques: Expressing, Activity Assay, Negative Control, Staining, Western Blot, Control, Transfection, Luciferase, Construct, Binding Assay

Journal: Scientific reports

Article Title: Up-regulation of ST18 in pemphigus vulgaris drives a self-amplifying p53-dependent pathomechanism resulting in decreased desmoglein 3 expression.

doi: 10.1038/s41598-022-09951-x

Figure Lengend Snippet: Figure 4. ST18 drives a p53-dependent self-amplifying process promoting autoantibody-mediated membranal DSG3 down-regulation in pemphigus vulgaris. Genetically determined ST18 overexpression (1) enhances autoantibodies-induced DSG3 down-regulation in keratinocytes (2, orange arrow) which triggers p38MAPK- dependent (3) p53 activity (4), which in turn up-regulates ST18 promoter activity (5), thus setting the stage for a self-amplifying pathogenetic cycle in PV.

Article Snippet: Briefly, primary keratinocytes were co-transfected with the same pGL2 vectors, Renilla expression vector and control siRNA (Life Technologies, Carlsbad, CA, 452002) or a specific p38 alpha MAPK14-specific siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, SC-29433).

Techniques: Over Expression, Activity Assay

Journal: Biochemistry

Article Title: Engineering a Protein Binder Specific for p38α with Interface Expansion

doi: 10.1021/acs.biochem.8b00408

Figure Lengend Snippet: A comparison of D motif binding sites on p38α and ERK2. (A) A crystal structure (PDB ID: 2y8o) of a docking peptide derived from MKK6 (purple) bound to p38α (gray). p38α residues adjacent to the binding site that differ from ERK2 are shown in green. (B) A superposition of the MKK6 D-motif (purple) on a crystal structure of ERK2 (gray, PDB ID: 2y9q). Residues in green differ from those on p38α. *Residue numbers from ERK2 are shifted up by one to match structurally equivalent positions on p38α.

Article Snippet: Plasmids for cell-culture work Monobodies were cloned into the pLEX-MCS vector (Thermo Scientific) between BamHI and XhoI restriction sites. pDONR223-MAPK14 (p38α) was a gift from William Hahn & David Root (Addgene plasmid # 23865). pLX302 was a gift from David Root (Addgene plasmid # 25896) 20 .

Techniques: Binding Assay, Derivative Assay

Journal: Biochemistry

Article Title: Engineering a Protein Binder Specific for p38α with Interface Expansion

doi: 10.1021/acs.biochem.8b00408

Figure Lengend Snippet: Competitive Fluorescence Polarization (FP) binding assays to determine epitope specificity and binding affinity. (a) Schematic representation of the FP assay. Pep-MKK6 labeled with TAMRA dye was first equilibrated with p38α in solution. Subsequently, αp38mb was titrated into the solution of pep-MKK6/p38α complex, thereby displacing pep-MKK6 from its binding site on p38α. (b) FP values were recorded (circular data points) and fit with a competitive binding model (solid line). Loss of polarization with increasing amounts of αp38mb indicates that αp38mb competes for binding to p38α with pep-MKK6 at the same binding site, confirming the epitope-specificity of αp38mb. KD values from three independent experiments were 600±298 nM.

Article Snippet: Plasmids for cell-culture work Monobodies were cloned into the pLEX-MCS vector (Thermo Scientific) between BamHI and XhoI restriction sites. pDONR223-MAPK14 (p38α) was a gift from William Hahn & David Root (Addgene plasmid # 23865). pLX302 was a gift from David Root (Addgene plasmid # 25896) 20 .

Techniques: Fluorescence, Binding Assay, FP Assay, Labeling

Journal: Biochemistry

Article Title: Engineering a Protein Binder Specific for p38α with Interface Expansion

doi: 10.1021/acs.biochem.8b00408

Figure Lengend Snippet: Overview of anchor-guided engineering (A) Structure of a monobody (1fna) with the FG loop removed to allow insertion of the anchor peptide and the BC loop colored in cyan. (B) Crystal structure of p38α (gray) bound to the MKK6 D motif (PDB ID: 2y8o). (C) Model generated with the AnchorDesign protocol in Rosetta showing that insertion of the D motif in the FG loop of the monobody is predicted to place the BC loop adjacent to a patch of residues (green) that differ between p38α and ERK2.

Article Snippet: Plasmids for cell-culture work Monobodies were cloned into the pLEX-MCS vector (Thermo Scientific) between BamHI and XhoI restriction sites. pDONR223-MAPK14 (p38α) was a gift from William Hahn & David Root (Addgene plasmid # 23865). pLX302 was a gift from David Root (Addgene plasmid # 25896) 20 .

Techniques: Generated

Journal: Biochemistry

Article Title: Engineering a Protein Binder Specific for p38α with Interface Expansion

doi: 10.1021/acs.biochem.8b00408

Figure Lengend Snippet: Expression and screening analysis of monobody yeast library (Lib2). (a) Yeast cells displaying monobody mutants were labeled with an anti-cmyc antibody followed by a secondary antibody conjugated to Alexa Fluor-633 (red histogram) or the secondary antibody only (blue histogram). Approximately 40% of the cells expressed the cmyc tag and therefore full-length monobody mutants as cell surface fusions. (b), (c) and (d) show flow cytometry plots from the screening of monobody yeast library by FACS. Yeast cells displaying monobody mutants were simultaneously labeled with a chicken anti-cmyc antibody and 0 μM (b, no p38α control), 1 μM (c, sort 1) or 5 nM (d, sort 6) biotinylated p38α followed by secondary labeling with a goat anti-chicken antibody conjugated to Alexa Fluor 633 (to detect expression) and streptavidin conjugated to PE (to detect binding) and analyzed by flow cytometry. Cells in the polygon in (c) were sorted from round 1, grown and further sorted by labeling at successively lower concentrations of p38α until sort 6 (d). Yeast cells from round 6 were plated to isolate individual clones for further analysis.

Article Snippet: Plasmids for cell-culture work Monobodies were cloned into the pLEX-MCS vector (Thermo Scientific) between BamHI and XhoI restriction sites. pDONR223-MAPK14 (p38α) was a gift from William Hahn & David Root (Addgene plasmid # 23865). pLX302 was a gift from David Root (Addgene plasmid # 25896) 20 .

Techniques: Expressing, Labeling, Flow Cytometry, Binding Assay, Clone Assay

Journal: Biochemistry

Article Title: Engineering a Protein Binder Specific for p38α with Interface Expansion

doi: 10.1021/acs.biochem.8b00408

Figure Lengend Snippet: Affinity and specificity analysis of αp38mb by ITC. ITC experiments are reported for the titration of αp38mb into solutions of 12.5 μM p38α (a), 13 μM ERK2 (b), and 15 μM JNK1 (c). The binding affinity (KD) of αp38mb for p38α was measured to be 649±100 nM, whereas no specific binding was detected of αp38mb to either ERK2 or JNK1.

Article Snippet: Plasmids for cell-culture work Monobodies were cloned into the pLEX-MCS vector (Thermo Scientific) between BamHI and XhoI restriction sites. pDONR223-MAPK14 (p38α) was a gift from William Hahn & David Root (Addgene plasmid # 23865). pLX302 was a gift from David Root (Addgene plasmid # 25896) 20 .

Techniques: Titration, Binding Assay

Journal: Biochemistry

Article Title: Engineering a Protein Binder Specific for p38α with Interface Expansion

doi: 10.1021/acs.biochem.8b00408

Figure Lengend Snippet: The binding interaction between the engineered monobody and p38α is retained in cell culture. (a) Monobodies were co-expressed with p38α in cells. Wildtype monobody (WTmb) or engineered monobody (αp38mb) containing the HA-epitope tag was transiently co-transfected with V5-tagged p38α in HEK293 cells. Immunoblotting shows co-expression of p38α with either WTmb or αp38mb. (b) Constructs from (a) were used in a pull-down assay where p38α was immunoprecipitated with anti-V5 antibody followed by Immunoblotting analysis of V5 and HA-epitope tags. Co-immunoprecipitation of αp38mb with p38α was observed, but WTmb was not co-immunoprecipitated.

Article Snippet: Plasmids for cell-culture work Monobodies were cloned into the pLEX-MCS vector (Thermo Scientific) between BamHI and XhoI restriction sites. pDONR223-MAPK14 (p38α) was a gift from William Hahn & David Root (Addgene plasmid # 23865). pLX302 was a gift from David Root (Addgene plasmid # 25896) 20 .

Techniques: Binding Assay, Cell Culture, Transfection, Western Blot, Expressing, Construct, Pull Down Assay, Immunoprecipitation

Journal: bioRxiv

Article Title: PI31 is an adaptor protein for proteasome transport in axons and required for synaptic development and function

doi: 10.1101/364463

Figure Lengend Snippet: (A) A candidate-based kinase RNAi screen identified p38β as the kinase that phosphorylates PI31 at S168. Tub-GAL4, Tub-GAL80 ts >UAS-HA-PI31 flies were crossed with RNAi transgenic fly strains targeting each kinase of MAPK or CDK family. HA-PI31 protein was immunoprecipitated and digested. The peptides containing the phosphorylated and non-phosphorylated S168 (EVTTQTTN 168 SPRPIGSDPDPLR) were quantified by MS analysis. Phospho-peptide signal versus total signal was used to estimate the phosphorylation occupancy percentage (POP). The POP distribution of kinase RNAi strains is presented as a box plot with the following percentiles: 10% and 90% (bottom and top whiskers), 25% and 75% (bottom and top of box) and 50% (middle line). The POPs for control RNAi strains are shown as grey dots, and the POPs for replicates of the P38β RNAi are shown as red and yellow dots, suggesting ∼50% decrease of phosphorylation. See Table S5 for the summary of MS analysis. (B) The S153 phosphorylation of human PI31 (pS153-hPI31) increased in response to osmotic stress, a classical condition to activate p38. HEK293 cells were treated with 0.3M sorbitol for indicated time. See for the generation and validation of the pS153-hPI31 antibody. (C) The increase of pS153-hPI31 under osmotic stress was suppressed by p38 inhibitors. HEK293 cells were pretreated with indicated compounds (SB203580, SB202190 and BIRB796) at 10 µM for 2 hours, followed by the treatment of 0.3M sorbitol for 1 hour. The results show that SB203580 and SB202190, which inhibit p38α and p38β, only mildly suppressed pS153-hPI31, whereas BIRB796 that targets all four isoforms of p38, dramatically reduced pS153-hPI31. This suggests that PI31 phosphorylation requires p38 activity. (D) In vitro kinase assays using recombinant proteins generated in E coli. demonstrate that p38 can directly phosphorylate hPI31 at S153. (E) Model for PI31-mediated transport of proteasomes in axons. Proteasomes can move rapidly along microtubules to fulfill dynamic local demands for protein degradation in different cellular compartments( ; ; ; ; ; ). PI31 binds directly to both proteasomes and dynein light chain LC8-type proteins (DYNLL1/2). Inactivation of PI31 severely reduces proteasome movement and leads to the accumulation of Poly-Ub aggregates in the periphery of neurons. This suggests that PI31 functions as an adaptor to directly couple proteasomes to microtubule-based motors. PI31 phosphorylation by the stress-activated p38 MAP kinase regulates the PI31-dDYNLL1 interaction and thereby modulates proteasome transport in axons. This may serve to dynamically adjust the spatio-temporal distribution of proteasomes in response to stress to match changing demands for protein breakdown. If this mechanism is impaired, mis-regulation of proteasome distribution occurs and leads to a failure of localized protein degradation. See also and Table S5.

Article Snippet: Purified recombinant human p38α (MAPK14, GST-tagged, Thermo Fisher Scientific, #PV3304), p38β (MAPK11, His-tagged, Thermo Fisher Scientific, #PV3679), p38γ (MAPK12, His-tagged Thermo Fisher Scientific, #PV3654), p38d (MAPK13, His-tagged, Thermo Fisher Scientific, #PV3656) and hPI31 (UBPBio, #A3901) were used for in vitro kinase assay.

Techniques: Transgenic Assay, Immunoprecipitation, Activity Assay, In Vitro, Recombinant, Generated

Journal: Advanced Science

Article Title: Targeting TTK Inhibits Tumorigenesis of T‐Cell Lymphoma Through Dephosphorylating p38α and Activating AMPK/mTOR Pathway

doi: 10.1002/advs.202413990

Figure Lengend Snippet: TTK interacted with p38α through C‐terminal and phosphorylated p38α in TCL. A) GO enrichment analysis of differentially phosphorylated proteins. The circle area indicated the number of genes, while the circle color represented the range of the corrected P values. B) 12 potential kinase substrates of TTK were identified by integrating the results of motif prediction percentile and phosphoproteomics quantification. Bar plot showed the fold change of differentially phosphorylated proteins. C) The predicted binding mode of TTK (blue) and p38α (green). D) Co‐IP assay showed the interaction between TTK and p38α in TCL. E) Immunofluorescence co‐staining of TTK and p38α in TCL. Bar = 20 µm. F) Immunofluorescence distribution showed the co‐localization of TTK (red) and p38α (green). G) The schematic of TTK truncated fragments. H) Co‐IP assay confirmed the interaction between TTK 525–857aa and p38α in TCL. I) WB analysis showed the expression level of p38α and p‐p38α after TTK knockdown in TCL. J) WB analysis showed the expression level of p38α and p‐p38α after CFI‐402257 treatment (10 µM, 48 h) in TCL. K) WB analysis showed the expression level of p38α and p‐p38α after transfected with TTK kinase inactivated mutation plasmid. L‐M) CCK8 assay showed that the proliferation of TCL cells was inhibited by TTK kinase inactivation and p38 phosphorylation inactivation plasmids, which was reversed by p38 phosphorylation activation plasmid (n = 3). N) Detection of cell apoptosis in TCL cells after TTK knockdown and p38α phosphorylation activation plasmid transfection by flow cytometry (n = 3). O) Detection of cell cycle in TCL cells after TTK knockdown and p38α phosphorylation activation plasmid transfection by flow cytometry (n = 3). Data are shown as the mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The primary antibodies included TTK (A2500, Abclonal), p38α (66234‐1‐Ig, Proteintech), AKT (60203‐2‐Ig, Proteintech), Flag (66008‐4‐Ig, Proteintech), Rabbit Control IgG (AC005, Abclonal), Mouse Control IgG (AC011, Abclonal).

Techniques: Phospho-proteomics, Binding Assay, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Expressing, Knockdown, Transfection, Mutagenesis, Plasmid Preparation, CCK-8 Assay, Activation Assay, Flow Cytometry

Journal: Advanced Science

Article Title: Targeting TTK Inhibits Tumorigenesis of T‐Cell Lymphoma Through Dephosphorylating p38α and Activating AMPK/mTOR Pathway

doi: 10.1002/advs.202413990

Figure Lengend Snippet: Inhibiting TTK activated the AMPK/mTOR pathway through p38α to restrain TCL development. A) KEGG enrichment analysis of differentially phosphorylated proteins. The circle area indicates the number of genes in the pathway, while the circle color represents the range of the corrected P values. B,C) WB analysis showed the expression level of p‐AMPK and p‐mTOR after TTK knockdown and CFI‐402257 treatment (10 µM, 48 h). D) WB analysis showed the expression level of p‐AMPK and p‐mTOR after transfection with p38α phosphorylation inactivation plasmid in TCL. E) WB analysis showed the expression level of p‐AMPK and p‐mTOR after TTK knockdown and transfection with p38α phosphorylation activation plasmid. F) CCK8 assay showed that the combined treatment with Rapamycin (mTOR inhibitor, 10 µM, 48 h) enhanced anti‐tumor effects of CFI‐402257 (10 µM, 48 h) in TCL (n = 3). G) WB analysis showed the expression level of p‐AMPK and p‐mTOR after TTK knockdown and Dorsomorphin (AMPK inhibitor) treatment (10 µM, 48 h). H) CCK8 assay showed that Dorsomorphin (AMPK inhibitor, 10 µM, 48 h) and MHY1485 (mTOR activator, 20 µM, 48 h) rescued the inhibitory effect of TTK knockdown on TCL cell proliferation (n = 3). I) Mechanism diagram summarized that TTK contributed to TCL development through regulating the p38α/AMPK/mTOR axis. Data are shown as the mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The primary antibodies included TTK (A2500, Abclonal), p38α (66234‐1‐Ig, Proteintech), AKT (60203‐2‐Ig, Proteintech), Flag (66008‐4‐Ig, Proteintech), Rabbit Control IgG (AC005, Abclonal), Mouse Control IgG (AC011, Abclonal).

Techniques: Expressing, Knockdown, Transfection, Phospho-proteomics, Plasmid Preparation, Activation Assay, CCK-8 Assay

Journal: Advanced Science

Article Title: Targeting TTK Inhibits Tumorigenesis of T‐Cell Lymphoma Through Dephosphorylating p38α and Activating AMPK/mTOR Pathway

doi: 10.1002/advs.202413990

Figure Lengend Snippet: TTK regulated the autophagy in TCL via modulating p38α phosphorylation. A) IF analysis of LC3B autophagic vesicles (red) after CFI‐402257 treatment (10, 20µM, 48 h) in TCL cells. Bar = 20 µm. B) IF analysis of LC3B autophagic vesicles (red) after TTK knockdown in TCL cells. Bar = 20 µm. C) IF analysis of LC3B autophagic vesicles (red) after transfection with p38α phosphorylation inactivation plasmid in TCL. Bar = 20 µm. D) WB analysis showed the expression level of p62, Beclin and LC3B‐II after CFI‐402257 treatment (10 µM, 48 h) in TCL cells. E) WB analysis showed the expression level of p62, Beclin, and LC3B‐II after TTK knockdown in TCL. F) WB was used to determine the expression level of p62, Beclin, and LC3B‐II after TTK knockdown and chloroquine treatment (10µM, 12 h) in TCL. G) WB analysis showed the expression level of p62, Beclin, and LC3B‐II after transfection with p38α phosphorylation inactivation plasmid in TCL. H) WB analysis showed the expression level of p62, Beclin and LC3B‐II after TTK knockdown and transfection with p38α phosphorylation activation plasmid in TCL. Data are shown as the mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The primary antibodies included TTK (A2500, Abclonal), p38α (66234‐1‐Ig, Proteintech), AKT (60203‐2‐Ig, Proteintech), Flag (66008‐4‐Ig, Proteintech), Rabbit Control IgG (AC005, Abclonal), Mouse Control IgG (AC011, Abclonal).

Techniques: Phospho-proteomics, Knockdown, Transfection, Plasmid Preparation, Expressing, Activation Assay