Journal: EBioMedicine

Article Title: Subcellular compartmentalization of docking protein-1 contributes to progression in colorectal cancer

doi: 10.1016/j.ebiom.2016.05.003

Figure Lengend Snippet: Expression of DOK1 protein in CRC cells and tissues. (A) Top: Detection of DOK1 variants in human transformed and CRC cell lines (1 = SW480, 2 = HCT116, 3 = HT29, 4 = HEK293T, 5 = Caco2) using domain-specific antibodies (Abs). Total cell lysates (TCLs) were subjected to Western blotting with anti-N (A3) or anti-C (M19) DOK1 Abs. Bottom: O.D. values of bands in gels were normalized to HSP90 and calculated as -fold ± S.E. vs. normal colon (NC) tissue ( n = 3 per cell line, *p < 0.05 vs. NC, Kruskal Wallis test). NC control is the mean of n = 5 healthy individuals. (B) DOK1 variants in intestinal tissues. Top: Whole tissue lysates from frozen samples of matched tumor (TU) and normal colon (NC) specimens from CRC patients (or C57BL6J mice as control) were analysed as in A. Bottom: Quantitative data are -fold ± S.E. ( n = 15 cases, *p < 0.05 vs. p62, two-way ANOVA). (C) Scheme of human DOK1 protein variants. Legend: FL = full-length p62 DOK1, DC = p44 C-terminal part of DOK1 , DN = p33–37 N-terminal part of DOK1, DEL = small p19–22 deletion variant of DOK1 , C = C-terminus, N = N-terminus, BCCL13 = leukemia variant ( , ). Subcellular localization and Ab binding epitopes are depicted.

Article Snippet: Mouse monoclonal DOK1 (A3) Ab was diluted 1:200, and staining was processed according to the protocol from Vectastain ABC (HRP) kit (Vectorlabs).

Techniques: Expressing, Transformation Assay, Western Blot, Variant Assay, Binding Assay

Journal: EBioMedicine

Article Title: Subcellular compartmentalization of docking protein-1 contributes to progression in colorectal cancer

doi: 10.1016/j.ebiom.2016.05.003

Figure Lengend Snippet: Subcellular localization of DOK1 protein variants. (A) Anti-N Ab (a-N) recognizes nuclear DOK1, anti-C Ab (a-C) cytoplasmic DOK1. Cycling cells were fixed and stained for immunofluorescence microscopy. Data show enrichment of DOK1 signals detected by a-N Ab in the nucleus (NUC) as -fold ± S.E. compared to a-C Ab ( n = 3 per cell line, > 30 nuclei per field, n = 5 fields, *p < 0.05 a-N vs. a-C; Mann Whitney test). Colors: green = DOK1, blue = nuclei; Magnification 630 ×. (B) Anti-N Ab detects p37 DOK1 in the nucleus of malignant cells. Cells were subjected to subcellular fractionation and Western blotting. O.D. values from bands in gels are means ± S.E. of DOK1 protein per fraction ( n = 3 per cell line, *p < 0.05 vs. NUC; Kruskal Wallis test). Legend: CYT = cytoplasm, NUC = nucleus, INS = insoluble membrane and matrix fraction (cytoskeleton, chromatin e.a.). (C) Anti-C Ab detects colocalization of cytoplasmic DOK1 with calnexin (ER marker) but not with POM121 (ENV marker) in the perinuclear area of the endoplasmic reticulum (ER) and the nuclear envelope (ENV). Anti-N Ab detects overlay of nuclear DOK1 with nuclei (DAPI). HEK293T cells were analysed as in A. Colors: green = organelle marker, red = DOK1, blue = nuclei; Magnification 630 ×. (D) Anti-C Ab detects FL p62 and p44 DOK1 in the cytoplasm and insoluble fraction of non-malignant cells. Fresh-frozen normal colon (NC) tissue from patients or C57BL6J mice was subjected to fractionation as in B. Results are shown as in B ( n = 3 per species, *p < 0.05 vs. NUC, Kruskal Wallis test).

Article Snippet: Mouse monoclonal DOK1 (A3) Ab was diluted 1:200, and staining was processed according to the protocol from Vectastain ABC (HRP) kit (Vectorlabs).

Techniques: Staining, Immunofluorescence, Microscopy, MANN-WHITNEY, Fractionation, Western Blot, Marker

Journal: EBioMedicine

Article Title: Subcellular compartmentalization of docking protein-1 contributes to progression in colorectal cancer

doi: 10.1016/j.ebiom.2016.05.003

Figure Lengend Snippet: N- and C-terminal domains determine subcellular localizations of DOK1. (A) Ectopic DOK1 mutants mimic compartmentalization of endogenous DOK1 protein variants. HEK293T cells were transfected with expression plasmids encoding FL p62 DOK1 or the FLAG-tagged truncation mutants p33 DN and p44 DC for 48 h before immunofluorescence staining using FLAG Ab. Colors: green = FLAG-DOK1, blue = nuclei; Magnification 630 ×. Data show enrichment of DN in the nucleus (NUC) as -fold ± S.E. compared to DC ( n = 3 per mutant, > 50 nuclei per field, n = 5 fields, *p < 0.05 DN vs. DC, Mann Whitney test). (B) DN is targeted to the nucleus, DC to the cytoplasm. HEK293T cells were transfected as in A. TCLs were subjected to subcellular fractionation and Western blotting using anti-N (FLAG) and anti-C DOK1 Abs. O.D. values normalized to HSP90 or lamin AC are -fold ± S.E. of DOK1 protein per fraction ( n = 3, *p < 0.05 mutant vs. FL, Kruskal Wallis test).

Article Snippet: Mouse monoclonal DOK1 (A3) Ab was diluted 1:200, and staining was processed according to the protocol from Vectastain ABC (HRP) kit (Vectorlabs).

Techniques: Transfection, Expressing, Immunofluorescence, Staining, Mutagenesis, MANN-WHITNEY, Fractionation, Western Blot

Journal: EBioMedicine

Article Title: Subcellular compartmentalization of docking protein-1 contributes to progression in colorectal cancer

doi: 10.1016/j.ebiom.2016.05.003

Figure Lengend Snippet: DOK1 forms a complex with and increases transcriptional activity of PPARγ. (A) CoIP of FL p62 DOK1 and PPARγ from whole tissue lysates of matched frozen human TU and NC specimens. Proteins were immunoprecipitated (“IP”) with PPARγ (H100) or no Ab (bead control). Coprecipitated proteins were detected (“IB”) with anti-N DOK1 Ab. Results are means ± S.E. ( n = 3 patients, *p < 0.05 NC vs. TU, two-way ANOVA). (B) Proximity ligation assay (PLA). HEK293T cells were serum-deprived for 16 h followed by incubation with rosi (at 10 μM) or vehicle (DMSO) for 24 h. Immunofluorescence stainings were done with anti-N DOK1 and PPARγ (#2435) Abs or single Ab (negative control). Results are -fold ± S.E. ( n = 3 per treatment, > 50 nuclei per field, n = 5 fields, *p < 0.05 vs. rosi, Kruskal Wallis test). Colors: pink dots = DOK1–PPARγ colocalization, green = actin (phalloidin or “no dye” negative control), blue = nuclei; Magnification 630 × and 400 ×. (C) DOK1 promotes transcriptional activity of PPARγ. HCT116 cells were cotransfected with DOK1 and PPRE reporter plasmids for 24 h followed by treatment with 1 μM rosi for 24 h. Luciferase activity in TCLs normalized to protein content was calculated as -fold ± S.E. ( n = 3, *p < 0.05 DOK1 + rosi vs. EV + rosi, Kruskal Wallis test). PTB and C-terminal domains, but not the N-terminal part of DOK1, are required for PPARγ activation. (D) DOK1 increases PPARγ-target gene expression. Cells were transfected and treated as above. Normalized CT-values from RT-qPCRs are -fold ± S.E. ( n = 3, *p < 0.05 rosi vs. vehicle, Friedmann test). (E) DOK1 knock-down decreases PPARγ-activity. Cells were cotransfected with PPRE reporter plasmid and siRNA (at 100 nM) for 24 h and treated with 1 μM rosi for additional 24 h. Data are shown as in C ( n = 3 per cell line, *p < 0.05 DOK1- vs. control-siRNA; two-way ANOVA). (F) DOK1 knock-down reduces PPARγ-target gene expression. Cells were treated as in E and data are presented as in D ( n = 3, *p < 0.05 DOK1- vs. control-siRNA, Friedmann test).

Article Snippet: Mouse monoclonal DOK1 (A3) Ab was diluted 1:200, and staining was processed according to the protocol from Vectastain ABC (HRP) kit (Vectorlabs).

Techniques: Activity Assay, Immunoprecipitation, Proximity Ligation Assay, Incubation, Immunofluorescence, Negative Control, Luciferase, Activation Assay, Expressing, Transfection, Plasmid Preparation

Journal: EBioMedicine

Article Title: Subcellular compartmentalization of docking protein-1 contributes to progression in colorectal cancer

doi: 10.1016/j.ebiom.2016.05.003

Figure Lengend Snippet: DOK1 inhibits c - FOS promoter transcription. (A) Western blots of pulldown assays detecting active GTP-bound pan-RAS proteins in TCLs compared to total pan-RAS (input) using RALGDS-GST as a bait. Results are -fold ± S.E. ( n = 3 per cell line, *p < 0.05 vs. beads, Kruskal Wallis test). (B) DOK1 does not inhibit mutant RAS. Cells were transfected with EV or FL p62 DOK1 for 48 h before pulldown. Data are presented as in A ( n = 3, n.s., two-way ANOVA). (C) DOK1 does not inhibit ERK1/2 phosphorylation. HEK293T, SW480 and HCT116 were transfected with DOK1 plasmids for 24 h, serum deprived for 16 h and restimulated with EGF (10 ng/ml) for the times indicated. Data from Western blots of TCLs are shown as in A ( n = 3 per cell line, n.s., two-way ANOVA). (D) DOK1 inhibits the human c - FOS promoter. HCT116 cells were cotransfected with DOK1 and SRE reporter plasmids for 24 h followed by incubation with EGF (10 ng/ml) in serum-free medium for additional 24 h. Luciferase activity in TCLs normalized to protein content was calculated as -fold ± S.E. ( n = 3, * p < 0.05 vs. EV, two-way ANOVA).

Article Snippet: Mouse monoclonal DOK1 (A3) Ab was diluted 1:200, and staining was processed according to the protocol from Vectastain ABC (HRP) kit (Vectorlabs).

Techniques: Western Blot, Mutagenesis, Transfection, Incubation, Luciferase, Activity Assay

Journal: EBioMedicine

Article Title: Subcellular compartmentalization of docking protein-1 contributes to progression in colorectal cancer

doi: 10.1016/j.ebiom.2016.05.003

Figure Lengend Snippet: DOK1 augments PPARγ-ligand-mediated inhibition of cell proliferation. (A) PTB and C-terminal domains, but not the N-terminal part of DOK1, are required for growth inhibition. HCT116 were transfected with DOK1 plasmids, and proliferation was measured after 5 days in presence of rosi (3 μM). O.D. values from MTT assays were calculated as % ± S.E. ( n = 3, *p < 0.05 DOK1 + rosi vs. EV + rosi; two-way ANOVA). (B) DOK1 knock-down enhances cell growth. Cells were transfected with siRNA, and proliferation was determined as in A ( n = 3, *p < 0.05 DOK1- vs. control-siRNA, two-way ANOVA). (C) Growth inhibition is PPARγ-receptor dependent. HEK293T cells were transfected with PPARγ plasmids for 72 h in absence of ligand. Data are shown as in A ( n = 3, *p < 0.05 vs. WT, one-way ANOVA).

Article Snippet: Mouse monoclonal DOK1 (A3) Ab was diluted 1:200, and staining was processed according to the protocol from Vectastain ABC (HRP) kit (Vectorlabs).

Techniques: Inhibition, Transfection

Journal: EBioMedicine

Article Title: Subcellular compartmentalization of docking protein-1 contributes to progression in colorectal cancer

doi: 10.1016/j.ebiom.2016.05.003

Figure Lengend Snippet: Compartmentalization of DOK1 predicts prognosis of CRC patients. (A) Immunohistochemistry (IHC) with DOK1 anti-C Ab (ab8112) on tissue microarrays (TMAs) with tumor (TU, n = 1648) specimens from CRC patients. Representative images are shown. DOK1 (brown color) was localized to the tumor cells in the cytoplasm ( i ), the apical perinuclear area ( ii ) or the nucleus ( iii ). Note the mixed pattern of DOK1 staining (positive control) in the normal colon glands both in epithelial (enterocytes) and stromal cells of the lamina propria ( iv ). DOK1 positivity was lost in a subset of tumors ( v ) and was absent in the secondary Ab only (negative control) staining ( vi ). Magnifications 200 ×. (B) Loss of DOK1 protein expression predicts poor survival in CRC patients. Kaplan–Meier curves of patient overall survival (OS). Upper panels: OS for DOK1 tumor and stroma expression; Lower panels: OS for nuclear and cytoplasmic DOK1 expression in the tumor. Nuclear localization of DOK1 correlates with poor, cytoplasmic DOK1 with improved prognosis. Data are surviving proportions vs. months ( n = 1492 ,*p < 0.05 log-rank tests). Values for tumor-specific survival (TSS) and median survival are presented in Table S4.

Article Snippet: Mouse monoclonal DOK1 (A3) Ab was diluted 1:200, and staining was processed according to the protocol from Vectastain ABC (HRP) kit (Vectorlabs).

Techniques: Immunohistochemistry, Staining, Positive Control, Negative Control, Expressing

Journal: EBioMedicine

Article Title: Subcellular compartmentalization of docking protein-1 contributes to progression in colorectal cancer

doi: 10.1016/j.ebiom.2016.05.003

Figure Lengend Snippet: Signaling model at a glance. In cells with FL p62 DOK1 at the membrane or cytoplasmic p44 DC mutant, oncogenic signaling of many target proteins is inhibited (such as RTK, CTK, SMAD, RAS, ELK1 e.a.) leading to reduction of SRE transcription (which drives cells into G1-S phase) and activation of PPRE transcription (which dampens cell proliferation), two exemplary read-outs used in the present study. PPARγ-agonist (star) further augments PPARγ-mediated growth inhibition, resulting in a good prognosis for patients' survival. Mechanistically, DOK1 may act as a “co-chaperone” for ligand-dependent nuclear translocation of PPARγ across the ER and nuclear pores. In tumor cells with loss of FL p62 DOK1 or accumulation of nuclear p33 DN mutant, oncogenic signaling (depicted here exemplary for the RAS-ERK1/2-ELK1-SRF pathway) is unimpeded leading to enhanced SRE and reduced PPRE transcription, resulting in tumor cell proliferation and a poor prognosis. Mechanistically, MEK1 and ERK1/2 inhibit nuclear translocation of PPARγ by promoting its export to the cytoplasm and inactivation by phosphorylation. Legend: red = active oncogene; blue black = active tumor suppressor; grey = inactive protein.

Article Snippet: Mouse monoclonal DOK1 (A3) Ab was diluted 1:200, and staining was processed according to the protocol from Vectastain ABC (HRP) kit (Vectorlabs).

Techniques: Mutagenesis, Activation Assay, Inhibition, Translocation Assay

Journal:

Article Title: Inhibition of the Motility and Growth of B16F10 Mouse Melanoma Cells by Dominant Negative Mutants of Dok-1

doi: 10.1128/MCB.21.16.5437-5446.2001

Figure Lengend Snippet: Adhesion-induced tyrosine phosphorylation of endogenous Dok-1 and FAK in established B16F10 cell lines expressing recombinant Dok-1 proteins. (A) B16F10 cells were detached from culture dishes and either maintained in suspension (Susp) or replated on fibronectin-coated dishes (Adh). After incubation for 30 min at 37°C, cells were lysed and subjected to immunoprecipitation (IP) with MAb A-3 to Dok-1 or with normal mouse immunoglobulin G (NMG). The immunoprecipitates were then subjected to immunoblot analysis with HRP-conjugated MAb PY20 to phosphotyrosine (αPY). Duplicate immunoprecipitates were probed with polyclonal antibodies to Dok-1 (αDok) to verify the presence of equal amounts of Dok-1 in each sample. (B) The indicated cell lines (Cont [control], WT28, and ΔPH20) were treated as in panel A, and the resulting cell lysates were subjected to immunoprecipitation with MAb 9E10 to the Myc tag (αMyc). The phosphotyrosine content of each recombinant Dok-1 protein was assessed as in panel A. (C) The extent of adhesion-induced tyrosine phosphorylation of endogenous Dok-1 in the indicated cell lines was determined as in panel A. (D) The phosphotyrosine content of endogenous Dok-1 in the experiment shown in panel C was quantified by scanning densitometry with the NIH Image program, normalized for the amount of Dok-1 protein in each sample, and was expressed as a percentage of the value for control cells transfected with the empty vector. (E) The indicated cell lines were treated as in panel A and the resulting detargent-solubilized membrane fraction was subjected to immunoprecipitation with MAb B4F8 to RasGAP (αRasGAP). The immunoprecipitates were then subjected to immunoblot analysis with HRP-conjugated MAb PY20 to detect tyrosine-phosphorylated Dok-1 bound to RasGAP. Duplicate immunoprecipitates were probed with the MAb to RasGAP to verify the presence of equal amounts of RasGAP in each sample. Aliquots of each membrane fraction (Membrane) were also directly probed with the MAb to RasGAP. (F) The indicated cell lines were treated as in panel A and subjected to immunoprecipitation with polyclonal antibody C-20 to FAK (αFAK). The immunoprecipitates were then subjected to immunoblot analysis with HRP-conjugated MAb PY20 to phosphotyrosine. Duplicate immunoprecipitates were probed with polyclonal antibody 06-543 to FAK (αFAK) to verify the presence of equal amounts of FAK in each sample. The positions of endogenous Dok-1, exogenous Dok-1, RasGAP, FAK, and the phosphorylated forms of these various proteins [Dok(endo)-P, Myc-DokWT-P, and FAK-P] are indicated.

Article Snippet: Horseradish peroxidase (HRP)-conjugated monoclonal antibody (MAb) PY20 to phosphotyrosine, mouse MAbs to Dok-1 (A-3), RasGAP (B4F8), and RhoA, and rabbit polyclonal antibody to focal adhesion kinase (FAK) (C-20) were obtained from Santa Cruz Biotechnology; mouse MAbs to H-Ras and to Shc were from Transduction Laboratories; rabbit polyclonal antibody to FAK (06-543) was from Upstate Biotechnology; and rabbit polyclonal antibodies that react specifically with tyrosine-phosphorylated (activated) ERK or with total ERK protein were from New England Biolabs.

Techniques: Expressing, Recombinant, Incubation, Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation