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The homophilic receptor PTPRK selectively dephosphorylates multiple junctional regulators to promote cell–cell adhesion

Research Article
Cite this article as: eLife 2019;8:e44597 doi: 10.7554/eLife.44597
9 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
The homophilic receptor PTPRK is stabilized by cell-cell contact.

(A) Schematic of full length PTPRK. The extracellular MAM, Ig and fibronectin domains mediate homophilic interactions. The intracellular domain comprises a juxtamembrane domain and two PTP domains; one active (D1) and one inactive (D2). (B) Structured illumination microscopy images of MCF10As immunostained for PTPRK (F4 clone; magenta) and E-Cadherin (green). Graphs indicate fluorescence intensity through the Z-axis in indicated boxed regions. Scale bars = 10 µm. (C) Fluorescence microscopy images from co-cultures of wildtype and nuclear mApple-expressing PTPRK knockout MCF10As that were immunostained for PTPRK (magenta) and E-Cadherin (green). Nuclei were stained with Hoechst (blue). mApple positive PTPRK KO cells are indicated by orange asterisks. Cell junctions where PTPRK is absent are highlighted by white arrows. Scale bars = 20 µm. (D) MCF10As were plated at indicated densities and analyzed by immunoblot after 3 days in culture. Arrows indicate full length (top) and furin-cleaved PTPRK (bottom). See also Figure 1—figure supplement 1.

https://doi.org/10.7554/eLife.44597.002
Figure 1—figure supplement 1
Generation and validation of PTPRK antibodies and interaction screen.

(A) mRNA expression of R2B PTPs in Human tissues. Raw data obtained from Genotype-tissue expression portal (GTExportal.org; GTEx Consortium et al., 2015). (B) 15 rabbit monoclonal antibodies were screened for their ability to specifically detect PTPRK by immunoblot. Immunoblot analysis of lysates generated from HEK293 cells transiently transfected with HA-PTPRK or an siRNA pool targeting PTPRK mRNA. Left: The indicated Rabbit monoclonal antibodies detected bands at 215 kDa (full length PTPRK) and 95 kDa (Furin-cleaved PTPRK), which are depleted by siRNA and increased with PTPRK overexpression. This suggests the antibodies recognize epitopes C-terminal to the Furin-cleavage site. Right panel: immunoblot probed with a commercial mouse monoclonal antibody raised against a PTPRK extracellular fragment (Sc-374315). No bands corresponding to predicted sizes (Full length 215 kDa; Furin-cleaved extracellular fragment:~120 kDa.) or modulated by siRNA depletion or overexpression were observed. (C) PTPRK antibody recognition based on (B). (D) 15 monoclonal antibodies were screened for their ability to specifically detect PTPRK by immunofluorescence. Immunostaining of MCF10As transfected with a non-targeting or PTPRK siRNA using PTPRK rabbit monoclonal antibody clone 1 .F4 (Magenta). F-actin (green) and nuclei (blue) were stained with phalloidin and Hoechst, respectively. Scale bars = 50 µm. (E) MCF10As were transfected with plasmids for the expression of Cas9, eGFP and single guide RNAs targeting PTPRK exons 1 and 2. eGFP-positive cells were cloned and expanded. Lysates from three clones grown individually or pooled were analyzed by immunoblot. PTPRK was detected using a mix of 2 .G6, 2 .H4 and 4 .H5 monoclonal antibodies. (F) Quantitative PCR analysis of confluent MCF10A cDNA using the indicated probes. Ct values relative to the housekeeping gene RPL19 were normalized using 2-ΔCt. The means of technical duplicates are shown. (G) Summary of secreted protein microarray with two purified protein libraries representing more than 1500 genes (≈50% single transmembrane receptors and partial secreted factor coverage) against the recombinant Fc-tagged PTPRK extracellular domain. Each intersection plot represents two independent microarray screens and dots represent average scores for each protein in the library. The lower left square represents all non-hit proteins with a cut-off of 10. Data analysis for hit calling is described in the Materials and methods section.

https://doi.org/10.7554/eLife.44597.003
Figure 2 with 3 supplements
The interactome of the homophilic adhesion receptor PTPRK.

(A) Experimental schematic of PTPRK interactome and substrate trapping studies. DA = D1057A, CS = C1089S. (B–D) Statistically enriched (p<0.05, n = 4) proteins after pull downs from pervanadate treated MCF10A lysates are displayed on volcano plots comparing PTPRK-ICD to beads control (B), PTPRK-ICD-DA to PTPRK-ICD (C) and PTPRK-ICD-CS to PTPRK-ICD (D). (E) GO term analysis of proteins statistically enriched (p<0.05) on PTPRK-ICD domains using Metascape. (F–G) Selected PTPRK interactors identified by mass spectrometry were validated by immunoblot analysis. Input and supernatants reveal the extent of protein depletion by recombinant proteins. Arrow indicates relevant band. See also Figure 2—figure supplements 1, 2 and 3. (H) Confluent, pervanadate-treated MCF10A lysates were used for pull downs with PTPRK D1057A ICD. Where indicated, pull downs were incubated with and without 20 mM vanadate for 30 min. 4% inputs (I), 4% supernatants (S), 4% eluates (E; following vanadate treatment) and pull downs (P) were subjected to immunoblot analysis. (I) Confluent, pervanadate-treated MCF10A lysates were treated with or without CIP to remove protein phosphorylation and were used for pull downs with PTPRK C1089S ICD. 4% inputs (I), 4% supernatants (S) and pull downs (P) were subjected to immunoblot analysis.

https://doi.org/10.7554/eLife.44597.004
Figure 2—source data 1

Raw and processed PTPRK interactome proteomic data.

Spreadsheet of all raw Maxquant output files (raw) and Peruses-generated processed data (processed) for the PTPRK pull down proteomic experiments). p values were determined using a two-sample, two-sided t test performed with truncation by a permutation-based FDR (threshold value 0.05; n ≥ 3).

https://doi.org/10.7554/eLife.44597.008
Figure 2—source data 2

PTPRK domain-interaction summary.

Spreadsheet of proteins that were statistically-enriched (p<0.05;>2 fold enrichment) on different PTPRK domains after pull downs and mass spectrometry. p values were determined using a two-sample, two-sided t test performed with truncation by a permutation-based FDR (threshold value 0.05; n ≥ 3).

https://doi.org/10.7554/eLife.44597.009
Figure 2—figure supplement 1
Purification of biotinylated recombinant PTPRK domains.

(A) His- and Avi-tagged PTPRK domains were expressed in E. coli cultured in biotin-supplemented media and purified using Nickel-NTA beads, followed by size exclusion chromatography (SEC). DA = D1057A mutant. CS = C1089S mutant. (B) SEC-purified proteins bound to streptavidin resin were eluted and resolved by SDS-PAGE followed by Coomassie staining. In; input, B; beads. (C) The phosphatase activity of indicated amounts of purified proteins was assessed using the Biomol green assay with two tyrosine phosphorylated peptides as substrates and was quantified at 620 nm. (D) Recombinant proteins bound to streptavidin resin were used in pull down assays from pervanadate treated Hs27 fibroblast lysates. After extensive washing, bound proteins were eluted in sample buffer and analyzed by immunoblot.

https://doi.org/10.7554/eLife.44597.005
Figure 2—figure supplement 2
PTPRK interactome from Hs27 cell lysates and vanadate competition.

(A–C) Volcano plots showing statistically enriched (p<0.05, n = 3) proteins bound to the indicated recombinant proteins after pull downs from pervanadate-treated confluent Hs27 cell lysates comparing PTPRK-ICD to beads control (A), PTPRK-ICD-D1057A to PTPRK-ICD (B) and PTPRK-ICD-C1089S to PTPRK-ICD (C). Grey points that appear significant were consistently found on the beads-only control. (D) Comparison of proteins enriched on the PTPRK-ICD after pulldowns from MCF10A and Hs27 lysates. Protein lists were used as inputs for BioVenn (Hulsen et al., 2008). (E) Pull downs using PTPRK-ICD D1057A and PTPRK-D2 domains from confluent, pervanadate-treated MCF10A lysates were incubated with and without 20 mM vanadate for 30 min. 4% inputs (I), 4% supernatants (S), 4% eluates (E; following treatment) and pull downs (P) were subjected to immunoblot analysis.

https://doi.org/10.7554/eLife.44597.006
Figure 2—figure supplement 3
PTPRK-D2 interactome and PTPRM pull downs.

(A) Volcano plot showing statistically enriched (p<0.05, n = 3) proteins bound to the indicated recombinant proteins after pull downs from pervanadate-treated confluent MCF10A cell lysates comparing PTPRK-D2 to beads control. (B) Comparison of proteins enriched on PTPRK-D2 and PTPRK-ICD domains after pulldowns from MCF10A lysates. Protein lists were used as inputs for BioVenn (Hulsen et al., 2008). (C) After size exclusion chromatography (SEC), purified protein was incubated with or without streptavidin and subjected to SDS PAGE followed by Coomassie staining to determine the extent of biotinylation. Arrows indicate the purified domains and the respective streptavidin-induced mobility shift. (D–E) PTPRK interactors identified by mass spectrometry were validated using pull downs with the specified PTPRK and PTPRM protein domains from pervanadate-treated, confluent MCF10A cell lysates followed by immunoblot analysis. Input and supernatants reveal the extent of protein depletion by recombinant proteins.

https://doi.org/10.7554/eLife.44597.007
Figure 3 with 2 supplements
The PTPRK dependent tyrosine phosphoproteome.

(A) Schematic of workflow to enrich and identify phosphotyrosine peptides from SILAC-labeled wildtype and PTPRK KO MCF10As. Equal amounts of wildtype and PTPRK KO cell lysates were combined prior to trypsinization. A 10% sample was reserved for total proteome analysis. Tyrosine phosphorylated peptides were enriched using anti-phosphotyrosine antibodies and SH2 domain ‘superbinders’. (B) Volcano plot of tyrosine phosphosites detected in PTPRK KO and wildtype MCF10As. Phosphosites > 50% enriched in (p<0.05; n = 3) in PTPRK KO cells are labeled red and those enriched in wildtype are blue. FDR = 0.01, two valid values required. (C) Volcano plot of protein abundance. Proteins > 50% more abundant (p<0.05; n = 3) in PTPRK KO MCF10As are shown in red, and wildtype in blue. FDR = 0.01, two valid values required. (D) Overview of proteins with at least one tyrosine phosphorylation site increased in PTPRK KO cells as determined by quantitative proteomics (FDR = 0.01, one valid value required). Tyrosine phosphosite change in PTPRK KO cells compared to wildtype is indicated by colored circles:>3 fold up; purple,>1.5 fold up; red,<1.5 fold up or down (no change); grey. Proteins identified as interactors by AP-MS or immunoblotting in this study are highlighted in bold and italics. *Denotes proteins enriched on substrate traps. See also Figure 3—figure supplements 1 and 2.

https://doi.org/10.7554/eLife.44597.010
Figure 3—source data 1

Quantitative total and tyrosine phosphoproteomics.

Spreadsheet of all raw Maxquant output files (raw) and Peruses-generated processed data (processed; requiring either 1 or two valid values) for the total and tyrosine phosphoproteomic experiments. p values were determined using a one-sample, two-sided t test performed with truncation by a Benjamini Hochberg FDR (threshold value 0.05; n = 3).

https://doi.org/10.7554/eLife.44597.013
Figure 3—source data 2

Statistically upregulated proteins and phosphotyrosine sites in PTPRK KO cells following quantitative proteomics.

Spreadsheet of proteins that were statistically-enriched (≥50% + p<0.05) for the total and tyrosine phosphoproteomic experiments (1 and 2 valid values). p values were determined using a one-sample, two-sided t test performed with truncation by a Benjamini Hochberg FDR (threshold value 0.05; n = 3).

https://doi.org/10.7554/eLife.44597.014
Figure 3—figure supplement 1
The PTPRK-dependent tyrosine phosphoproteome.

(A) After SEC, proteins were incubated with or without streptavidin and subjected to SDS PAGE followed by Coomassie staining to determine the extent of biotinylation. Arrows indicate the purified domains and the respective streptavidin-induced mobility shift. (B) Volcano plot of tyrosine phosphosites detected in PTPRK KO and wildtype MCF10As. Phosphosites > 50% enriched in (p<0.05; n = 3) in PTPRK KO cells are labeled red and those enriched in wildtype are blue. FDR = 0.01, one valid value required. (C) Proteins with upregulated phosphotyrosine sites in PTPRK KO cells that were also identified as PTPRK interactors, either by proteomics or immunoblotting. (D) Weblogo representation of amino acids surrounding phosphotyrosine from candidate substrates in (C). (E) Surface charge representation of PTPRK-D1 (left; PDB: 2C7S Eswaran et al., 2006) showing acetate bound to the active site. Yellow line represents the approximate binding location for a ~ five amino acid phosphopeptide. Scale indicates kcal/mol·e. (F) Phopshopeptide motifs derived from PTPRK candidate substrates ([STEADNR][NPDHLRTY][INSEPGV]pY[VADIEFGSY][DTEGIKNQRS][LFPSTANR]) were used to search the phosphosite plus database. Scramble 1 and 2 correspond to the following searches: [LFPSTANR][DTEGIKNQRS][VADIEFGSY]pY[INSEGPV][INSEGPV][STEADNR] and [INSEGPV][NPDHLRTY][STEADNR]pY[LFPSTANR][DTEGIKNQRS], respectively. Listed are PTPRK-interacting proteins with phosphosites predicted by the consensus. Number of overlapping proteins between phosphosite plus searches and interactors are shown on Venn diagrams.

https://doi.org/10.7554/eLife.44597.011
Figure 3—figure supplement 2
The PTPRK-dependent tyrosine phosphoproteome is enriched for cell junction organization proteins.

(A–B) PTPRK interactors identified by mass spectrometry were validated using pull downs with the specified PTPRK and PTPRM protein domains from pervanadate-treated, confluent MCF10A cell lysates followed by immunoblot analysis. Input and supernatants reveal the extent of protein depletion by recombinant proteins. (C) GO term analysis using Metascape of proteins with increased tyrosine phosphorylation in PTPRK KO MCF10As. FDR = 0.01, one valid value required.

https://doi.org/10.7554/eLife.44597.012
Figure 4 with 1 supplement
PTPRK interacts with candidate substrates in confluent MCF10A cells.

(A) Representative immunoblot analysis of biotin pull downs from MCF10As expressing tGFP or PTPRK BioID constructs. See Materials and methods for details. Red and blue arrows indicate exogenous and endogenous PTPRK, respectively. (B) Quantification of BioID immunoblots. Green bars indicate the number of times a protein was enriched on PTPRK-C1089S.BirA*-Flag, compared to PTPRK.ECD +TMD.BirA*-Flag in separate experiments. Purple bars indicate the number of times a protein was not enriched or was not detected in any pull downs. n ≥ 1. (C) Schematic representation of PTPRK proximity-labeling by BioID. PTPRK extracellular domain homology model is based on PTPRM (PDB: 2V5Y; Aricescu et al., 2007). Proteins within the dotted lines were detected in pull downs from indicated BioID lysates. Proteins not detectably biotinylated are listed on the left. Proteins in bold and italics were previously identified as PTPRK interactors using BioID in HEK293 cells (St-Denis et al., 2016). See also Figure 4—figure supplement 1.

https://doi.org/10.7554/eLife.44597.015
Figure 4—figure supplement 1
Localization of PTPRK BioID proteins.

(A) MCF10As with stably integrated doxycycline-inducible expression constructs (PTPRK-ECD +TMD-BirA*-Flag and PTPRK-C1089s-BirA*-Flag) were treated with 150 ng/ml and 500 ng/ml doxycycline, respectively, and immunostained using an anti-Flag antibody. F-actin and nuclei were stained with phalloidin and Hoechst, respectively. Scale bars = 50 µm. (B) Representative immunoblot analysis of biotin pull downs from MCF10As expressing tGFP or PTPRK BioID constructs. See Materials and methods for details. Red and blue arrows indicate exogenous and endogenous PTPRK, respectively. * residual ABLIM3 signal.

https://doi.org/10.7554/eLife.44597.016
Figure 5 with 2 supplements
PTPRK directly and selectively dephosphorylates cell junction regulators.

(A) Workflow of in-lysate dephosphorylation assay. Recombinant PTPRK and PTPRM domains were incubated with pervanadate-treated MCF10A lysates for 1.5 hr at 4°C, followed by immunoprecipitation of tyrosine phosphorylated proteins. (B) Pervanadate-treated MCF10A lysates were incubated with the indicated domains at an amount pre-determined to give equal phosphatase-activity prior to phosphotyrosine immunoprecipitation and immunoblot analysis. (C) Pull downs using chimeric RPTPs from confluent, pervanadate-treated MCF10A lysates were subjected to immunoblot analysis. (D) Pervanadate-treated MCF10A lysates were incubated with the indicated domains prior to phosphotyrosine immunoprecipitation and immunoblot analysis. See also Figure 5—figure supplements 1 and 2.

https://doi.org/10.7554/eLife.44597.017
Figure 5—figure supplement 1
In vitro dephosphorylation assays and generation of RPTP chimeras.

(A) The indicated PTPRK and PTPRM domains were assayed for phosphatase activity using the pNPP colorimetric assay. Control wells contained pNPP only. Protein amounts used are shown. (B) Pervanadate-treated MCF10A lysates were incubated with predetermined amounts of the indicated domains to give equal phosphatase-activity, prior to phosphotyrosine immunoprecipitation and immunoblot analysis. (C) Recombinant proteins consisting of combinations of PTPRK and PTPRM D1 and D2 domains were expressed in and using Ni-NTA affinity resin. Purified proteins were then subjected to size exclusion chromatography. (D) Recombinant His- and Avi-tagged PTPRK and PTPRM chimeric domains were purified from E. coli cultured in biotin-supplemented media, incubated ±streptavidin and subjected to SDS-PAGE and Coomassie staining, to determine the extent of biotinylation. Arrows indicate the purified domains and the respective streptavidin-induced mobility shift. (E) The indicated recombinant PTPRK and PTPRM chimeric domains were incubated were assayed for phosphatase activity using the pNPP colorimetric assay. Control wells contained pNPP. Protein amounts used are shown.

https://doi.org/10.7554/eLife.44597.018
Figure 5—figure supplement 2
Analysis of PTPRK-D2 domain interactions.

(A) Pull downs using PTPRK-ICD D1057A and PTPRK-D2 domains from confluent, pervanadate-treated MCF10A lysates were incubated with and without 20 mM vanadate for 30 min. 4% inputs (I), 4% supernatants (S), 4% eluates (E; following treatment) and final pull downs (P) were subjected to immunoblot analysis. (B) Confluent, pervanadate-treated MCF10A lysates were treated with or without 20 U/ml CIP at 4°C for 16 hr to remove protein phosphorylation and were used for pull downs with PTPRK D2 domain. 4% inputs (I), 4% supernatants (S) and pull downs (P) were subjected to immunoblot analysis. (C) Clustal Omega alignment of PTPRK-D1 vs PTPRK-D2 vs PTPRK-D2 triple mutant generated using Jalview. (D) The indicated PTPRK domains were assayed for phosphatase activity using the pNPP colorimetric assay. Activity levels relative to PTPRK ICD are shown. Error bars denote ±SEM of technical triplicates. (E) Pull downs using indicated wildtype and mutant PTPRK domains from confluent, pervanadate-treated MCF10A lysates were analyzed by immunoblot. (F) Ribbon and surface representations of CD45 (PTPRC; PDB: 1YGR; Nam et al., 2005). The corresponding ‘active site’ cysteine residues (C828S for D1 and C1144 for D2) are highlighted in red. (G) Surface charge representation of PTPRK-D1 (left; PDB: 2C7S [Eswaran et al., 2006]) and PTPRK-D2 (right; homology model based on PDB: 6D3F (PTPRE-D2; [Lountos et al., 2018])).

https://doi.org/10.7554/eLife.44597.019
Figure 6 with 1 supplement
PTPRK dephosphorylates p120Cat Y228 and Y904 in MCF10A cells.

(A) Pervanadate-treated MCF10A lysates were incubated with and without the indicated recombinant PTPRK-D1, PTPRK-ICD or PTPRK-C1089S-ICD for 1.5 hr at 4°C, prior to immunoblot analysis. (B–C) Lysates from confluent wildtype and PTPRK KO MCF10As were analyzed by immunoblot and quantified by densitometry. Error bars denote ±SEM (n ≥ 3). Unpaired, two-tailed t test: *p<0.05, **p<0.005. (D) Wildtype or PTPRK KO MCF10As, with stably-integrated doxycycline-inducible tGFP, PTPRK or PTPRK-C1089S, were cultured for 6 days with indicated concentrations of doxycycline then lysed and subjected to immunoblot analysis. (E) Densitometric quantification of p120Cat phosphorylation normalized against total p120Cat. Error bars denote ±SEM (n = 5). Two-way ANOVA (Tukey’s multiple comparisons test): *p<0.005**, p<0.005, ***p<0.0005. See also Figure 6—figure supplement 1.

https://doi.org/10.7554/eLife.44597.020
Figure 6—source data 1

Densitometric analysis of immunoblots.

Spreadsheet of densitometric quantification of p120Cat phosphorylation (normalized against total p120Cat) from Figure 6C and Figure 6E. p values were determined using a two-way ANOVA.

https://doi.org/10.7554/eLife.44597.022
Figure 6—figure supplement 1
PTPRK dephosphorylates p120Cat-Y228 and Y904.

(A–B) Pervanadate-treated MCF10A lysates were incubated with and without the indicated recombinant PTPRK-ICD or PTPRM-ICD protein domains, with or without pervanadate (10 mM) for 1.5 hr at 4°C, prior to immunoblot analysis.

https://doi.org/10.7554/eLife.44597.021
Figure 7 with 2 supplements
PTPRK promotes junction integrity and organization in epithelial cells.

(A) Wildtype (Left) and PTPRK KO (Right) MCF10As were cultured on transwell filters before being fixed and prepared for conventional electron microscopy (EM). Scale bar = 5 µm. (B) Quantification of cell height relative to transwell filter. Three measurements per image were averaged. Each data point relates to one EM image. Error bars denote ±SEM. Unpaired, two tailed t test ***p<0.0005. (C) Stable PTPRK KO MCF10As were grown to confluence with or without 250 ng/ml doxycycline on 0.4 µm transwell filters prior to TEER analysis. Error bars denote ±SEM (n = 3). Two-way ANOVA (Sidak's multiple comparisons test): *p<0.05. (D) Confluent PTPRK KO MCF10As, with stably-integrated doxycycline-inducible PTPRK or PTPRK-C1089S, were cultured for 6 days with or without 250 ng/ml doxycycline then fixed and stained for E-Cadherin and F-actin. A representative confocal microscopy image is shown. Scale bar = 20 µm. (E) Quantification of relative F-actin staining intensity. 10 random fields/replicate were averaged. Error bars denote ±SEM (n ≥ 3). Two-way ANOVA (Sidak's multiple comparisons test): *p<0.05 (F) Quantification of colocalization (Pearson coefficient) between E-Cadherin and F-actin staining. 10 random fields/biological replicate were averaged. Error bars denote ±SEM (n = 3). Two-way ANOVA (Sidak's multiple comparisons test): *p<0.05. See also Figure 7—figure supplement 1.

https://doi.org/10.7554/eLife.44597.023
Figure 7—source data 1

Source data used in graphs.

Spreadsheet of normalized data from Figure 7B,C,E and F. p values were determined using a two-way ANOVA.

https://doi.org/10.7554/eLife.44597.026
Figure 7—figure supplement 1
Loss of PTPRK compromises cell junction integrity.

(A) Wildtype and PTPRK KO MCF10As grown to confluence on 0.4 µm transwell filters were subjected to a media change 24 hr prior to TEER analysis. Error bars denote ±SEM (n = 3). Unpaired, two-tailed t test: *p<0.05. (B) Fluorescence intensity of the lower chamber of 0.4 µm transwell filters with wildtype and PTPRK KO MCF10As after incubation with 3 mg/ml 250 kDa FITC Dextran (added to the upper chamber) for 24 hr. (C–D) Confluent wildtype and PTPRK KO MCF10As were fixed and stained for E-Cadherin (C), DSG3 (D) or p120Cat (E) and F-actin. A representative confocal microscopy image is shown. Scale bar = 20 µm. (F) Quantification of relative F-actin, E-Cadherin, DSG3 and p120Cat staining intensity comparing wildtype and PTPRK KO MCF10As. 10 random fields/biological replicate were averaged. Error bars denote ±SEM (n ≥ 3). Two-way ANOVA (Sidak's multiple comparisons test): **p<0.005, ***p<0.0005. (G–H) Quantification of co-localization (Pearson coefficient) between E-Cadherin (F) or DSG3 (G) and F-actin staining comparing wildtype and PTPRK KO MCF10As. Error bars denote ±SEM (n = 3). Unpaired, two-tailed t test: *p<0.05. (I) Immunoblot analysis of confluent wildtype and PTPRK KO MCF10A.

https://doi.org/10.7554/eLife.44597.024
Figure 7—figure supplement 2
Both PTPRK and PTPRK-C1089S partially rescue E-Cadherin intensity.

(A) Quantification of relative E-Cadherin staining intensity. 10 random fields/replicate were averaged. Error bars denote ±SEM (n ≥ 3). Two-way ANOVA (Sidak's multiple comparisons test): *p<0.05.

https://doi.org/10.7554/eLife.44597.025
Figure 8 with 1 supplement
PTPRK promotes organization in epithelial cells.

(A) Phase contrast images of wildtype and PTPRK KO. MCF10A spheroids after 14 day culture in Matrigel. Scale bar = 200 µm. (B) Frequency of aberrant acini observed in six independent wells each of wildtype and PTPRK KO MCF10A spheroids. Unpaired, two-tailed t test: *p<0.05. (C) Representative images of MCF10A spheroids stained for the Golgi marker GM130, F-actin and nuclei (Hoechst), after removal from Matrigel. Scale bar = 20 µm. (D) Circles were traced over cross sections, based on the Hoechst channel, for a total of 563 WT and 551 PTPRK KO immunostained spheroids from three entire slides per genotype and diameters calculated in Zen Pro. Unpaired, two-tailed t test: ***p<0.0005. See also Figure 8—figure supplement 1.

https://doi.org/10.7554/eLife.44597.027
Figure 8—source data 1

Source data used in graphs.

Spreadsheet of normalized data from Figure 8B and Figure 8D. p values were determined using an unpaired, two tailed t test.

https://doi.org/10.7554/eLife.44597.029
Figure 8—figure supplement 1
PTPRK loss perturbs epithelial organization.

(A) Phase contrast images of wildtype and PTPRK KO MCF10A spheroids after 14 day culture in Matrigel. Scale bars = 200 µm. (B) BrdU incorporation assay performed on subconfluent WT and PTPRK KO MCF10As (n = 3). Unpaired, two-tailed t test: ns = not significant.

https://doi.org/10.7554/eLife.44597.028

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Gene (Homo sapiens)PTPRKENSEMBL: ENST00000368213.9
Cell line (H. sapiens)MCF10AATCCCRL-10317
Cell line (H. sapiens)HEK293TD RonN/A
Cell line (H. sapiens)HEK293Sigma (ECACC)85120602-1VL
Cell line (H. sapiens)Hs27 FibroblastsSigma (ECACC)94041901-1VL
Cell line (H. sapiens)MCF10A PTPRK KO A4This studyCRISPR/Cas9 and
clonal selection
Cell line (H. sapiens)MCF10A PTPRK KO E3This studyCRISPR/Cas9 and
clonal selection
Cell line (H. sapiens)MCF10A PTPRK KO H1This studyCRISPR/Cas9 and
clonal selection
Cell line (H. sapiens)MCF10A PTPRK KO pooledThis study
Transfected
construct (H. sapiens)
MCF10A PTPRK KO pooled.tGFPThis studyLentivirally transduced
stable cell line
Transfected
construct (H. sapiens)
MCF10A PTPRK
KO pooled.tGFP.P2A.PTPRK
This studyLentivirally transduced
stable cell line
Transfected
construct (H. sapiens)
MCF10A PTPRK KO pooled.tGFP.P2A.PTPRK.C1089SThis studyLentivirally transduced
stable cell line
Transfected
construct (H. sapiens)
MCF10A.tGFPThis studyLentivirally transduced
stable cell line
Transfected
construct (H. sapiens)
MCF10A.tGFP.P2A.PTPRK.ECD-TMD.BirA*-FlagThis studyLentivirally transduced
stable cell line
Transfected
construct (H. sapiens)
MCF10A.tGFP.P2A.PTPRK.C1089S.BirA*-FlagThis studyLentivirally transduced
stable cell line
Transfected
construct (H. sapiens)
MCF10A PTPRK KO pooled.nuclear mAppleThis studyLentivirally transduced
stable cell line
Transfected
construct (H. sapiens)
MCF10A.nuclear mAppleThis studyLentivirally transduced
stable cell line
AntibodyRabbit monoclonal anti-PTPRKThis study2 .G6Western blot: 1:1000
AntibodyRabbit monoclonal anti-PTPRKThis study2 .H4Western blot: 1:1000
AntibodyRabbit monoclonal anti-PTPRKThis study2 .H5Western blot: 1:1000
AntibodyRabbit monoclonal anti-PTPRKThis study1 .F4FACS (1:200)
and Immunofluorescence
(IF; 1:200)
AntibodyMouse anti-PTPRKSanta Cruz BiotechnologyCat#Sc- 374315Western blot: 1:1000 (note: we did not observe any specific signal for PTPRK with this antibody)
AntibodyRabbit anti-PARD3SigmaCat#HPA030443 (lot: C105765)Western blot: 1:1000
AntibodyRabbit anti-PARD3Merck MilliporeCat#07–330Western blot: 1:1000
AntibodyMouse anti-RAPGEF6Santa Cruz
Biotechnology
Cat#sc-398642 (F-8)Western blot: 1:1000
AntibodyMouse anti-AfadinBD Transduction LabsCat#610732Western blot: 1:1000
AntibodyMouse anti-DLG5Santa Cruz
Biotechnology
Cat#SC374594 (A-11)Western blot: 1:1000
AntibodyMouse anti-PTPN14R and D SystemsCat#MAB4458Western blot: 1:1000
AntibodyMouse anti-E-CadherinBD Transduction
Labs
Cat#610181Western blot:
1:1000 IF: 1:100
AntibodyRabbit anti-b-CateninCell Signaling
Technology
Cat#9562SWestern blot: 1:1000
AntibodyRabbit anti-Phospho-EGFR (Y1068)Cell Signaling
Technology
Cat#3777SWestern blot: 1:1000
AntibodyRabbit anti-EGFRCell Signaling
Technology
Cat#4267SWestern blot: 1:1000
AntibodyRabbit anti-phospho-tyrosine(P-Tyr-1000)Cell Signaling
Technology
Cat#8954Western blot: 1:2000
AntibodyRabbit anti-MAP4K4Cell Signaling
Technology
Cat#5146Western blot: 1:1000
AntibodyRabbit anti-NUFIP2Bethyl
Laboratories, Inc
Cat#A301-600AWestern blot: 1:1000
AntibodyRabbit anti-FMRP1ThermoFisher
Scientific
Cat#MA5-15499Western blot: 1:1000
AntibodyRabbit anti-MINK1/MAP4K6ThermoFisher
Scientific
Cat#PA5-28901Western blot: 1:1000
AntibodyRabbit anti-PKP4Bethyl
Laboratories, Inc
Cat#A304-649AWestern blot: 1:1000
AntibodyMouse anti-P120 cateninBD Transduction
Laboratories
Cat#610133Western blot:
1:1000 IF: 1:100
AntibodyMouse anti-GM130BD Transduction
Laboratories
Cat#610822Western blot: 1:1000
AntibodyRabbit anti-STAT3Cell Signaling
Technology
Cat#4904SWestern blot: 1:1000
AntibodyRabbit anti-PaxillinCell Signaling
Technology
Cat#12065 (D9G12)Western blot: 1:1000
AntibodyMouse anti-Tubulin (Alpha)SigmaCat#T6199Western blot: 1:1000
AntibodyMouse anti-PTPRMSanta CruzCat#sc-56959Western blot: 1:1000
AntibodyRabbit anti-PKP3AbcamCat#AB109441Western blot: 1:10000
AntibodyRabbit-anti-ABLIM3SigmaCat#HPA003245Western blot: 1:1000
AntibodyRabbit-Anti-ZO2ThermoFisher
Scientific
Cat#711400Western blot: 1:1000
AntibodyRabbit-anti-Phospho-P120 catenin (Y904)Cell Signaling
Technology
Cat#2910Western blot: 1:1000
AntibodyRabbit-anti-Phospho-P120 catenin (Y228)Cell Signaling
Technology
Cat#2911Western blot: 1:1000
AntibodyRabbit polyclonal anti-Phospho-Paxillin (Y118)Cell Signaling
Technology
Cat#2541Western blot: 1:1000
AntibodyRabbit-anti-b-ActinSIGMACat#A2066Western blot: 1:1000
AntibodyMouse-anti-DSG3Bio-RadCat#MCA2273TWestern blot: 1:5000
AntibodyHRP conjugated-Donkey
anti-Rabbit IgG
Jackson
Immuno-Research
Cat#711-035-152Western blot: 1:5000
AntibodyHRP conjugated- Donkey anti-Mouse IgGJackson
Immuno-Research
Cat#711-035-152Western blot: 1:5000
AntibodyHRP conjugated- Mouse anti-Rabbit IgG (Conformation specific)Cell Signaling
Technology
Cat#5127SWestern blot: 1:2000
AntibodyAtto-488 Goat Anti-mouse IgGSigmaCat#62197IF: 1:400
AntibodyAtto-488 Goat Anti-mouse IgGSigmaCat#62197IF: 1:400
AntibodyAlexa Fluor-647 Goat Anti Rabbit IgGJackson
Immuno-Research
Cat#111-605-003IF: 1:400
Recombinant
DNA reagent
pCW57.tGFP.P2A.MCSAddgeneCat#71783
Recombinant
DNA reagent
pRK.HA.PTPRK.flagGenentechCorresponds to
Uniprot identifier:
Q15262-3
Recombinant
DNA reagent
pRK.PTPRK(1-752).IgG1Genentech
Recombinant
DNA reagent
pET15bJ. Deane
Recombinant
DNA reagent
pSP.Cas9.(BB).eGFPD Ron
Recombinant
DNA reagent
pMD2.GAddgeneCat#12259
Recombinant DNA reagentpsPAX2AddgeneCat#12260
Recombinant
DNA reagent
pLenti-puroAddgeneCat#39481
Recombinant
DNA reagent
PTPRK-BirA-R118G-FlagA-C Gingras
Recombinant
DNA reagent
pCW57.tGFP.P2A.PTPRKThis study
Recombinant
DNA reagent
pCW57.tGFP.P2A.PTPRK.C1089SThis study
Recombinant
DNA reagent
pCW57.tGFP.P2A.PTPRK(1-785).BirA-R118G.FlagThis study
Recombinant
DNA reagent
pCW57.tGFP.P2A.PTPRK.C1089S.BirA-R118G.FlagThis study
Recombinant
DNA reagent
pET15b.His.TEV.AviThis study
Recombinant
DNA reagent
pET15b.His.TEV.Avi.PTPRK.ICDThis study
Recombinant
DNA reagent
pET15b.His.TEV.Avi.PTPRK.ICD.D1057AThis study
Recombinant
DNA reagent
pET15b.His.TEV.Avi.PTPRK.ICD.C1089SThis study
Recombinant
DNA reagent
pET15b.His.TEV.Avi.PTPRK.D1This study
Recombinant
DNA reagent
pET15b.His.TEV.Avi.PTPRK.D2This study
Recombinant
DNA reagent
pET15b.His.TEV.Avi.
PTPRK.D2.triple
This studyMutations: A1346P,
S1347D, L1384S,
E1427Q, A1428T
Recombinant
DNA reagent
pET15b.His.TEV.Avi.PTPRM.ICDThis study
Recombinant
DNA reagent
pET15b.His.TEV.Avi.PTPRM.D1This study
Recombinant
DNA reagent
pET15b.His.TEV.Avi. PTPRK-D1_K-D2.This study
Recombinant
DNA reagent
pET15b.His.TEV.Avi.PTPRM-D1_M-D2This study
Recombinant
DNA reagent
pET15b.His.TEV.Avi.PTPRK-D1_M-D2.This study
Recombinant
DNA reagent
pET15b.His.TEV.Avi.PTPRM-D1_K-D2.This study
Recombinant
DNA reagent
pET15b.His.TEV.Avi.Src.sbSH2This study
Recombinant
DNA reagent
pET15b.His.TEV.Avi.Grb2.sbSH2This study
Recombinant
DNA reagent
pSP.Cas9.PTPRK.sgRNA1This study
Recombinant
DNA reagent
pSP.Cas9.PTPRK.sgRNA2This study
Sequence-based reagentON-TARGETplus Human PTPRK siRNADharmacon,
GE Healthcare
Cat#J-004204–06
Sequence-
based reagent
ON-TARGETplus Non-targeting pool siRNADharmacon,
GE Healthcare
Cat#D-001810-10-05
Sequence-
based reagent
PTPRK CRISPR, BbsI.PTPRKgRNA1.FwdSIGMACACCGCATGGATACGACTGCGGCGG
Sequence-
based reagent
PTPRK CRISPR, BbsI.PTPRKgRNA1.RevSIGMAAAACCCGCCGCAGTCGTATCCATGC
Sequence-
based reagent
PTPRK CRISPR, BbsI.PTPRKgRNA2.FwdSIGMACACCGATCTCGGGTGGTAGATAATG
Sequence-
based reagent
PTPRK CRISPR, BbsI.PTPRKgRNA2.RevSIGMAAAACCATTATCTACCACCCGAGATC
Sequence-
based reagent
TaqMan probe: Hs02338565_gH (RPL19)Thermo Fisher
Scientific
Cat#4331182
Sequence-
based reagent
TaqMan probe: Hs00267788_m1 (PTPRK)Thermo Fisher
Scientific
Cat#4331182
Sequence-
based reagent
TaqMan probe: Hs00267809_m1 (PTPRM)Thermo Fisher
Scientific
Cat#4331182
Sequence-
based reagent
TaqMan probe: Hs00179247_m1 (PTPRT)Thermo Fisher
Scientific
Cat#4331182
Sequence-
based reagent
TaqMan probe: Hs00963911_m1 (PTPRU)Thermo Fisher
Scientific
Cat#4351372
Peptide,
recombinant
protein
DADE-pTyr-LIPQQG-
phospho-peptide
Cambridge
Research
Biochemicals
Cat#crb1000746
Peptide,
recombinant protein
END-pTyr-INASL-phospho-peptideCambridge
Research
Biochemicals
Cat#crb1000745
Peptide,
recombinant protein
CatalaseSigmaCat#C134514
Peptide,
recombinant protein
Cholera ToxinSigmaCat#C-8052
Peptide,
recombinant protein
InsulinSigmaCat#I-1882
Peptide,
recombinant protein
Epidermal Growth FactorPeprotechCat#AF-100-15-1MG
Peptide,
recombinant protein
Lysyl endopeptidase (LysC)WakoCat#129–02541
Peptide,
recombinant protein
Trypsin (proteomics grade)Thermo Fisher
Scientific
Cat#90058
Commercial
assay or kit
BIOMOL Green reagentENZOCat#BML-AK111-0250
Commercial
assay or kit
Phosphate standardENZOCat#BML-KI102-0001
Commercial
assay or kit
Q5 High-Fidelity DNA PolymeraseNew England
Biolabs
Cat#M0491S
Commercial
assay or kit
Phusion Hot Start II DNA polymeraseThermo Fisher
Scientific
Cat#F549L
Commercial
assay or kit
EZ-ECL substrateGeneflowCat#K1-0170
Commercial
assay or kit
NuPAGE MES
(2-ethanesulfonic acid) SDS running buffer
ThermoFisher
Scientific
Cat#NP0002
Commercial
assay or kit
InstantBlueExpedeonCat#ISB1L
Commercial
assay or kit
Phosphatase inhibitor cocktailRocheCat#04906845001
Commercial
assay or kit
TaqMan Universal Master Mix IIApplied BiosystemsCat#4440040
Commercial
assay or kit
MycoAlertTM PLUS Mycoplasma Detection KitLonza#LT07-705
Commercial
assay or kit
MycoProbe Mycoplasma Detection KitR and D Systems#CUL001B
Chemical
compound, drug
Hydrogen peroxideThermo Fisher
Scientific
Cat#H/1750/15
Chemical
compound, drug
Sodium orthovanadateAlfa AesarCat#J60191
Chemical
compound, drug
250 kDa-FITC-dextranSigmaCat#FD250S-100MG
Chemical
compound, drug
Para-Nitrophenol-phosphate (pNPP)New England BiolabsCat#P0757
Chemical
compound, drug
IPTGGeneronCat#GEN-S-02122
Chemical
compound, drug
D-biotinSigmaCat#B4639
Chemical
compound, drug
L-glutamineSigmaCat#G7513
Chemical
compound, drug
HydrocortisoneSigmaCat#H-0888
Chemical
compound, drug
PuromycinThermo Fisher
Scientific
Cat#A11138-03
Chemical
compound, drug
Phosphate free H2OThermo Fisher
Scientific
Cat#10977–035
Chemical
compound, drug
8M Guanidine HClThermo Fisher
Scientific
Cat#24115
Chemical
compound, drug
EPPS pH 8.5Alfa AesarCat#561296
Chemical
compound, drug
Trifluoroacetic Acid (TFA)Thermo Fisher
Scientific
Cat#28904
Chemical
compound, drug
AcetonitrileVWRCat#8364.290
Chemical
compound, drug
Sodium phosphate
dibasic (Na2HPO4)
Acros OrganicsCat#343811000
Chemical
compound, drug
NH4OHAcros OrganicsCat#460801000
Chemical
compound, drug
Methanol-free 16%
(w/v) paraformaldehyde (PFA)
Thermo Fisher
Scientific
Cat#28906
Software, algorithmMaxquantComputational
Systems
Biochemistry
Max Planck Institute
of Biochemistry
Software, algorithmPerseusComputational
Systems
Biochemistry
Max Planck Institute
of Biochemistry
Software, algorithmFIJI/ImageJLaboratory for
Optical and
Computational
Instrumentation
University of
Wisconsin-Madison
Software, algorithmZen BlueZeiss
Software, algorithmZen BlackZeiss
Software, algorithmGraphpadPrism
Software, algorithmChimeraUCSF
OtherHRP-conjugated
Streptavidin
Thermo Fisher
Scientific
Cat#434323
OtherSTABLE competent E. coliNEBCat#C3040I
OtherDH5alpha
competent E. coli
InvitrogenCat#18265017
OtherBL21 DE3 Rosetta E. coliJ DeaneN/A
OtherDMEMThermo Fisher
Scientific
Cat#41965–039
OtherHam's F-12SigmaCat#N4888
OtherHorse SerumThermo Fisher
Scientific
Cat#16050–122
OtherFibroblast growth
medium (FGM)
PromocellCat#C-23010
OtherFetal Bovine SerumSigmaCat#F7524-500ml
OtherTrypsin-EDTA solutionSigmaCat#T3924
OtherGeneJuice
transfection reagent
Merck MilliporeCat#70967–3
OtherEDTA-free
protease inhibitors
RocheCat#11836170001
OtherLipofectamine RNAiMaxInvitrogenCat#13778075
OtherOptiMEMThermo Fisher
Scientific
Cat#31985070
OtherLipofectamine LTXThermoFisher
Scientific
Cat#15338100
OtherProtein G agarose beadsMerck MilliporeCat#16–266
OtherNi-NTA agaroseQIAGENCat#1018244
OtherStreptavidin-coated
magnetic beads
New England
Biolabs
Cat#S1420S
OtherStreptavidin agaroseThermoFisher
Scientific
Cat#20357
OtherDMEM SILAC mediaThermo Fisher
Scientific
Cat#PI89985
OtherHam's F-12 SILAC mediaThermo Fisher
Scientific
Cat#88424
OtherHeavy Arginine + 10SigmaCat#608033–250 mg
 otherHeavy Lysine + 8SigmaCat#608041–100 mg
OtherProlineSigmaCat#P0380
OtherLight ArginineSigmaCat#A5006
OtherLight LysineSigmaCat#L5501
OtherHoechst 33342Thermo Fisher
Scientific
Cat#62249
OtherBODIPY 558/568 phalloidinInvitrogenCat#B3475IF: 1:400
OtherProLong Gold antifadeInvitrogenCat#P36934
OtherNormal Serum BlockBioLegendCat#927502
OtherMatrigelCorningCat#356231
Other0.2 mm nitrocellulose
membrane
GE HealthcareCat#15289804
Other0.4 mm pore size
Transwell filter
CorningCat#353095
Other24-well companion
plates
for Transwell filters
CorningCat#353504
OtherMillicell ERS-2
Volt/Ohm meter
Merck MilliporeCat#MERS00002
OtherSuperdex 200
16/600 column
GE HealthcareCat#28-9893-35
OtherSuperdex 75
16/600 column
GE HealthcareCat#28-9893-33
OtherUltracel-3K regenerated
cellulose centrifugal filter
Merck MilliporeCat#UFC900324
OtherUltracel-10 K
regenerated
cellulose centrifugal filter
Merck MilliporeCat#UFC901024
OtherUltracel-30 K regenerated
cellulose centrifugal filter
Merck MilliporeCat#UFC903024
OtherNuPAGE 4–12% Bis-Tris gelThermo Fisher
Scientific
Cat#NP0321BOX
Other1.5 ml low protein
binding centrifuge tubes
EppendorfCat#0030 108. 116
Other1cc/50 mg Sep-Pak
Vac tC18 cartridges
WatersCat#WAT054960,
Other1.5 ml Diagenode sonicator
tubes
DiagenodeCat#C30010010
Other5 ml low protein
binding centrifuge tubes
EppendorfCat#0030 108.302
Other2 ml low protein
binding centrifuge tubes
Thermo Fisher
Scientific
Cat#88379
OtherGraphite spin columnsThermo Fisher
Scientific
Cat#88302
OtherTitansphere Phos-TiO
Tips (200 ml/3 mg)
GL Sciences IncCat#5010–21311
Other18 mm x 18 mm,1.5
mm thick high-
performance coverslips
ZeissCat#474030-9000-000

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