ER-Bound Protein Tyrosine Phosphatase PTP1B Interacts with Src at the Plasma Membrane/Substrate Interface

PTP1B is an endoplasmic reticulum (ER) anchored enzyme whose access to substrates is partly dependent on the ER distribution and dynamics. One of these substrates, the protein tyrosine kinase Src, has been found in the cytosol, endosomes, and plasma membrane. Here we analyzed where PTP1B and Src physically interact in intact cells, by bimolecular fluorescence complementation (BiFC) in combination with temporal and high resolution microscopy. We also determined the structural basis of this interaction. We found that BiFC signal is displayed as puncta scattered throughout the ER network, a feature that was enhanced when the substrate trapping mutant PTP1B-D181A was used. Time-lapse and co-localization analyses revealed that BiFC puncta did not correspond to vesicular carriers; instead they localized at the tip of dynamic ER tubules. BiFC puncta were retained in ventral membrane preparations after cell unroofing and were also detected within the evanescent field of total internal reflection fluorescent microscopy (TIRFM) associated to the ventral membranes of whole cells. Furthermore, BiFC puncta often colocalized with dark spots seen by surface reflection interference contrast (SRIC). Removal of Src myristoylation and polybasic motifs abolished BiFC. In addition, PTP1B active site and negative regulatory tyrosine 529 on Src were primary determinants of BiFC occurrence, although the SH3 binding motif on PTP1B also played a role. Our results suggest that ER-bound PTP1B dynamically interacts with the negative regulatory site at the C-terminus of Src at random puncta in the plasma membrane/substrate interface, likely leading to Src activation and recruitment to adhesion complexes. We postulate that this functional ER/plasma membrane crosstalk could apply to a wide array of protein partners, opening an exciting field of research

PTP1B triggers integrin-mediated repression of myosin activity and modulates cell contractility

ABSTRACT Cell contractility and migration by integrins depends on precise regulation of protein tyrosine kinase and Rho-family GTPase activities in specific spatiotemporal patterns. Here we show that protein tyrosine phosphatase PTP1B cooperates with β3 integrin to activate the Src/FAK signalling pathway which represses RhoA-myosin-dependent contractility. Using PTP1B null (KO) cells and PTP1B reconstituted (WT) cells, we determined that some early steps following cell adhesion to fibronectin and vitronectin occurred robustly in WT cells, including aggregation of β3 integrins and adaptor proteins, and activation of Src/FAK-dependent signalling at small puncta in a lamellipodium. However, these events were significantly impaired in KO cells. We established that cytoskeletal strain and cell contractility was highly enhanced at the periphery of KO cells compared to WT cells. Inhibition of the Src/FAK signalling pathway or expression of constitutive active RhoA in WT cells induced a KO cell phenotype. Conversely, expression of constitutive active Src or myosin inhibition in KO cells restored the WT phenotype. We propose that this novel function of PTP1B stimulates permissive conditions for adhesion and lamellipodium assembly at the protruding edge during cell spreading and migration

P120-Catenin Regulates Early Trafficking Stages of the N-Cadherin Precursor Complex.

It is well established that binding of p120 catenin to the cytoplasmic domain of surface cadherin prevents cadherin endocytosis and degradation, contributing to cell-cell adhesion. In the present work we show that p120 catenin bound to the N-cadherin precursor, contributes to its anterograde movement from the endoplasmic reticulum (ER) to the Golgi complex. In HeLa cells, depletion of p120 expression, or blocking its binding to N-cadherin, increased the accumulation of the precursor in the ER, while it decreased the localization of mature N-cadherin at intercellular junctions. Reconstitution experiments in p120-deficient SW48 cells with all three major isoforms of p120 (1, 3 and 4) had similar capacity to promote the processing of the N-cadherin precursor to the mature form, and its localization at cell-cell junctions. P120 catenin and protein tyrosine phosphatase PTP1B facilitated the recruitment of the N-ethylmaleimide sensitive factor (NSF), an ATPase involved in vesicular trafficking, to the N-cadherin precursor complex. Dominant negative NSF E329Q impaired N-cadherin trafficking, maturation and localization at cell-cell junctions. Our results uncover a new role for p120 catenin bound to the N-cadherin precursor ensuring its trafficking through the biosynthetic pathway towards the cell surface

Combinatorial analysis of p120 and NSF in N-cadherin processing.

<p>(A) SW48 cells expressing pro-WT in combination with empty vector (pcDNA) or p120-1, and NSF-WT (WT) or NSF-E329Q (E329Q). Shown is a representative blot probed with anti-N-cadherin to detect the precursor and mature form of N-cadherin, anti-p120, anti-NSF, and anti-tubulin. (B, C) Quantification of N-cadherin precursor/ mature ratio (B) or p120/ NSF ratio (C) from blot scans expressed in arbitrary units (A. U.). Graph bars represent means ± S.E.M. from three independent experiments. Statistical significance (p < 0.05) was determined using one-way ANOVA, followed by the Dunnett´s multiple comparison <i>post hoc</i> test (B), or a two-tailed Student´s <i>t</i>-test (C). Different letters in (B) indicate statistically different means.</p

Function of NSF in N-cadherin trafficking.

<p>(A) Western blotting analysis of HeLa cells co-transfected with pro-WT, and empty vector or vector encoding NSF-WT or NSF-E329Q. Precursor and mature forms of pro-WT were detected with anti-N-cadherin. The lower size of the endogenous N-cadherin is not depicted. Visualization of ectopic NSF-WT and NSF-E329Q signals under non-saturating conditions did not allow detection of endogenous NSF (first lane). At least three independent experiments were used for quantification of precursor and mature forms of pro-WT (right graph). Bars represent means ± S.E.M. of precursor/mature ratios normalized to tubulin. Statistical significance (p < 0.05) was determined using one-way ANOVA, followed by the Dunnett´s multiple comparison <i>post hoc</i> test. Different letters indicate statistically different means. (B-G) Distribution of N-cadherin-GFP and ectopic NSF constructs. HeLa cells were co-transfected with N-cadherin-GFP and either NSF-WT (B-D) or NSF-E329Q (E-G). (H-M) Colocalization of N-cadherin precursor (HA, red label) and the ER marker calnexin (green label) in cells co-transfected with pro-WT2 and NSF-WT (H-J) or NSF-E329Q (K-M). The expression of exogenous NSF constructs was verified in triple label samples (not shown). (N) Manders colocalization coefficients of the HA/calnexin signal overlapping in NSF-WT (n = 15 cells) and NSF-E329Q (n = 23 cells) conditions. Bars represent means ± S.E.M. Statistical significance was determined by a two-tailed Mann-Whitney test. Cells were analyzed by confocal microscopy and representative images projected along the z-axis are shown. Enlarged views (4x) from selected regions (yellow arrowheads) are shown as insets. Scale bar in (E), 35 μm. (O) HeLa cells were co-transfected with proWT and non-targeting siRNA duplexes (Control) or NSF siRNA duplexes (NSF siRNA). After 48 h, precursor and mature forms of the proWT construct, NSF and tubulin were analyzed by Western blotting. Quantification of NSF levels (NSF siRNA values relativized to control siRNA) and precursor/mature ratios in control and NSF siRNA conditions is shown at the right. Bars represent means ± S.E.M. from three independent experiments. Statistical significance (asterisks, p < 0.05) was determined by a two-tailed Student´s <i>t</i>-test.</p

Distribution of N-cadherin precursor in p120KD HeLa cells.

<p>HeLa cells were co-transfected with pro-WT2 and either control (A-C, G-I) or p120KD (D-F, J-L) shRNAi. Cells were analyzed by confocal microscopy and representative images projected along the z-axis are shown. (A-F) Colocalization of N-cadherin precursor (HA, red label) with the ER marker calnexin (green label), detected by immunofluorescence. (G-L) Colocalization of the N-cadherin precursor (HA, red label) with transfected GFP-ERGIC-53 (green signal). Enlarged views from selected regions (white dotted boxes) are shown at the right. The expression of the control or p120KD shRNAi constructions in the analyzed cells is revealed by the modified nuclear-targeted GFP encoded by the vector (green channel). Scale bar in (L), 35 μm. (M) Number of HA puncta per cell in control and p120KD conditions (n = 27 cells per condition). Horizontal lanes indicate the position of means (control = 87,8; p120KD = 162.1). (N) Manders colocalization coefficients of the HA puncta overlapping calnexin in peripheral regions (control, n = 9 cells, total 422 puncta; p120KD, n = 7 cells, total 617 puncta). (O, P) Manders colocalization coefficients of the HA label overlapping GFP-ERGIC-53; independent analysis of the punctate (O) and clustered perinuclear distribution (P) of the HA label was performed (n = 24 cells per condition). Bars represent means ± S.E.M. Statistical significance was determined by a two-tailed Mann-Whitney test.</p

Expression and distribution of BiFC constructs.

<p>(A) Diagram of fusion proteins. YN (residues 1–154) and YC (residues 155–238) fragments of enhanced YFP were fused to the N-terminus of wild type (WT) and substrate trap mutant D181A (DA) PTP1B. The same fragments were fused at the C-terminus of mouse Src and Fyn. The amino acids of the linker region are indicated in italics. (B) Constructs were transiently expressed in CHO-K1 cells and probed in Western blots with anti-PTP1B (left panel), anti-Src (middle panel), and anti-Fyn (right panel) antibodies. YC and YN fragments of YFP add ∼10 and 18 kDa, respectively, to the partner fused proteins (PTP1B, Src and Fyn). Arrowheads indicate the migration of the endogenous proteins. Anti-PTP1B does not recognize the endogenous CHO-K1 protein; thus, a cell extract from PTP1B knockout cells reconstituted with human PTP1B was probed with this antibody and shown in the lane marked as PTP1BWT. Subcellular distribution of constructs used for BiFC was assessed by fluorescence microscopy. CHO-K1 cells expressing YC-PTP1BWT (C, C′, D), YC-PTP1BDA (E, E′, F), and SYF cells expressing Fyn-YN (G, H), and Src-YN (I, J) were immunolabeled with anti-PTP1B (C, Ć, E, É), anti-Fyn (G) and anti-Src (I) followed by secondary antibodies conjugated with Alexa Fluor 568 nm. Images on the red channel show that YC-PTP1BWT (C, Ć) and YC-PTP1BDA (E, É) localize in the ER, as expected (Ćand É are magnifications of regions within boxes in C and E, respectively). In addition, YCPTP1BDA accumulates in small puncta (arrows in É). Fyn-YN (G) and Src-YN (I) are enriched at the cell margin and in a perinuclear compartment. All constructs display background fluorescence at the green channel in which BiFC is analyzed (D, F, H, J). (K-N) Starved SYF cells expressing Src-YN were plated for 30 min in the absence (K, L) and in the presence (M, N) of serum. Note that Src-YN localizes in a perinuclear compartment in the absence of serum (L) and redistributes to peripheral, radial focal adhesions in the presence of serum (N), as expected. (K, M) Surface reflectance interference contrast images showing the membrane in contact with the substrate. Dashed lines indicate the perimeter of cells. Scale bar, 40 µm.</p

N-cadherin distribution in SW48 colon carcinoma cells reconstituted with p120.

<p>(A) Cells were co-transfected with pro-WT and p120 isoforms 1, 3 or 4. Soluble protein extracts from parental (P) and reconstituted cells (1, 3 and 4) were analyzed in Western blotting probed with anti-N-cadherin to recognize the precursor and mature forms of pro-WT, the p120 isoforms (clone 15D2), and tubulin. The graph shows quantification of precursor/mature ratios normalized to tubulin. (B-H) Confocal analysis of representative cells reconstituted with isoform 1 (B-D), 3 (E-G) and 4 (H). Cells were labeled for p120 (B, E, H) and F-actin (C, F). Arrows point intercellular junctions. "<i>nt</i>" in (B) indicates non-transfected cells. (I-L) Distribution of precursor and mature forms of N-cadherin in parental cells expressing pro-WT and empty vector (I), or vectors encoding p120-1 (J), p120-3 (K) or p120-4 (L). The distribution of ectopic N-cadherin precursor (red label) and total N-cadherin (green label) was detected by anti-HA and GFP fluorescence, respectively. (M-P) Colocalization of the N-cadherin precursor with ER and Golgi markers. Cells expressing pro-WT2 in parental (M) and reconstituted with p120-1 (O) conditions were double-labeled for HA (red signal) and calnexin (green signal). The expression of p120-1 construct was verified in triple label samples (not shown). Note the redistribution of the HA signal to the perinuclear compartment in p120-1 reconstituted cells. The Golgi apparatus was revealed by GalNacT-DsRed transfection in parental (N) and p120-1 reconstituted (P) cells. Note the tight overlapping of the HA label (shown in red) with GalNacT-DsRed (shown in green) in the Golgi. (Q) Manders colocalization coefficients calculated from HA and calnexin (n = 13 cells), and HA and GalNacT (n = 13 cells) signals. Bars represent means ± S.E.M. Statistical significance was determined by a two-tailed Mann-Whitney test. All cell images represent equatorial confocal planes and white arrows point intercellular junctions. Scale bar in (P), 14 μm.</p

Distribution of the BiFC signal.

<p>CHO-K1 cells were co-transfected with BiFC pairs and analyzed by fluorescence microscopy. (A-C) Representative BiFC distribution of YC-PTP1BWT/Src-YN is shown. Most cells show the BiFC signal as bright fluorescence puncta associated with a network pattern of lower fluorescence intensity (A, magnifications in B and C). (D–G) Representative BiFC distribution of YC-PTP1BDA/Src-YN is shown. Note that BiFC is exclusively seen as bright puncta (magnifications in F, G), sometimes more dense in the perinuclear region (arrow in D). Scale bar in A: 25 µm. Magnifications in B, C, F, and G are at 200% of the original images (E) Image taken under surface reflection interference contrast. (H–J) Representative CHO-K1 cell co-transfected with the YC-PTP1BWT/Src-YN pair and then fixed and processed for immunofluorescence detection of calnexin, using Alexa Fluor 568-conjugated secondary antibodies. (H) Calnexin labeling, (I) BiFC, (J) merge of both channels. (K) Cytofluorogram showing the high correlation between red/green pixels corresponding to the calnexin and BiFC images, respectively. Arbitrary units (a.u.) represent grey level values from 12-bit images. Pearson’s correlation coefficient close to 1 reveals positive correlation. Manders’ coefficients M1 and M2 estimate the amount of co-localizing signal from the calnexin image to the BiFC image and viceversa, respectively. Both M1 and M2 coefficients are close to 100% indicating an almost perfect co-localization. Dashed lines indicate the perimeter of cells.</p

BiFC in ventral membranes.

<p>CHO-K1 cells were co-transfected with YC-PTP1B and either Src-YN (A–D) or SrcT-YN (E–H). Membrane preparations were obtained by sonication of the cells previously exposed to hypotonic conditions. After fixation with paraformaldehyde, Src-YN (A) and YC-PTP1B (E) were detected by specific primary antibodies and Alexa Fluor 568-conjugated secondary antibodies. The BiFC signal was displayed in puncta that tightly colocalized with the Src-YN staining (C). In contrast, BiFC signal was undetectable when using SrcT-YN (F, G). SRIC analysis showed dark/light patterns of the ventral membrane in contact with the substrate (D, H). Dashed lines indicate the perimeter of cells. Scale bar: 25 µm.</p