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

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

Schematic view of BiFC results.

<p>ER membranes (green) are positioned close to the plasma membrane (brown) by microtubules (black). PTP1B anchored to the cytosolic surface of the ER membrane (green pins) can interact with Src (red pins) associated to the cytosolic side of the plasma membrane. The substrate trap mutant PTP1BDA enhances the interaction leading to the visualization of large puncta (inset, and green beads on the ER in the upper/lower view). TIRF microscopy reveals BiFC puncta at the ventral membrane and SRIC shows that some of BiFC puncta occur in close contact with the substrate (SRIC dark spots).</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 analysis by TIRFM.

<p>CHO-K1 cells were co-transfected with YC-PTP1BWT/Src-YN and then fixed and processed for immunofluorescence detection of Src (A) and PTP1B (D) by wide field (wf). The BiFC signal of the same cells was observed by wide field (B, E) and under TIRF illumination (C, F). Yellow boxes in D and F were magnified in G and H. Note that BiFC puncta visualized under TIRF illumination (H) overlap with the ER network seen with anti-PTP1B (G). Also note that some puncta (white arrows in H and insets) display a comet-like appearance with a grading intensity of fluorescence brightest at the tip, suggesting a “dipping down” toward the substrate and into the region of exponentially increasing excitation of the evanescent field. Several BiFC puncta (yellow arrowheads) coincide with dark spots visualized by SRIC (I). Scale bar in A, 25 µm; scale bar in I, 2.5 µm.</p

Expression of the BiFC signal in wild type and mutant forms of Src and PTP1B.

<p>BiFC signal was assessed in cells that were positive for the detection, by immunofluorescence, of a member of the BiFC pair. Three categories were observed in the BiFC channel: background (not shown), ER + punctate, and only punctate. n: number of analyzed 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