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

Abstract

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 promotes focal complex maturation, lamellar persistence and directional migration

“…To directly visualize catalytic PTP1B-a-actinin complexes in adhesions of intact WT cells we performed bimolecular fluorescence complementation (BiFC) analysis (Hu et al, 2002). Catalytic PTP1B-substrate complexes can be visualized only when their steady state concentration is significantly increased by using substrate trapping mutants of PTP1B, such as the PTP1B D181A (DA) (Haj et al, 2002;Boute et al, 2003;Monteleone et al, 2012). Indeed, co-expression of YN-PTP1BWT/a-actinin-YC (YN, amino acids 1-154; YC, amino acids 155-238 of EYFP) did not reveal a detectable BiFC signal (supplementary material Fig.…”

“…Each BiFC pair transfected individually showed the expected subcellular distribution and did not display detectable fluorescence in the BiFC channel (supplementary material Fig. S2) (Monteleone et al, 2012). We analyzed the presence of the BiFC signal in adhesions by immunofluorescence detection of vinculin and by surface reflection interference contrast (SRIC) imaging.…”

Protein tyrosine phosphatase PTP1B in cell adhesion and migration

“…3,4,12,18,[45][46][47] Using a combination of time lapse, total internal reflection fluorescence microscopy, and BiFC we were able to visualize ER-bound PTP1B/ Src complexes as small fluorescent puncta distributed uniformly at the plasma membrane in contact with the substrate. 8 The substrate trap mutation (D181A) in PTP1B and the tyrosine 529 at the C-tail of Src were both required for BiFC to occur, as well as the plasma membrane targeting motif of Src. 8 This study also shows dynamic projections of ER tubules toward the plasma membrane, suggesting the assembly of catalytic PTP1B/Src BiFC complexes at random point locations in the plasma membrane/substrate interface.…”

“…8 The substrate trap mutation (D181A) in PTP1B and the tyrosine 529 at the C-tail of Src were both required for BiFC to occur, as well as the plasma membrane targeting motif of Src. 8 This study also shows dynamic projections of ER tubules toward the plasma membrane, suggesting the assembly of catalytic PTP1B/Src BiFC complexes at random point locations in the plasma membrane/substrate interface. Nevertheless, the possibility that Src could also be targeted by PTP1B at typical adhesion structures cannot be excluded (Fig.…”

“…Extension of the ER toward the cell periphery allows PTP1B to dephosphorylate substrates located at the plasma membrane, for example Src, EphA3, and proteins associated to cadherin and integrin complexes. [2][3][4][5][6][7][8] Alternatively, PTP1B can dephosphorylate the cytosolic domains of endocytosed receptors, which are brought into contact with the ER, as, for example, activated platelet-derived growth factor and epidermal growth factor receptors. 9 Through the proline-rich motif, PTP1B has also been shown to interact directly with components of focal adhesions, such as the SH3 domain of adaptor protein p130Cas.…”