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是一种锚定在内质网（endoplasmic reticulum, ER）上的酶，其接触底物的能力部分依赖于内质网的分布与动态特性。其底物之一——蛋白酪氨酸激酶Src（protein tyrosine kinase Src）已被发现存在于细胞质、内体与细胞质膜中。本研究通过结合时空高分辨显微镜的双分子荧光互补（bimolecular fluorescence complementation, BiFC）技术，分析了完整细胞内PTP1B与Src发生物理互作的位点；同时还解析了二者互作的结构基础。
我们发现BiFC信号呈点状分散于整个内质网网络中，当使用底物捕获突变体PTP1B-D181A时，该信号特征会得到增强。延时成像与共定位分析显示，BiFC荧光点并非对应囊泡载体，而是定位于动态内质网管状结构的尖端。经细胞揭膜术处理后，BiFC荧光点可保留于腹侧膜制备物中；同时在全内反射荧光显微镜（total internal reflection fluorescent microscopy, TIRFM）的隐失场中，也能在完整细胞的腹侧质膜处检测到此类荧光点。此外，BiFC荧光点常与表面反射干涉对照（surface reflection interference contrast, SRIC）观察到的暗斑共定位。
去除Src的肉豆蔻酰化与多碱性基序会消除BiFC信号。此外，PTP1B的活性位点以及Src上的负调控酪氨酸529是决定BiFC信号产生的主要因素，尽管PTP1B上的SH3结合基序也发挥了一定作用。我们的研究结果表明，锚定在内质网上的PTP1B会在质膜/底物界面的随机点状位点上，与Src羧基端的负调控位点发生动态互作，这一过程可能介导Src的激活并将其招募至黏着斑复合物中。我们推测，这种功能性的内质网-质膜串扰可能适用于众多蛋白质互作对，由此开辟了一个极具前景的研究领域。