Cancer Research Unit, Research Division, Saskatchewan Cancer Agency, 20 Campus Drive, Saskatoon, Saskatchewan, Canada S7N 4H4.
Cell Signal. 2010 Oct;22(10):1562-75. doi: 10.1016/j.cellsig.2010.05.025. Epub 2010 Jun 4.
Activated receptor tyrosine kinases recruit many signaling proteins to activate downstream cell proliferation and survival pathways, including phosphatidylinositol 3-kinase (PI3K) consisting of a p85 regulatory protein and a p110 catalytic protein. We have recently shown the p85alpha protein also has in vitro GTPase activating protein (GAP) activity towards Rab5 and Rab4, small GTPases that regulate vesicle trafficking events for activated receptors. Expression of a GAP-defective mutant, p85R274A, resulted in sustained levels of activated platelet-derived growth factor receptors (PDGFRs) and enhanced downstream signaling. In this report we have characterized Rab5- and Rab4-mediated PDGFR trafficking in cells expressing wild type p85 and GAP-defective mutant p85R274A. Wild type p85 overexpressing cells had slower PDGFR trafficking consistent with enhanced GAP activity deactivating Rab5 and Rab4 to block their vesicle trafficking functions. Mutant p85R274A expression increased the internalization rate of PDGFRs, a Rab5-dependent process, without preventing PDGFR ubiquitination. Immunofluorescence studies further demonstrated that p85R274A-expressing cells showed Rab5 accumulation at intracellular locations. Pull-down and FRAP (fluorescence recovery after photobleaching) experiments indicate this is likely membrane-associated Rab5-GTP, sustained due to decreased p85 GAP activity for the p85R274A mutant. These cells also had substantial amounts of activated PDGFRs in Rab4-positive recycling endosomes, a compartment that usually contains primarily deactivated/dephosphorylated receptors. Our results suggest that the PDGFR-associated GAP activity of p85 regulates both Rab5 and Rab4 functions in cells to influence the movement of activated PDGFR through endosomal compartments. Disruption of this regulation by p85R274A expression impacts PDGFR phosphorylation/dephosphorylation, degradation kinetics and downstream signaling by altering the time receptors spend in specific intracellular endosomal compartments. These results demonstrate that the p85alpha protein is an important regulator of Rab-mediated PDGFR trafficking, which significantly impacts receptor signaling and degradation.
激活的受体酪氨酸激酶募集许多信号蛋白来激活下游细胞增殖和存活途径，包括由 p85 调节蛋白和 p110 催化蛋白组成的磷脂酰肌醇 3-激酶 (PI3K)。我们最近表明，p85alpha 蛋白还具有针对 Rab5 和 Rab4 的体外 GTP 酶激活蛋白 (GAP) 活性，Rab5 和 Rab4 是调节激活受体的囊泡运输事件的小 GTP 酶。表达 GAP 缺陷突变体 p85R274A 会导致激活的血小板衍生生长因子受体 (PDGFR) 的持续水平，并增强下游信号。在本报告中，我们已经描述了表达野生型 p85 和 GAP 缺陷突变体 p85R274A 的细胞中 Rab5 和 Rab4 介导的 PDGFR 运输。野生型 p85 过表达细胞的 PDGFR 运输速度较慢，这与增强的 GAP 活性失活 Rab5 和 Rab4 以阻断其囊泡运输功能一致。突变体 p85R274A 的表达增加了 PDGFR 的内化率，这是一个 Rab5 依赖性过程，而不会阻止 PDGFR 泛素化。免疫荧光研究进一步表明，表达 p85R274A 的细胞显示 Rab5 在细胞内位置的积累。拉下和 FRAP（光漂白后荧光恢复）实验表明，这可能是膜相关的 Rab5-GTP，由于 p85R274A 突变体的 p85 GAP 活性降低而持续存在。这些细胞还在 Rab4 阳性再循环内体中含有大量激活的 PDGFR，这是一个通常主要包含失活/去磷酸化受体的隔室。我们的结果表明，p85 调节 PDGFR 相关 GAP 活性，以调节细胞中 Rab5 和 Rab4 的功能，从而影响激活的 PDGFR 通过内体隔室的运动。通过改变受体在特定细胞内内体隔室中花费的时间，p85R274A 表达对 PDGFR 磷酸化/去磷酸化、降解动力学和下游信号的破坏会影响 PDGFR 磷酸化/去磷酸化、降解动力学和下游信号。这些结果表明，p85alpha 蛋白是 Rab 介导的 PDGFR 运输的重要调节剂，这显著影响受体信号和降解。
