Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, Dayton, Ohio 45435, USA.
Mol Cell Biol. 2011 Nov;31(22):4524-37. doi: 10.1128/MCB.05684-11. Epub 2011 Sep 19.
Phagocytosis is a primary innate response of both macrophages and neutrophils involving the formation of filamentous actin (F-actin)-rich protrusions that are extended around opsonized pathogens to form a phagocytic cup, resulting in their subsequent internalization. The molecular mechanism for this is still not completely understood. We now show for the first time that phospholipase D2 (PLD2) binds to growth factor receptor-bound protein 2 (Grb2) and to the Wiskott-Aldrich syndrome protein (WASp) to form a heterotrimer complex, PLD2-Grb2-WASp, and present the mechanism of interaction. Grb2 binds to the Y169/Y179 residues of PLD2 using its only SH2 domain, and it interacts with the poly-proline region of WASp using its two SH3 domains. The PLD2-Grb2-WASp heterotrimer can be visualized in early phagocytic cups of macrophages ingesting opsonized red blood cells, where it associates with polymerized actin. Cup colocalization and phagocytosis are disrupted with mutants that alter binding at either of the two proteins or by silencing Grb2 with RNA interference (RNAi). WASp association to PLD2-K758R, a lipase-inactive mutant, still occurs, albeit at lower levels, indicating that PLD2 plays a second role in phagocytosis, which is the production of phosphatidic acid (PA) and activation of phosphatidylinositol 5-kinase (PI5K) with subsequent synthesis of phosphatidylinositol 4,5-bisphosphate (PIP(2)). The latter can be blocked with RNAi, which negates phagocytosis. Lastly, a constitutively "open" active form of WASp (WASp-L270P) brings phagocytosis to its maximum level, which can be mimicked with WASp-WT plus PLD2 or plus PA. Since neither a protein-protein disruption nor lack of PLD activity completely negates cup formation or phagocytosis, we posit a two-step mechanism: PLD2 anchors WASp at the phagocytic cup through Grb2 following protein-protein interactions and also activates it, making key lipids available locally. The heterotrimer PLD2-Grb2-WASp then enables actin nucleation at the phagocytic cup and phagocytosis, which are at the center of the innate immune system function.
吞噬作用是巨噬细胞和中性粒细胞的主要先天反应，涉及形成富含丝状肌动蛋白 (F-actin) 的突起，这些突起围绕着调理病原体延伸，形成吞噬杯，从而导致随后的内化。其分子机制仍不完全清楚。我们现在首次表明，磷脂酶 D2 (PLD2) 与生长因子受体结合蛋白 2 (Grb2) 和 Wiskott-Aldrich 综合征蛋白 (WASp) 结合形成异三聚体复合物 PLD2-Grb2-WASp，并提出了相互作用的机制。Grb2 通过其唯一的 SH2 结构域与 PLD2 的 Y169/Y179 残基结合，并通过其两个 SH3 结构域与多脯氨酸区的 WASp 相互作用。PLD2-Grb2-WASp 异三聚体可在吞噬调理红细胞的巨噬细胞的早期吞噬杯中可视化，在那里它与聚合肌动蛋白相关联。用改变两种蛋白质中任一种的结合的突变体或用 RNA 干扰 (RNAi) 沉默 Grb2 可破坏杯状共定位和吞噬作用。PLD2-K758R（脂酶失活突变体）与 WASp 的结合仍然发生，尽管水平较低，这表明 PLD2 在吞噬作用中发挥第二个作用，即产生磷脂酸 (PA) 和激活磷脂酰肌醇 5-激酶 (PI5K)，随后合成磷脂酰肌醇 4,5-二磷酸 (PIP(2))。可以用 RNAi 阻断后者，从而阻止吞噬作用。最后，一种组成型“开放”的活性形式的 WASp (WASp-L270P) 将吞噬作用带到其最大水平，这可以通过 WASp-WT 加上 PLD2 或加上 PA 来模拟。由于既没有蛋白质-蛋白质的破坏也没有 PLD 活性完全否定杯状形成或吞噬作用，因此我们提出了一个两步机制：PLD2 通过 Grb2 锚定吞噬杯中的 WASp，随后通过蛋白质-蛋白质相互作用使其激活，从而使关键脂质在局部可用。然后，PLD2-Grb2-WASp 异三聚体使肌动蛋白在吞噬杯中发生核化并吞噬作用，这是先天免疫系统功能的核心。
