Inhibition of phosphatidylinositol-4-phosphate 5-kinase Iα impairs localized actin remodeling and suppresses phagocytosis
Inhibition of phosphatidylinositol-4-phosphate 5-kinase Iα impairs localized actin remodeling and suppresses phagocytosis
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DOI：
10.1074/jbc.m209046200
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发表时间：
2002-11-15
影响因子：
4.8
通讯作者：
Grinstein, S
中科院分区：
文献类型：
--
作者：
Coppolino, MG;Dierckman, R;Grinstein, S
肌动蛋白聚合驱动吞噬作用所需的伪足的延伸。磷脂酰肌醇4，5-二磷酸（PIP 2）被认为在这一过程中发挥核心作用，因为它与几种肌动蛋白调节蛋白相互作用，并在吞噬作用部位发生急性和局部变化。因此，我们研究了是否磷脂酰肌醇-4-磷酸5-激酶（PIPK），负责从磷脂酰肌醇4-磷酸产生PIP 2的酶，参与控制吞噬作用。发现PIPKIalpha在形成吞噬体时瞬时积累。为了测试PIPKIalpha在颗粒吞噬中的功能参与，我们产生了缺乏激酶活性的双突变体（D309 N/R427 Q）。当在培养细胞中异位表达时，该突变体靶向质膜并在颗粒吞噬期间在吞噬体杯中积累。PIP 5 KI α D309 N/R427 Q的表达损害RAW264.7巨噬细胞和通过转染中国仓鼠卵巢细胞中的Fc受体产生的工程化吞噬细胞中的吞噬作用。吞噬作用的抑制不能归因于颗粒结合或受体聚集的缺陷，这是使用绿色荧光蛋白标记的Fc γ受体监测的。相反，非活性激酶的表达减少了PIP 2和F-肌动蛋白在吞噬体杯中的积累。这些数据表明，PIPKIalpha活性参与肌动蛋白重塑，这是有效吞噬的先决条件。PIPKIalpha似乎有助于PIP 2水平的瞬时变化，这与肌动蛋白调节蛋白的募集和调节相关，并且可能是所需的。
Actin polymerization drives the extension of pseudopods required for phagocytosis. Phosphatidylinositol 4,5-bisphosphate (PIP2) is thought to play a central role in this process, because it interacts with several actin-regulatory proteins and undergoes acute and localized changes at sites of phagocytosis. We therefore studied whether phosphatidylinositol-4-phosphate 5-kinase (PIPK), the enzyme responsible for the generation of PIP2 from phosphatidylinositol 4-phosphate, is involved in the control of phagocytosis. PIPKIalpha was found to accumulate transiently on forming phagosomes. To test the functional involvement of PIPKIalpha in particle engulfment, we generated a double mutant (D309N/R427Q) that lacks kinase activity. When ectopically expressed in cultured cells, this mutant is targeted to the plasma membrane and accumulates at the phagosomal cup during particle engulfment. Expression of PIP5KIalpha D309N/R427Q impaired phagocytosis in RAW264.7 macrophages and in engineered phagocytes generated by transfection of Fc receptors in Chinese hamster ovary cells. Inhibition of phagocytosis could not be attributed to defects in particle binding or receptor clustering, which was monitored using green fluorescent protein-tagged Fcy receptors. Instead, expression of the inactive kinase diminished the accumulation of PIP2 and of F-actin in the phagosomal cup. These data suggest that PIPKIalpha activity is involved in the actin remodeling that is a prerequisite for efficient phagocytosis. PIPKIalpha appears to contribute to the transient changes in PIP2 levels that are associated with, and likely required for, the recruitment and regulation of actin-modulating proteins.