Localization of phospholipase D1 to caveolin-enriched membrane via palmitoylation: Implications for epidermal growth factor signaling
Localization of phospholipase D1 to caveolin-enriched membrane via palmitoylation: Implications for epidermal growth factor signaling
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DOI：
10.1091/mbc.e02-02-0100
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发表时间：
2002-11-01
影响因子：
3.3
通讯作者：
Ryu, SH
中科院分区：
文献类型：
--
作者：
Han, JM;Kim, Y;Ryu, SH
磷脂酶 D (PLD) 被认为可以介导表皮生长因子 (EGF) 信号传导。然而，EGF诱导PLD激活的分子机制尚未阐明。我们研究了 PLD1 磷酸化和区室化在 EGF 信号传导中的重要性。 EGF 处理瞬时表达 PLD1 的 COS-7 细胞可刺激 PLD1 活性并诱导 PLD1 磷酸化。 EGF诱导的苏氨酸147磷酸化被完全阻断，并且PLD1磷酸化位点的点突变(S2A/T147A/S561A)减弱了PLD1的活性。在 PLD1 转染的 COS-7 细胞中，通过腺病毒介导的基因转移表达显性失活 PKCα 突变体，极大地抑制了 EGF 诱导的 PLD1 磷酸化和激活。 EGF 诱导的 PLD1 磷酸化主要发生在富含洞穴油的膜 (CEM) 部分，以及 PLD1 磷酸化的动力学。 CEM 中的 PLD1 磷酸化与全膜中的 PLD1 磷酸化密切相关。有趣的是，在棕榈酰化缺陷的 C240S/C241S 突变体中，EGF 诱导的 PLD1 磷酸化和激活以及 PLD1 与 CEM 中的 Caveohn-1 和 EGF 受体的免疫共沉淀显着减弱，该突变体未定位于 CEM。免疫细胞化学分析显示，野生型PLD1与caveohn-1和EGF受体共定位，磷酸化PLD1仅定位于质膜，尽管在囊泡结构中也检测到了一些PLD1。转染野生型 PLD1 但不转染 C240S/C241S 突变体会增加 EGF 诱导的 raf-1 易位至 CEM 和 ERK 磷酸化。这项研究首次表明，EGF 诱导的 PLD1 磷酸化和激活发生在 CEM 中，并且 PLD1 通过棕榈酰化正确定位到 CEM 对于 EGF 信号转导至关重要。
Phospholipase D (PLD) has been suggested to mediate epidermal growth factor (EGF) signaling. However, the molecular mechanism of EGF-induced PLD activation has not yet been elucidated. We investigated the importance of the phosphorylation and compartmentalization of PLD1 in EGF signaling. EGF treatment of COS-7 cells transiently expressing PLD1 stimulated PLD1 activity and induced PLD1 phosphorylation. The EGF-induced phosphorylation of threonine147 was completely blocked and the activity of PLD1 attenuated by point mutations (S2A/T147A/S561A) of PLD1 phosphorylation sites. The expression of a dominant negative PKCalpha mutant by adenovirus-mediated gene transfer greatly inhibited the phosphorylation and activation of PLD1 induced by EGF in PLD1-transfected COS-7 cells. EGF-induced PLD1 phosphorylation occurred primarily in the caveoil-enriched membrane (CEM) fraction, and the kinetics of PLD1 phosphorylation. in the CEM were strongly correlated with PLD1 phosphorylation in the total membrane. Interestingly, EGF-induced PLD1 phosphorylation and activation and the coimmunoprecipitation of PLD1 with caveohn-1 and the EGF receptor in the CEM were significantly attenuated in the palmitoylation-deficient C240S/C241S mutant, which did not localize to the CEM. Immunocytochemical analysis revealed that wild-type PLD1 colocalized with caveohn-1 and the EGF receptor and that phosphorylated PLD1 was localized exclusively in the plasma membrane, although some PLD1 was also detected in vesicular structures. Transfection of wild-type PLD1 but not of C240S/C241S mutant increased EGF-induced raf-1 translocation to the CEM and ERK phosphorylation. This study shows, for the first time, that EGF-induced PLD1 phosphorylation and activation occur in the CEM and that the correct localization of PLD1 to the CEM via palmitoylation is critical for EGF signaling.