[논문]Pleckstrin homology domain of phospholipase D2 is a negative regulator of focal adhesion kinase

Kim, Mi Kyoung; Hwang, Won Chan; Min, Do Sik

doi:10.5483/bmbrep.2021.54.2.154

[국내논문] Pleckstrin homology domain of phospholipase D2 is a negative regulator of focal adhesion kinase 원문보기

BMB reports, v.54 no.2, 2021년, pp.112 - 117

Kim, Mi Kyoung (Department of Molecular Biology, College of Natural Science, Pusan National University) , Hwang, Won Chan (Department of Molecular Biology, College of Natural Science, Pusan National University) , Min, Do Sik (College of Pharmacy, Yonsei University)

Abstract ▼ AI-Helper

Phospholipase D2 (PLD2) has been implicated in the tyrosine kinase-mediated signaling pathways, but the regulation events are yet to be identified. Herein, we demonstrate that pleckstrin homology (PH) domain of PLD2 (PLD2-PH) exerts an antitumorigenic effect via the suppression of PLD2 and focal adhesion kinase (FAK). The kinase domain of FAK interacts with PLD2-PH and induces tyrosine phosphorylation and activation of PLD2. Furthermore, PLD2 increased tyrosine phosphorylation of FAK. However, ectopic expression of the PLD2-PH competes for binding to FAK and reduces the interaction between PLD2 and FAK, thereby suppressing FAK-induced PLD activation and tyrosine phosphorylation of FAK. The PLD2-PH suppressed the migration and invasion of glioblastoma cells, as well as tumor formation in a xenograft mouse model. This study uncovers a novel role of PLD2-PH as a negative regulator of PLD2 and FAK.

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표/그림 (4)

그림 Fig. 1. FAK preferentially binds PLD2 and induces tyrosine phosphorylation. (A-D) HEK293 cells were co-transfected with the indicated constructs, and the cell lysates were immunoprecipitated and/or immunoblotted with the indicated antibodies. (E) HN33 cells were treated with LPA (100 μM) for the indicated time, and the interaction of PLD2 with FAK was analyzed by immunoprecipitation and immunoblotting. The data are representative of three independent experiments.
그림 Fig. 2. The PLD2-PH domain suppresses FAK-induced PLD2 activation by reducing the interaction between FAK and PLD2. (A) Schematic representation of full-length WT FAK, as well as its FERN, kinase, and FAT domains. HEK293 cells were transfected with the indicated constructs, and the lysates were subjected to immunoprecipitation and/or immunoblotting with the indicated antibodies. (B) Schematic representation of the structure of PLD2, including its PX, PH, and I-IV domains (conserved domains in the PLD family). A GST pull-down assay using equal amounts of GST or GSTPLD2 fragment fusion proteins immobilized on Glutathione Sepharose 4B beads was performed on the lysates of HEK293 cells. The amount of GST fusion proteins was visualized by Coomassie Brilliant Blue (CBB) staining. (C) The effect of PLD2-F1 or PLD2- F2 on the interaction between PLD2 and FAK. (D) The effect of the PX or PH domains of PLD2 on its binding to FAK. Domain mapping for the interaction site between PLD2 and FAK was performed using a GST-pull down assay. (E) The effect of the PLD2-PH domain on the interaction between PLD2 and FAK. (F-H) The effect of the transfection of the indicated constructs on PLD2 activity in HN33 cells. *P < 0.05 compared to the cells transfected with the empty vector. The results are shown as the mean ± SEM. Immunoblotting data are representative of three independent experiments.
그림 Fig. 3. The PLD2-PH domain suppresses the tyrosine phosphorylation of FAK that is normally induced by PLD2. (A) The lysates of U87MG cells expressing WT PLD2 and catalytically inactive PLD2 (PLD2-KRM) were analyzed by immunoblotting with the indicated antibodies. (B) The effect of the PLD2-PH domain on the activation of FAK, as analyzed through immunoblotting. The data are representative of three independent experiments.
그림 Fig. 4. The PLD2-PH domain suppresses the migration and invasion of cancer cells, as well as tumor growth. (A) Cell migration assays were conducted using Transwell plates for 24 h after transfection of the indicated constructs into U87MG cells. (B) Invasion assays were performed for 24 h using Matrigel-coated Transwell plates. The extent of cell migration and invasion was expressed as the average number of cells per field of view among the different experimental groups. Bar, 100 μm. *P < 0.05 compared to the empty vector. (C) U87MG cells expressing the empty vector or the PLD2-PH domain were injected into nude mice, and the tumor volume was then analyzed. Data are expressed as the mean ± SD of eight different mice per group. (D) Immunohistochemical staining of Ki67 in the tumor tissues. Bar, 100 μm. Data are representative of three independent experiments. (E) Schematic representation for role of PLD2-PH. FAK and PLD2 are positively regulated with each other via tyrosine phosphorylation. PLD2-PH inhibits their interaction and activities, followed by suppression of migration, invasion and tumor formation.

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문제 정의

of this feedback loop. This study aimed to better define the molecular link between PLD2 and FAK and the role of this interaction in cancer. FAK induces tyrosine phosphorylation of PLD2 via the interaction of its kinase domain with PLD2.
It is speculated that under situation of high expression levels of PLD2 and FAK such as cancers, PLD2-PH domain might play a dominant role in the disruption of their interaction and activities. In conclusion, this study advances the understanding of the crosstalk that occurs between PLD2 and FAK during their activation via a positive feedback loop.

가설 설정

These results indicate that the F1 fragment, but not the F2 fragment, of PLD2 likely competes with FAK for the binding of WT PLD2. Therefore, we hypothesized that the F1 region of PLD2 containing the PX and PH domains are important for the interaction with FAK. We next wanted to determine whether FAK binds to the PX or PH domain of PLD2.

데이터처리

indicated. Significant differences among the means were identified by ANOVA. A P-value < 0.

이론/모형

(D) The effect of the PX or PH domains of PLD2 on its binding to FAK. Domain mapping for the interaction site between PLD2 and FAK was performed using a GST-pull down assay. (E) The effect of the PLD2-PH domain on the interaction between PLD2 and FAK.

후속연구

One possibility is that PLD2 can recruit specific protein kinases to FAK that induce the tyrosine phosphorylation of FAK. Further studies will be needed to fully investigate the mechanism of FAK-mediated PLD activation. Recently, it was reported that the PLD1-PH domain interacts with and promotes the degradation of HIF-1 even under hypoxic conditions, thus suppressing tumor progression (14).

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