Regulation of microtubule nucleation in glioblastoma cells by ARF GTPase-activating proteins GIT1 and GIT2 and protein kinase C.

IF 5.3 2区医学 Q1 ONCOLOGY

Cancer Cell International Pub Date : 2025-04-02 DOI:10.1186/s12935-025-03740-y

Vadym Sulimenko, Eduarda Dráberová, Vladimíra Sládková, Tetyana Sulimenko, Věra Vosecká, Omar Skalli, Pavel Dráber

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引用次数: 0

Abstract

Background: G protein-coupled receptor kinase-interacting proteins (GITs) function as GTPase-activating proteins (GAPs) for small GTPases of the ADP-ribosylation factor (Arf) family. While GIT proteins (GIT1 and GIT2) regulate both cell migration and microtubule organization, their corresponding regulatory mechanisms in glioblastoma cells remain largely unknown. To further investigate their role in microtubule modulation, we examined the function of GITs in microtubule nucleation and the involvement of protein kinase C (PKC) in this process.

Methods: Glioblastoma cell lines with depleted GIT protein levels were generated using shRNA lentiviral vectors. The cellular localization of GITs was visualized by immunofluorescence microscopy, microtubule nucleation was analyzed using time-lapse imaging, and cell migration was assessed through a wound healing assay. Phosphomimetic and non-phosphorylatable variants of GIT2 were prepared by site-directed mutagenesis. Immunoprecipitation, pull-down experiments, and kinase assays in the presence of PKC inhibitors were used to study protein interactions.

Results: Both GIT1 and GIT2 associate with proteins of the γ-tubulin ring complexes (γTuRCs), the primary microtubule nucleators, and localize to centrosomes. Depletion of GIT2 enhances centrosomal microtubule nucleation and has a more pronounced, yet opposite, effect on this process compared to GIT1. In contrast, the depletion of both GIT1 and GIT2 similarly affects cell migration. The N-terminal ArfGAP domain of GIT2 associates with centrosomes, regulates microtubule nucleation, and is phosphorylated by PKC, which modulates this process. We identified serine 46 (S46) on the ArfGAP domain as a PKC phosphorylation site and demonstrated that phosphorylation of GIT2 at S46 promotes microtubule nucleation.

Conclusions: We propose that GIT2 phosphorylation provides a novel regulatory mechanism for microtubule nucleation in glioblastoma cells, contributing to their invasive properties.

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ARF gtpase激活蛋白GIT1、GIT2和蛋白激酶C对胶质母细胞瘤细胞微管成核的调控

背景：G蛋白偶联受体激酶相互作用蛋白（GITs）是ADP-核糖基化因子（Arf）家族小GTP酶的GTP酶激活蛋白（GAPs）。虽然 GIT 蛋白（GIT1 和 GIT2）可调控细胞迁移和微管组织，但它们在胶质母细胞瘤细胞中的相应调控机制在很大程度上仍然未知。为了进一步研究它们在微管调节中的作用，我们研究了GITs在微管成核中的功能以及蛋白激酶C（PKC）在这一过程中的参与：方法：利用 shRNA 慢病毒载体生成了 GIT 蛋白水平降低的胶质母细胞瘤细胞系。用免疫荧光显微镜观察 GITs 的细胞定位，用延时成像分析微管成核，用伤口愈合试验评估细胞迁移。通过定点突变制备了GIT2的拟磷酸化和非磷酸化变体。免疫沉淀、下拉实验以及在PKC抑制剂存在下的激酶测定被用来研究蛋白质的相互作用：结果：GIT1和GIT2都与γ-微管蛋白环复合物（γTuRCs）的蛋白质（主要的微管成核因子）结合，并定位于中心体。与 GIT1 相比，GIT2 的缺失会增强中心体微管的成核，并对这一过程产生更明显但相反的影响。相比之下，GIT1 和 GIT2 的缺失同样会影响细胞迁移。GIT2 的 N 端 ArfGAP 结构域与中心体结合，调节微管成核，并被 PKC 磷酸化，从而调节这一过程。我们发现 ArfGAP 结构域上的丝氨酸 46 (S46) 是 PKC 的磷酸化位点，并证明 GIT2 在 S46 处的磷酸化能促进微管成核：我们认为 GIT2 磷酸化为胶质母细胞瘤细胞的微管成核提供了一种新的调控机制，有助于其侵袭特性的形成。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Cancer Cell International ONCOLOGY-

CiteScore

10.90

自引率

1.70%

发文量

360

审稿时长

1 months

期刊介绍： Cancer Cell International publishes articles on all aspects of cancer cell biology, originating largely from, but not limited to, work using cell culture techniques. The journal focuses on novel cancer studies reporting data from biological experiments performed on cells grown in vitro, in two- or three-dimensional systems, and/or in vivo (animal experiments). These types of experiments have provided crucial data in many fields, from cell proliferation and transformation, to epithelial-mesenchymal interaction, to apoptosis, and host immune response to tumors. Cancer Cell International also considers articles that focus on novel technologies or novel pathways in molecular analysis and on epidemiological studies that may affect patient care, as well as articles reporting translational cancer research studies where in vitro discoveries are bridged to the clinic. As such, the journal is interested in laboratory and animal studies reporting on novel biomarkers of tumor progression and response to therapy and on their applicability to human cancers.