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

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F68378050%3A_____%2F25%3A00619012" target="_blank" >RIV/68378050:_____/25:00619012 - isvavai.cz</a>

Výsledek na webu

<a href="https://doi.org/10.1186/s12935-025-03740-y" target="_blank" >https://doi.org/10.1186/s12935-025-03740-y</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1186/s12935-025-03740-y" target="_blank" >10.1186/s12935-025-03740-y</a>

Jazyk výsledku

angličtina

Název v původním jazyce

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

Popis výsledku v původním jazyce

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 gamma-tubulin ring complexes (gamma TuRCs), 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.

Název v anglickém jazyce

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

Popis výsledku anglicky

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 gamma-tubulin ring complexes (gamma TuRCs), 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.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10601 - Cell biology

Projekt

Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2025

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Cancer Cell International

ISSN

1475-2867

e-ISSN

1475-2867

Svazek periodika

25

Číslo periodika v rámci svazku

1

Stát vydavatele periodika

GB - Spojené království Velké Británie a Severního Irska

Počet stran výsledku

21

Strana od-do

125

Kód UT WoS článku

001458536900001

EID výsledku v databázi Scopus

2-s2.0-105001692427