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.

Department of Biological Sciences The University of Memphis 101 Life Science Building Memphis TN 38152 USA

Department of Biology of Cytoskeleton Institute of Molecular Genetics Czech Academy of Sciences 142 20 Prague 4 Czech Republic

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Ohgaki H, Kleihues P. Population-based studies on incidence, survival rates, and genetic alterations in astrocytic and oligodendroglial gliomas. J Neuropathol Exp Neurol. 2005;64:479–89. 10.1093/jnen/64.6.479. PubMed

Louis DN, Perry A, Wesseling P, Brat DJ, Cree IA, Figarella-Branger D, Hawkins C, et al. The 2021 WHO classification of tumors of the central nervous system: a summary. Neuro Oncol. 2021;23:1231–51. 10.1093/neuonc/noab106. PubMed PMC

Thakur A, Faujdar C, Sharma R, Sharma S, Malik B, Nepali K, Liou JP. Glioblastoma: current status, emerging targets, and recent advances. J Med Chem. 2022;65:8596–685. 10.1021/acs.jmedchem.1c01946. PubMed PMC

Katsetos CD, Dráberová E, Legido A, Dráber P. Tubulin targets in the pathobiology and therapy of glioblastoma multiforme. II. γ-Tubulin. J Cell Physiol. 2009;221:514–20. 10.1002/jcp.21884. PubMed

Skalli O, Wilhelmsson U, Orndahl C, Fekete B, Malmgren K, Rydenhag B, Pekny M. Astrocytoma grade IV (glioblastoma multiforme) displays 3 subtypes with unique expression profiles of intermediate filament proteins. Hum Pathol. 2013;44:2081–8. 10.1016/j.humpath.2013.03.013. PubMed

Lv S, Chen Z, Mi H, Yu X. Cofilin acts as a booster for progression of malignant tumors represented by glioma. Cancer Manag Res. 2022;14:3245–69. 10.2147/CMAR.S389825. PubMed PMC

Oakley CE, Oakley BR. Identification of γ -tubulin, a new member of the tubulin superfamily encoded by mipA gene of Aspergillus nidulans. Nature. 1989;338:662–4. 10.1038/338662a0. PubMed

Wise DO, Krahe R, Oakley BR. The γ -tubulin gene family in humans. Genomics. 2000;67:164–70. 10.1006/geno.2000.6247. PubMed

Vinopal S, Černohorská M, Sulimenko V, Sulimenko T, Vosecká V, Flemr M, Dráberová E, et al. γ -Tubulin 2 nucleates microtubules and is downregulated in mouse early embryogenesis. PLoS ONE. 2012;7: e29919. 10.1371/journal.pone.0029919. PubMed PMC

Oegema K, Wiese C, Martin OC, Milligan RA, Iwamatsu A, Mitchison TJ, Zheng Y. Characterization of two related Drosophila γ -tubulin complexes that differ in their ability to nucleate microtubules. J Cell Biol. 1999;144:721–33. 10.1083/jcb.144.4.721. PubMed PMC

Zupa E, Liu P, Würtz M, Schiebel E, Pfeffer S. The structure of the γ -TuRC: a 25-years-old molecular puzzle. Curr Opin Struct Biol. 2021;66:15–21. 10.1016/j.sbi.2020.08.008. PubMed

Thawani A, Petry S. Molecular insight into how γ -TuRC makes microtubules. J Cell Sci. 2021;134:jcs245464. 10.1242/jcs.245464. PubMed PMC

Serna M, Zimmermann F, Vineethakumari C, Gonzalez-Rodriguez N, Llorca O, Lüders J. CDK5RAP2 activates microtubule nucleator γ TuRC by facilitating template formation and actin release. Dev Cell. 2024;59:3175–88. 10.1016/j.devcel.2024.09.001. PubMed

Sulimenko V, Dráberová E, Dráber P. γ -Tubulin in microtubule nucleation and beyond. Front Cell Dev Biol. 2022;10: 880761. 10.3389/fcell.2022.880761. PubMed PMC

Sztul E, Chen PW, Casanova JE, Cherfils J, Dacks JB, Lambright DG, Lee FS, et al. ARF GTPases and their GEFs and GAPs: concepts and challenges. Mol Biol Cell. 2019;30:1249–71. 10.1091/mbc.E18-12-0820. PubMed PMC

Premont RT, Claing A, Vitale N, Perry SJ, Lefkowitz RJ. The GIT family of ADP-ribosylation factor GTPase-activating proteins. Functional diversity of GIT2 through alternative splicing. J Biol Chem. 2000;275:22373–80. 10.1074/jbc.275.29.22373. PubMed

Zhou W, Li X, Premont RT. Expanding functions of GIT Arf GTPase-activating proteins, PIX Rho guanine nucleotide exchange factors and GIT-PIX complexes. J Cell Sci. 2016;129:1963–74. 10.1242/jcs.179465. PubMed PMC

Hoefen RJ, Berk BC. The multifunctional GIT family of proteins. J Cell Sci. 2006;119:1469–75. 10.1242/jcs.02925. PubMed

Webb DJ, Mayhew MW, Kovalenko M, Schroeder MJ, Jeffery ED, Whitmore L, Shabanowitz J, et al. Identification of phosphorylation sites in GIT1. J Cell Sci. 2006;119:2847–50. 10.1242/jcs03044. PubMed

Zhao ZS, Lim JP, Ng YW, Lim L, Manser E. The GIT-associated kinase PAK targets to the centrosome and regulates Aurora-A. Mol Cell. 2005;20:237–49. 10.1016/j.molcel.2005.08.035. PubMed

Sulimenko V, Hájková Z, Černohorská M, Sulimenko T, Sládková V, Dráberová L, Vinopal S, et al. Microtubule nucleation in mouse bone marrow-derived mast cells is regulated by the concerted action of GIT1/βPIX proteins and calcium. J Immunol. 2015;194:4099–111. 10.4049/jimmunol.1402459. PubMed

Sulimenko V, Sládková V, Sulimenko T, Dráberová E, Vosecká V, Dráberová L, Skalli O, et al. Regulation of microtubule nucleation in mouse bone marrow-derived mast cells by ARF GTPase-activating protein GIT2. Front Immunol. 2024;15:1321321. 10.3389/fimmu.2024.1321321. PubMed PMC

Hořejší B, Vinopal S, Sládková V, Dráberová E, Sulimenko V, Sulimenko T, Vosecká V, et al. Nuclear γ-tubulin associates with nucleoli and interacts with tumor suppressor protein C53. J Cell Physiol. 2012;227:367–82. 10.1002/jcp.22772. PubMed

Nováková M, Dráberová E, Schürmann W, Czihak G, Viklický V, Dráber P. γ -Tubulin redistribution in taxol-treated mitotic cells probed by monoclonal antibodies. Cell Motil Cytoskel. 1996;33:38–51. 10.1002/(SICI)1097-0169(1996)33:1%3c38::AID-CM5%3e3.0.CO;2-E. PubMed

Dráberová E, Sulimenko V, Vinopal S, Sulimenko T, Sládková V, D’Agostino L, Sobol M, et al. Differential expression of human γ -tubulin isotypes during neuronal development and oxidative stress points to a γ -tubulin-2 prosurvival function. FASEB J. 2017;31:1828–46. 10.1096/fj.201600846RR. PubMed

Dráberová E, D’Agostino L, Caracciolo V, Sládková V, Sulimenko T, Sulimenko V, Sobol M, et al. Overexpression and nucleolar localization of γ -tubulin small complex proteins GCP2 and GCP3 in glioblastoma. J Neuropathol Exp Neurol. 2015;74:723–42. 10.1097/NEN.0000000000000212. PubMed

Zíková M, Dráberová E, Sulimenko V, Dráber P. New monoclonal antibodies specific for microtubule-associated protein MAP2. Folia Biol (Praha). 2000;46:87–8. PubMed

Černohorská M, Sulimenko V, Hájková Z, Sulimenko T, Sládková V, Vinopal S, Dráberová E, et al. GIT1/βPIX signaling proteins and PAK1 kinase regulate microtubule nucleation. BBA Mol Cell Res. 2016;1863:1282–97. 10.1016/j.bbamcr.2016.03.016. PubMed

Macůrek L, Dráberová E, Richterová V, Sulimenko V, Sulimenko T, Dráberová L, Marková V, et al. Regulation of microtubule nucleation from membranes by complexes of membrane-bound γ -tubulin with Fyn kinase and phosphoinositide 3-kinase. Biochem J. 2008;416:421–30. 10.1042/BJ20080909. PubMed

Pan Y, Jing R, Pitre A, Williams BJ, Skalli O. Intermediate filament protein synemin contributes to the migratory properties of astrocytoma cells by influencing the dynamics of the actin cytoskeleton. FASEB J. 2008;22:3196–206. 10.1096/fj.08-106187. PubMed PMC

Kumari A, Panda D. Monitoring the disruptive effects of tubulin-binding agents on cellular microtubules. Methods Mol Biol. 2022;2430:431–48. 10.1007/978-1-0716-1983-4_27. PubMed

Mitchison TJ, Kirschner MW. Isolation of mammalian centrosomes. Methods Enzymol. 1986;134:261–8. 10.1016/0076-6879(86)34094-1. PubMed

Evans L, Mitchison T, Kirschner M. Influence of the centrosome on the structure of nucleated microtubules. J Cell Biol. 1985;100:1185–91. 10.1083/jcb.100.4.1185. PubMed PMC

Kukharskyy V, Sulimenko V, Macůrek L, Sulimenko T, Dráberová E, Dráber P. Complexes of γ -tubulin with non-receptor protein tyrosine kinases Src and Fyn in differentiating P19 embryonal carcinoma cells. Exp Cell Res. 2004;298:218–28. 10.1016/j.yexcr.2004.04.016. PubMed

Frangioni JV, Neel BG. Solubilization and purification of enzymatically active glutathione-S-transferase (pGEX) fusion proteins. Anal Biochem. 1993;210:179–87. 10.1006/abio.1993.1170. PubMed

Dráber P. Quantitation of proteins in sample buffer for sodium dodecyl sulfate-polyacrylamide gel electrophoresis using colloidal silver. Electrophoresis. 1991;12:453–6. 10.1002/elps.1150120617. PubMed

Pijuan J, Barceló C, Moreno DF, Maiques O, Sisó P, Marti RM, Macià A, et al. In vitro cell migration, invasion, and adhesion assays: from cell imaging to data analysis. Front Cell Dev Biol. 2019;7:107. 10.3389/fcell.2019.00107. PubMed PMC

Suarez-Arnedo A, Torres Figueroa F, Clavijo C, Arbelaez P, Cruz JC, Munoz-Camargo C. An image J plugin for the high throughput image analysis of in vitro scratch wound healing assays. PLoS ONE. 2020;15: e0232565. 10.1371/journal.pone.0232565. PubMed PMC

Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9:676–82. 10.1038/nmeth.2019. PubMed PMC

Frank SR, Hansen SH. The PIX-GIT complex: a G protein signaling cassette in control of cell shape. Semin Cell Dev Biol. 2008;19:234–44. 10.1016/j.semcdb.2008.01.002. PubMed PMC

Lim PS, Sutton CR, Rao S. Protein kinase C in the immune system: from signalling to chromatin regulation. Immunology. 2015;146:508–22. 10.1111/imm.12510. PubMed PMC

Way KJ, Chou E, King GL. Identification of PKC-isoform-specific biological actions using pharmacological approaches. Trends Pharmacol Sci. 2000;21:181–7. 10.1016/s0165-6147(00)01468-1. PubMed

Martiny-Baron G, Kazanietz MG, Mischak H, Blumberg PM, Kochs G, Hug H, Marmé D, et al. Selective inhibition of protein kinase C isozymes by the indolocarbazole Gö 6976. J Biol Chem. 1993;268:9194–7. 10.1016/S0021-9258(18)98335-3. PubMed

Webb DJ, Kovalenko M, Whitmore L, Horwitz AF. Phosphorylation of serine 709 in GIT1 regulates protrusive activity in cells. Biochem Biophys Res Commun. 2006;346:1284–8. 10.1016/j.bbrc.2006.06.036. PubMed

Katsetos CD, Reddy G, Dráberová E, Šmejkalová B, Del Valle L, Ashraf Q, Tradevosyan A, et al. Altered cellular distribution and subcellular sorting of γ -tubulin in diffuse astrocytic gliomas and human glioblastoma cell lines. J Neuropathol Exp Neurol. 2006;65:465–77. 10.1097/01.jnen.0000229235.20995.6e. PubMed

Ludueña RF. Multiple forms of tubulin: different gene products and covalent modifications. Int Rev Cytol. 1998;178:207–75. 10.1016/s0074-7696(08)62138-5. PubMed

Linhartová I, Dráber P, Dráberová E, Viklický V. Immunological discrimination of β-tubulin isoforms in developing mouse brain. Posttranslational modification of non-class III β-tubulins. Biochem J. 1992;288:919–24. 10.1042/bj2880919. PubMed PMC

Turn RE, Linnert J, Gigante ED, Wolfrum U, Caspary T, Kahn RA. Roles for ELMOD2 and Rootletin in ciliogenesis. Mol Biol Cell. 2021;32:800–22. 10.1091/mbc.E20-10-0635. PubMed PMC

Turn RE, East MP, Prekeris R, Kahn RA. The ARF GAP ELMOD2 acts with different GTPases to regulate centrosomal microtubule nucleation and cytokinesis. Mol Biol Cell. 2020;31:2070–91. 10.1091/mbc.E20-01-0012. PubMed PMC

Francis JW, Turn RE, Newman LE, Schiavon C, Kahn RA. Higher order signaling: ARL2 as regulator of both mitochondrial fusion and microtubule dynamics allows integration of 2 essential cell functions. Small GTPases. 2016;7:188–96. 10.1080/21541248.2016.1211069. PubMed PMC

Ma D, Lin KY, Suresh D, Lin J, Gujar MR, Aung HY, Tan YS, et al. Arl2 GTPase associates with the centrosomal protein Cdk5rap2 to regulate cortical development via microtubule organization. PLoS Biol. 2024;22: e3002751. 10.1371/journal.pbio.3002751. PubMed PMC

Frank SR, Adelstein MR, Hansen SH. GIT2 represses Crk- and Rac1-regulated cell spreading and Cdc42-mediated focal adhesion turnover. EMBO J. 2006;25:1848–59. 10.1038/sj.emboj.7601092. PubMed PMC

Gavina M, Za L, Molteni R, Pardi R, de Curtis I. The GIT-PIX complexes regulate the chemotactic response of rat basophilic leukaemia cells. Biol Cell. 2010;102:231–44. 10.1042/BC20090074. PubMed PMC

Montesinos MS, Dong W, Goff K, Das B, Guerrero-Given D, Schmalzigaug R, Premont RT, et al. Presynaptic deletion of GIT proteins results in increased synaptic strength at a mammalian central synapse. Neuron. 2015;88:918–25. 10.1016/j.neuron.2015.10.042. PubMed PMC

Yoo SM, Cerione RA, Antonyak MA. The Arf-GAP and protein scaffold Cat1/Git1 as a multifaceted regulator of cancer progression. Small GTPases. 2020;11:77–85. 10.1080/21541248.2017.1362496. PubMed PMC

Duan B, Cui J, Sun S, Zheng J, Zhang Y, Ye B, Chen Y, et al. EGF-stimulated activation of Rab35 regulates RUSC2-GIT2 complex formation to stabilize GIT2 during directional lung cancer cell migration. Cancer Lett. 2016;379:70–83. 10.1016/j.canlet.2016.05.027. PubMed

Frank SR, Kollmann CP, van de Lidth Jeude JF, Thiagarajah JR, Engelholm LH, Frodin M, Hansen SH. The focal adhesion-associated proteins DOCK5 and GIT2 comprise a rheostat in control of epithelial invasion. Oncogene. 2017;36:1816–28. 10.1038/onc.2016.345. PubMed PMC

Tanna CE, Goss LB, Ludwig CG, Chen PW. Arf GAPs as regulators of the actin cytoskeleton-an update. Int J Mol Sci. 2019;20:442. 10.3390/ijms20020442. PubMed PMC

Vitale N, Patton WA, Moss J, Vaughan M, Lefkowitz RJ, Premont RT. GIT proteins, A novel family of phosphatidylinositol 3,4, 5-trisphosphate-stimulated GTPase-activating proteins for ARF6. J Biol Chem. 2000;275:13901–6. 10.1074/jbc.275.18.13901. PubMed

Kong KF, Fu G, Zhang Y, Yokosuka T, Casas J, Canonigo-Balancio AJ, Becart S, et al. Protein kinase C- controls CTLA-4-mediated regulatory T cell function. Nat Immunol. 2014;15:465–72. 10.1038/ni.2866. PubMed PMC

Huck B, Kemkemer R, Franz-Wachtel M, Macek B, Hausser A, Olayioye MA. GIT1 phosphorylation on serine 46 by PKD3 regulates paxillin trafficking and cellular protrusive activity. J Biol Chem. 2012;287:34604–13. 10.1074/jbc.M112.374652. PubMed PMC

Rey O, Yuan J, Young SH, Rozengurt E. Protein kinase Cv/protein kinase D3 nuclear localization, catalytic activation, and intracellular redistribution in response to G protein-coupled receptor agonists. J Biol Chem. 2003;278:23773–85. 10.1074/jbc.M300226200. PubMed

Chen D, Purohit A, Halilovic E, Doxsey SJ, Newton AC. Centrosomal anchoring of protein kinase C βII by pericentrin controls microtubule organization, spindle function, and cytokinesis. J Biol Chem. 2004;279:4829–39. 10.1074/jbc.M311196200. PubMed

Wei SY, Lin TE, Wang WL, Lee PL, Tsai MC, Chiu JJ. Protein kinase C-δ and -β coordinate flow-induced directionality and deformation of migratory human blood T-lymphocytes. J Mol Cell Biol. 2014;6:458–72. 10.1093/jmcb/mju050. PubMed

Fanning A, Volkov Y, Freeley M, Kelleher D, Long A. CD44 cross-linking induces protein kinase C-regulated migration of human T lymphocytes. Int Immunol. 2005;17:449–58. 10.1093/intimm/dxh225. PubMed

Martini S, Soliman T, Gobbi G, Mirandola P, Carubbi C, Masselli E, Pozzi G, et al. PKCε controls mitotic progression by regulating centrosome migration and mitotic spindle assembly. Mol Cancer Res. 2018;16:3–15. 10.1158/1541-7786.MCR-17-0244. PubMed PMC

Passalacqua M, Patrone M, Sparatore B, Melloni E, Pontremoli S. Protein kinase C-θ is specifically localized on centrosomes and kinetochores in mitotic cells. Biochem J. 1999;337:113–8. 10.1024/bj3370113. PubMed PMC

Bouchet BP, Akhmanova A. Microtubules in 3D cell motility. J Cell Sci. 2017;130:39–50. 10.1242/jcs.189431. PubMed

Kim Y, Varn FS, Park SH, Yoon BW, Park HR, Lee C, Verhaak RGW, et al. Perspective of mesenchymal transformation in glioblastoma. Acta Neuropathol Commun. 2021;9:50. 10.1186/s40478-021-01151-4. PubMed PMC

Mandil R, Ashkenazi E, Blass M, Kronfeld I, Kazimirsky G, Rosenthal G, Umansky F, et al. Protein kinase Cα and protein kinase C δ play opposite roles in the proliferation and apoptosis of glioma cells. Cancer Res. 2001;61:4612–9. PubMed

Arcos-Montoya D, Wegman-Ostrosky T, Mejía-Pérez S, De la Fuente-Granada M, Camacho-Arroyo I, Garcia-Carrancá A, Velasco-Velázquez MA, et al. Progesterone receptor together with PKCα expression as prognostic factors for astrocytomas malignancy. Onco Targets Ther. 2021;14:3757–68. 10.2147/OTT.S280314. PubMed PMC

Totaro A, Astro V, Tonoli D, de Curtis I. Identification of two tyrosine residues required for the intramolecular mechanism implicated in GIT1 activation. PLoS ONE. 2014;9: e93199. 10.1371/journal.pone.0093199. PubMed PMC

Brown MC, Cary LA, Jamieson JS, Cooper JA, Turner CE. Src and FAK kinases cooperate to phosphorylate paxillin kinase linker, stimulate its focal adhesion localization, and regulate cell spreading and protrusiveness. Mol Biol Cell. 2005;16:4316–28. 10.1091/mbc.e05-02-0131. PubMed PMC

Frese S, Schubert WD, Findeis AC, Marquardt T, Roske YS, Stradal TE, Heinz DW. The phosphotyrosine peptide binding specificity of Nck1 and Nck2 Src homology 2 domains. J Biol Chem. 2006;281:18236–45. 10.1074/jbc.M512917200. PubMed