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.

• Keywords
• Centrosomes, G protein-coupled receptor kinase-interacting proteins (GITs), Glioblastoma cells, Microtubule nucleation, Protein kinase C (PKC),
• Publication type
• Journal Article MeSH

Anaplastic Large Cell Lymphoma (ALCL) is an aggressive T-cell lymphoma affecting children and young adults. About 30% of patients develop therapy resistance therefore new precision medicine drugs are highly warranted. Multiple rounds of structure-activity optimization of Caffeic Acid Phenethyl Ester have resulted in CM14. CM14 causes upregulation of genes involved in oxidative stress response and downregulation of DNA replication genes leading to G2/M arrest and subsequent apoptosis induction. In accordance with this, an unbiased proteomics approach, confocal microscopy and molecular modeling showed that TUBGCP2, member of the centrosomal γ-TuRC complex, is a direct interaction partner of CM14. CM14 overcomes ALK inhibitor resistance in ALCL and is also active in T-cell Acute Lymphoblastic Leukemia and Acute Myeloid Leukemia. Interestingly, CM14 also induced cell death in docetaxel-resistant prostate cancer cells thus suggesting an unexpected role in solid cancers. Thus, we synthesized and thoroughly characterized a novel TUBGCP2 targeting drug that is active in ALCL but has also potential for other malignancies.

• MeSH
• Apoptosis drug effects   MeSH
• Centrosome * drug effects   metabolism   MeSH
• Drug Resistance, Neoplasm drug effects   MeSH
• Phenylethyl Alcohol * pharmacology   analogs & derivatives   chemistry   MeSH
• Caffeic Acids * pharmacology   chemistry   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Antineoplastic Agents * pharmacology   chemistry   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• caffeic acid phenethyl ester MeSH Browser
• Phenylethyl Alcohol * MeSH
• Caffeic Acids * MeSH
• Antineoplastic Agents * MeSH

Aggregation of high-affinity IgE receptors (FcϵRIs) on granulated mast cells triggers signaling pathways leading to a calcium response and release of inflammatory mediators from secretory granules. While microtubules play a role in the degranulation process, the complex molecular mechanisms regulating microtubule remodeling in activated mast cells are only partially understood. Here, we demonstrate that the activation of bone marrow mast cells induced by FcϵRI aggregation increases centrosomal microtubule nucleation, with G protein-coupled receptor kinase-interacting protein 2 (GIT2) playing a vital role in this process. Both endogenous and exogenous GIT2 were associated with centrosomes and γ-tubulin complex proteins. Depletion of GIT2 enhanced centrosomal microtubule nucleation, and phenotypic rescue experiments revealed that GIT2, unlike GIT1, acts as a negative regulator of microtubule nucleation in mast cells. GIT2 also participated in the regulation of antigen-induced degranulation and chemotaxis. Further experiments showed that phosphorylation affected the centrosomal localization of GIT2 and that during antigen-induced activation, GIT2 was phosphorylated by conventional protein kinase C, which promoted microtubule nucleation. We propose that GIT2 is a novel regulator of microtubule organization in activated mast cells by modulating centrosomal microtubule nucleation.

• Keywords
• G protein-coupled receptor kinase-interacting protein 2 (GIT2), centrosome, mast cells, microtubule nucleation, protein kinase C (PKC),
• MeSH
• Centrosome metabolism   MeSH
• Bone Marrow * MeSH
• Mast Cells * metabolism   MeSH
• Microtubules * metabolism   MeSH
• Mice MeSH
• GTPase-Activating Proteins * metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Git2 protein, mouse MeSH Browser
• GTPase-Activating Proteins * MeSH

Microtubules composed of αβ-tubulin dimers are dynamic cytoskeletal polymers that play key roles in essential cellular processes such as cell division, organelle positioning, intracellular transport, and cell migration. γ-Tubulin is a highly conserved member of the tubulin family that is required for microtubule nucleation. γ-Tubulin, together with its associated proteins, forms the γ-tubulin ring complex (γ-TuRC), that templates microtubules. Here we review recent advances in the structure of γ-TuRC, its activation, and centrosomal recruitment. This provides new mechanistic insights into the molecular mechanism of microtubule nucleation. Accumulating data suggest that γ-tubulin also has other, less well understood functions. We discuss emerging evidence that γ-tubulin can form oligomers and filaments, has specific nuclear functions, and might be involved in centrosomal cross-talk between microtubules and microfilaments.

• Keywords
• microtubule nucleation, αβ-tubulin dimer, γ-tubulin functions, γ-tubulin isotypes, γ-tubulin ring complexes (γ-TuRC),
• Publication type
• Journal Article MeSH
• Review MeSH

ER distribution depends on microtubules, and ER homeostasis disturbance activates the unfolded protein response resulting in ER remodeling. CDK5RAP3 (C53) implicated in various signaling pathways interacts with UFM1-protein ligase 1 (UFL1), which mediates the ufmylation of proteins in response to ER stress. Here we find that UFL1 and C53 associate with γ-tubulin ring complex proteins. Knockout of UFL1 or C53 in human osteosarcoma cells induces ER stress and boosts centrosomal microtubule nucleation accompanied by γ-tubulin accumulation, microtubule formation, and ER expansion. C53, which is stabilized by UFL1, associates with the centrosome and rescues microtubule nucleation in cells lacking UFL1. Pharmacological induction of ER stress by tunicamycin also leads to increased microtubule nucleation and ER expansion. Furthermore, tunicamycin suppresses the association of C53 with the centrosome. These findings point to a novel mechanism for the relief of ER stress by stimulation of centrosomal microtubule nucleation.

• Keywords
• CDK5RAP3, ER stress, UFL1, microtubule nucleation, γ-tubulin,
• MeSH
• Humans MeSH
• Microtubules metabolism   MeSH
• Tumor Suppressor Proteins metabolism   MeSH
• Cell Cycle Proteins metabolism   MeSH
• Endoplasmic Reticulum Stress physiology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• CDK5RAP3 protein, human MeSH Browser
• Tumor Suppressor Proteins MeSH
• Cell Cycle Proteins MeSH

In cells, microtubules typically nucleate from microtubule organizing centers, such as centrosomes. γ-Tubulin, which forms multiprotein complexes, is essential for nucleation. The γ-tubulin ring complex (γ-TuRC) is an efficient microtubule nucleator that requires additional centrosomal proteins for its activation and targeting. Evidence suggests that there is a dysfunction of centrosomal microtubule nucleation in cancer cells. Despite decades of molecular analysis of γ-TuRC and its interacting factors, the mechanisms of microtubule nucleation in normal and cancer cells remains obscure. Here, we review recent work on the high-resolution structure of γ-TuRC, which brings new insight into the mechanism of microtubule nucleation. We discuss the effects of γ-TuRC protein dysregulation on cancer cell behavior and new compounds targeting γ-tubulin. Drugs inhibiting γ-TuRC functions could represent an alternative to microtubule targeting agents in cancer chemotherapy.

• Keywords
• cancers, microtubule nucleation, γ-tubulin complexes,
• Publication type
• Journal Article MeSH
• Review MeSH

Profilin 1 is a crucial actin regulator, interacting with monomeric actin and several actin-binding proteins controlling actin polymerization. Recently, it has become evident that this profilin isoform associates with microtubules via formins and interferes with microtubule elongation at the cell periphery. Recruitment of microtubule-associated profilin upon extensive actin polymerizations, for example, at the cell edge, enhances microtubule growth, indicating that profilin contributes to the coordination of actin and microtubule organization. Here, we provide further evidence for the profilin-microtubule connection by demonstrating that it also functions in centrosomes where it impacts on microtubule nucleation.

• MeSH
• Actins metabolism   MeSH
• Caco-2 Cells MeSH
• Centrosome metabolism   MeSH
• Formins metabolism   MeSH
• Gene Knockout Techniques MeSH
• Humans MeSH
• Melanoma, Experimental metabolism   pathology   MeSH
• Microfilament Proteins metabolism   MeSH
• Microtubules metabolism   MeSH
• Mice MeSH
• Skin Neoplasms metabolism   pathology   MeSH
• Polymerization MeSH
• Profilins genetics   metabolism   MeSH
• Signal Transduction genetics   MeSH
• Transfection MeSH
• Tubulin metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Actins MeSH
• Formins MeSH
• Microfilament Proteins MeSH
• PFN1 protein, human MeSH Browser
• Pfn1 protein, mouse MeSH Browser
• Profilins MeSH
• Tubulin MeSH

De novo heterozygous missense variants in the γ-tubulin gene TUBG1 have been linked to human malformations of cortical development associated with intellectual disability and epilepsy. Here, we investigated through in-utero electroporation and in-vivo studies, how four of these variants affect cortical development. We show that TUBG1 mutants affect neuronal positioning, disrupting the locomotion of new-born neurons but without affecting progenitors' proliferation. We further demonstrate that pathogenic TUBG1 variants are linked to reduced microtubule dynamics but without major structural nor functional centrosome defects in subject-derived fibroblasts. Additionally, we developed a knock-in Tubg1Y92C/+ mouse model and assessed consequences of the mutation. Although centrosomal positioning in bipolar neurons is correct, they fail to initiate locomotion. Furthermore, Tubg1Y92C/+ animals show neuroanatomical and behavioral defects and increased epileptic cortical activity. We show that Tubg1Y92C/+ mice partially mimic the human phenotype and therefore represent a relevant model for further investigations of the physiopathology of cortical malformations.

• MeSH
• Centrosome metabolism   MeSH
• Behavior, Animal MeSH
• Microscopy, Electron MeSH
• Embryo, Mammalian MeSH
• Epilepsy genetics   MeSH
• Fibroblasts cytology   metabolism   ultrastructure   MeSH
• Genetic Predisposition to Disease MeSH
• Gene Knock-In Techniques MeSH
• HeLa Cells MeSH
• Intravital Microscopy MeSH
• Microscopy, Confocal MeSH
• Humans MeSH
• Malformations of Cortical Development genetics   MeSH
• Microtubules genetics   metabolism   MeSH
• Mutation, Missense MeSH
• Disease Models, Animal MeSH
• Cerebral Cortex abnormalities   cytology   diagnostic imaging   MeSH
• Mice, Transgenic MeSH
• Mice MeSH
• Neurogenesis genetics   MeSH
• Neurons physiology   MeSH
• Cell Movement genetics   MeSH
• Tubulin genetics   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Video-Audio Media MeSH
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• TUBG1 protein, human MeSH Browser
• TUBG1 protein, mouse MeSH Browser
• Tubulin MeSH

The antigen-mediated activation of mast cells initiates signaling events leading to their degranulation, to the release of inflammatory mediators, and to the synthesis of cytokines and chemokines. Although rapid and transient microtubule reorganization during activation has been described, the molecular mechanisms that control their rearrangement are largely unknown. Microtubule nucleation is mediated by γ-tubulin complexes. In this study, we report on the regulation of microtubule nucleation in bone marrow-derived mast cells (BMMCs) by Src homology 2 (SH2) domain-containing protein tyrosine phosphatase 1 (SHP-1; Ptpn6). Reciprocal immunoprecipitation experiments and pull-down assays revealed that SHP-1 is present in complexes containing γ-tubulin complex proteins and protein tyrosine kinase Syk. Microtubule regrowth experiments in cells with deleted SHP-1 showed a stimulation of microtubule nucleation, and phenotypic rescue experiments confirmed that SHP-1 represents a negative regulator of microtubule nucleation in BMMCs. Moreover, the inhibition of the SHP-1 activity by inhibitors TPI-1 and NSC87877 also augmented microtubule nucleation. The regulation was due to changes in γ-tubulin accumulation. Further experiments with antigen-activated cells showed that the deletion of SHP-1 stimulated the generation of microtubule protrusions, the activity of Syk kinase, and degranulation. Our data suggest a novel mechanism for the suppression of microtubule formation in the later stages of mast cell activation.

• Keywords
• SHP-1 tyrosine phosphatase, bone marrow-derived mast cells, cell activation, microtubule nucleation, γ-tubulin complexes,
• MeSH
• Cell Degranulation MeSH
• HEK293 Cells MeSH
• Syk Kinase metabolism   MeSH
• Humans MeSH
• Mast Cells cytology   metabolism   MeSH
• MCF-7 Cells MeSH
• Microtubules metabolism   MeSH
• Mice MeSH
• Tubulin metabolism   MeSH
• Protein Tyrosine Phosphatase, Non-Receptor Type 6 antagonists & inhibitors   physiology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Syk Kinase MeSH
• Ptpn6 protein, mouse MeSH Browser
• Syk protein, mouse MeSH Browser
• Tubulin MeSH
• Protein Tyrosine Phosphatase, Non-Receptor Type 6 MeSH

γ-Tubulin is a conserved member of the tubulin superfamily with a function in microtubule nucleation. Proteins of γ-tubulin complexes serve as nucleation templates as well as a majority of other proteins contributing to centrosomal and non-centrosomal nucleation, conserved across eukaryotes. There is a growing amount of evidence of γ-tubulin functions besides microtubule nucleation in transcription, DNA damage response, chromatin remodeling, and on its interactions with tumor suppressors. However, the molecular mechanisms are not well understood. Furthermore, interactions with lamin and SUN proteins of the LINC complex suggest the role of γ-tubulin in the coupling of nuclear organization with cytoskeletons. γ-Tubulin that belongs to the clade of eukaryotic tubulins shows characteristics of both prokaryotic and eukaryotic tubulins. Both human and plant γ-tubulins preserve the ability of prokaryotic tubulins to assemble filaments and higher-order fibrillar networks. γ-Tubulin filaments, with bundling and aggregating capacity, are suggested to perform complex scaffolding and sequestration functions. In this review, we discuss a plethora of γ-tubulin molecular interactions and cellular functions, as well as recent advances in understanding the molecular mechanisms behind them.

• Keywords
• SUN proteins, filaments, gamma-tubulin, lamins, mechanosensing, nuclear functions, nucleation,
• MeSH
• Cell Nucleus metabolism   MeSH
• Cell Cycle MeSH
• Nuclear Proteins metabolism   MeSH
• Nuclear Envelope metabolism   MeSH
• Humans MeSH
• Microtubules metabolism   MeSH
• Tubulin metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Nuclear Proteins MeSH
• Tubulin MeSH