Overexpression and Nucleolar Localization of γ-Tubulin Small Complex Proteins GCP2 and GCP3 in Glioblastoma

Abstract

The expression, cellular distribution, and subcellular sorting of the microtubule (MT)-nucleating γ-tubulin small complex (γTuSC) proteins, GCP2 and GCP3, were studied in human glioblastoma cell lines and in clinical tissue samples representing all histologic grades of adult diffuse astrocytic gliomas (n = 54). Quantitative real-time polymerase chain reaction revealed a significant increase in the expression of GCP2 and GCP3 transcripts in glioblastoma cells versus normal human astrocytes; these were associated with higher amounts of both γTuSC proteins. GCP2 and GCP3 were concentrated in the centrosomes in interphase glioblastoma cells, but punctate and diffuse localizations were also detected in the cytosol and nuclei/nucleoli. Nucleolar localization was fixation dependent. GCP2 and GCP3 formed complexes with γ-tubulin in the nucleoli as confirmed by reciprocal immunoprecipitation experiments and immunoelectron microscopy. GCP2 and GCP3 depletion caused accumulation of cells in G2/M and mitotic delay but did not affect nucleolar integrity. Overexpression of GCP2 antagonized the inhibitory effect of the CDK5 regulatory subunit-associated tumor suppressor protein 3 (C53) on DNA damage G2/M checkpoint activity. Tumor cell GCP2 and GCP3 immunoreactivity was significantly increased over that in normal brains in glioblastoma samples; it was also associated with microvascular proliferation. These findings suggest that γTuSC protein dysregulation in glioblastomas may be linked to altered transcriptional checkpoint activity or interaction with signaling pathways associated with a malignant phenotype.

Differential expression of human γ‐tubulin isotypes during neuronal development and oxidative stress points to a γ‐tubulin‐2 prosurvival function

“…Immunolabeling was performed as described (31). Rabbit anti‐γ‐tubulin Abs (T3195 and T5912; Sigma‐Aldrich) were diluted 1: 75 and 1: 250, respectively.…”

“…Double‐label immunofluorescence on FFPE samples was performed as described (31, 32). After hydration, the FFPE tissue sections were treated with chilled sodium borohydride (1 mg/ml) in PBS for 50 min.…”

Gamma-tubulin coordinates nuclear envelope assembly around chromatin

“…γ-Tubulin is a cytosolic protein that regulates α- and β-tubulin nucleation to a growing microtubule ( Kollman et al, 2011 ). We and others have shown that nuclear γ-tubulin associates with Rad51, C53, GCP2, GCP3 and E2F1 ( Draberova et al, 2015 ; Ehlen et al, 2012 ; Hoog et al, 2011 ; Horejsi et al, 2012 ; Lesca et al, 2005 ) and inhibition of the nuclear activity of γ-tubulin does not interfere with microtubule dynamics ( Lindstrom et al, 2015 ). During the cell cycle, γ-tubulin has various functions, regulating centrosomal duplication and mitotic spindle formation and moderating transcriptional activities of E2Fs during S-phase ( Hoog et al, 2011 ).…”

γ-tubulin as a signal-transducing molecule and meshwork with therapeutic potential

“…γ-tubulin is a ubiquitously expressed protein that appears in abundance in both the cytosol and the nuclear compartments of cells in all mammalian tissues. 13 , 14 , 20 – 23…”

“…1 and 2 ). 13 , 14 , 20 – 22 , 24 , 27 This process implies that the cytosolic and nuclear γ-tubulin pools have different conformations, which could provide the basis for the development of therapeutic compounds that target the nuclear activity of γ-tubulin. Furthermore, studies of murine NIH3T3 embryonic fibroblasts and human U2OS osteosarcoma cells have indicated that γ-tubulin is the only member of the tubulin family that has a bipartite NLS on the C terminus (Fig.…”