Nuclear γ‐tubulin associates with nucleoli and interacts with tumor suppressor protein C53

Abstract

γ-Tubulin is assumed to be a typical cytosolic protein necessary for nucleation of microtubules from microtubule organizing centers. Using immunolocalization and cell fractionation techniques in combination with siRNAi and expression of FLAG-tagged constructs, we have obtained evidence that γ-tubulin is also present in nucleoli of mammalian interphase cells of diverse cellular origins. Immunoelectron microscopy has revealed γ-tubulin localization outside fibrillar centers where transcription of ribosomal DNA takes place. γ-Tubulin was associated with nucleolar remnants after nuclear envelope breakdown and could be translocated to nucleoli during mitosis. Pretreatment of cells with leptomycin B did not affect the distribution of nuclear γ-tubulin, making it unlikely that rapid active transport via nuclear pores participates in the transport of γ-tubulin into the nucleus. This finding was confirmed by heterokaryon assay and time-lapse imaging of photoconvertible protein Dendra2 tagged to γ-tubulin. Immunoprecipitation from nuclear extracts combined with mass spectrometry revealed an association of γ-tubulin with tumor suppressor protein C53 located at multiple subcellular compartments including nucleoli. The notion of an interaction between γ-tubulin and C53 was corroborated by pull-down and co-immunoprecipitation experiments. Overexpression of γ-tubulin antagonized the inhibitory effect of C53 on DNA damage G(2) /M checkpoint activation. The combined results indicate that aside from its known role in microtubule nucleation, γ-tubulin may also have nuclear-specific function(s).

The Nuclear Localization of γ-Tubulin Is Regulated by SadB-mediated Phosphorylation

“…Finally, only Ser 385 -Cγ-tubGFP and Ala 385 -Cγ-tubGFP form tubular structures and associate with α-tubulin and GCP2. Altogether, the results reported here support the existence of different γ-tubulin conformational states that may aid γ-tubulin to bind structurally distinct proteins and in this way provide γ-tubulin with the observed functional properties (8, 10–14, 27). …”

“…To visualize the cellular localization of γ-tubulin during cell cycle (8, 10, 11), we performed immunofluorescence analysis with previously characterized antibodies (8, 9) of a synchronized cell population (Fig. 1, A and B ).…”

“…1, A and B ). Upon S phase entry, γ-tubulin accumulated in the nucleus (8, 10, 11) and remained nuclear throughout S phase (Fig. 1 A ).…”

“…In the nucleus, γ-tubulin regulates E2 promoter-binding factor (E2F) transcriptional activity (8–11), ensuring a transient transcription of genes necessary for centrosomal duplication and DNA-replication (8, 9). Moreover, nuclear γ-tubulin also forms complexes with Rad51 and C53 (10, 11). …”

Human TUBG2 gene is expressed as two splice variant mRNA and involved in cell growth

“…γ‐tubulin has been analyzed in various cells and tissues, but in previous studies, distinction was rarely made between γ‐tubulin1 and γ‐tubulin2 at a protein level, mainly due to the lack of the easily applicable method . In this study, we established the method by which human γ‐tubulin1 and γ‐tubulin2 can be separated into two bands on SDS‐PAGE and clearly distinguished from each other by western blotting.…”