Nuclear localization of γ‐tubulin affects E2F transcriptional activity and S‐phase progression

We show that the centrosome‐ and microtubule‐regulating protein γ‐tubulin interacts with E2 promoter binding factors (E2Fs) to modulate E2F transcriptional activity and thereby control cell cycle progression. γ‐Tubulin contains a C‐terminal signal that results in its translocation to the nucleus during late G1 to early S phase. γ‐Tubulin mutants showed that the C terminus interacts with the transcription factor E2F1 and that the E2F1–γ‐tubulin complex is formed during the G1/S transition, when E2F1 is transcriptionally active. Furthermore, E2F transcriptional activity is altered by reduced expression of γ‐tubulin or by complex formation between γ‐tubulin and E2F1, E2F2, or E2F3, but not E2F6. In addition, the γ‐tubulin C terminus encodes a DNA‐binding domain that interacts with E2F‐regulated promoters, resulting in γ‐tubulin‐mediated transient activation of E2Fs. Thus, we report a novel mechanism regulating the activity of E2Fs, which can help explain how these proteins affect cell cycle progression in mammalian cells.—Höög, G., Zarrizi, R., von Stedingk, K., Jonsson, K., Alvarado‐Kristensson, M. Nuclear localization of γ‐tubulin affects E2F transcriptional activity and S‐phase progression. FASEB J. 25, 3815–3827 (2011). www.fasebj.org

During cell division, the point of no return is when a cell commits to divide at the G 1 /S transition. At the molecular level, during G 1 /S, the CDK2-cyclin E complex hyperphosphorylates retinoblastoma protein (RB), which leads to the dissociation of E2 promoter binding factor (E2F) from RB and initiation of E2F transcriptional activity. This event marks the onset of cell division, as E2F initiates the transcription of a number of genes necessary for cell cycle progression. Mutations in this RB-E2Fdependent cell cycle checkpoint have been implicated in the achievement of growth factor independence in multiple malignancies (1).
␥-Tubulin is a ubiquitously expressed protein that regulates interphase ␣␤-tubulin nucleation (2), centrosomal duplication (3), and spindle formation (4). ␥-Tubulin occurs in the cytosol (5), centrosomes (3), and nucleus (6 -8); in the latter location it associates with Rad51 during recombination repair (6). However, neither the mechanism underlying nuclear localization of ␥-tubulin nor the nuclear functions of this protein have been elucidated. The present study detected a pool of ␥-tubulin that is actively translocated to the nucleus of U2OS and NIH3T3 cells, where it modulates E2F transcriptional activity and cell cycle progression.

Cell culture, transfection, and cell cycle analysis
Murine NIH3T3 embryonic fibroblasts and human U2OS osteosarcoma cells were cultured as reported elsewhere (3) and were transfected with various cDNAs using Lipofectamine Plus (Gibco BRL, Gaithersburg, MD, USA) and Jet Pei (Q-Biogene, Carlsbad, Ca, USA). To obtain equal protein levels of the various ectopically expressed proteins, the following DNA amounts were used in transfection and cotransfection experiments: 960 ng of ␥-tubulin-green fluorescent protein (␥tubGFP), N-␥-tubulin 1-333 , or C-␥-tubulin 334 -452 and/or 40 ng of hemagglutinin (HA)-E2F1, HA-E2F2, HA-E2F3, HA-E2F6, GFP, or HA-database of rice transcription factor polypeptide 1 (Dp1) in U2OS cells and 875 ng of ␥tubGFP in NIH3T3 cells was cotransfected with 188 ng of the various E2Fs or GFP. To achieve phase synchronization, cells were arrested in the G 0 phase (3) and released for different periods of time. Phase distribution was examined by using propidium iodide to measure cell DNA content, as described previously (3).

Immunoprecipitation and Western blot analysis
Using previously described methodology (16), cell extracts were obtained, the remaining supernatants were precleared, and immunoprecipitation was performed using anti-HA IgG2a monoclonal antibody (Santa Cruz Biotechnology) or anti-E2F1 or anti-␥-tubulin IgG1 monoclonal antibody. Western blotting was performed as reported elsewhere (17).

Microscopy
NIH3T3 or U2OS cells were cultured and fixed as described previously (3). Staining of endogenous ␥-tubulin was performed with the following antibodies: rabbit anti-␥-tubulin T3320 or T5192 or mouse anti-␥-tubulin GTU-88. Fluorescence images were captured and processed using an Olympus Bx 51 microscope (Olympus, Tokyo, Japan). A minimum of 100 cells were examined in each sample.

Luciferase assays
U2OS cells were transfected with 170 ng of the desired luciferase reporter construct, 1 ng of PRL-CMV-Renilla luciferase reporter construct (Promega, Madison, WI, USA), 20 ng of an E2F construct, and 200 ng of a ␥-tubulin construct. If the total amount of plasmid DNA did not reach 400 ng, pCDNA 3.1 or pEGFP empty vectors were added, and an equal amount of plasmid DNA was used in the transfections. At 1 d after transfection, cells were harvested for determination of luciferase and Renilla activity. Double determination of luciferase activity was achieved using a dual-luciferase reporter assay system (Promega).

Gene expression analysis
Total RNA from transfected U2OS cells was extracted as previous described (3), followed by a cleaning step using the RNeasy Mini Kit (Qiagen). mRNA expression array analysis was performed using the human Illumina platform. The gene set enrichment analysis (GSEA) program (http://www. broadinstitute.org/gsea/) was used to generate a ranked gene list according to differential expression between control-shRNA-and ␥-TUBULIN-shRNA-transfected samples. Genes were ranked in descending order with regard to level of expression in the ␥-TUBULIN-shRNA-transfected cells. GSEA (19) was performed on the generated ranked gene list using an E2F1-upregulated gene set generated from the publically available data set (accession number GSE1562; http://www.ncbi.nlm.nih.gov/geo/) consisting of control (LacZ) and exogenous E2F1 expressing melanoma SK-MEL-2 cells (20). E2F1-upregulated genes were defined as all genes displaying Ն5-fold higher expression in the E2F1-expressing cells compared to control ( Table 1).
To examine ␥-tubulin-correlated gene expression in tumor material, two publically available datasets were examined including 315 breast tumors, 121 lung tumors [National Center for Biotechnology (NCBI) Gene Expression Omnibus (GEO) accession number GSE2109] and 290 colon tumors (GEO accession number GSE14333). For each tumor type, all genes were ranked according to correlation to ␥-tubulin expression. The top 100 correlated genes to ␥-tubulin in each case were used to perform a Gene Ontology (GO) analysis, using the Functional Annotation tool in the online Database for Annotation, Visualization and Integrated Discovery (DAVID; refs. 21,22).

Electrophoretic mobility shift assay (EMSA)
The double-stranded oligonucleotides used for EMSA were derived from the dihydrofolate reductase promoter, an E2F1 All genes displaying a Ն5-fold higher expression in E2F1 overexpressing melanoma SK-MEL-2 cells compared to control SK-MEL-2 cells (data set NCBI GEO accession number GSE1562). Genes are displayed in order of most positively enriched (genes  to the most negatively enriched (genes 37-77) in the ␥-Tubulin-shRNA-transfected cells. HOXD8 (gene 36) displayed no enrichment. consensus site, and the sequences were as follows (23): nonmutated 5ЈAGCTTCTAGTGCAATTTCGCGCCAAACTTGG3Ј and 5ЈAATTCCAAGTTTGGCGCGAAATTGCACTAGA3Ј (E2F site underscored); mutated 5ЈAGCTTCTAGTGCAATTGCTCGAC-CAACTTGG3Ј and 5ЈAATTCCAAGTTGGTCGAGCAATTG-CACTAGA3Ј (mutated bases underscored). The oligos were aligned and subcloned into HindIII/EcoRI sites of pcDNA3.1.

Microtubule regrowth assay
Cells were treated and analyzed as described previously (3).

Statistical evaluation
All data are expressed as means Ϯ sd, and Student's paired t test was used to analyze the differences. Cell cycle profiles were assessed using FlowJo (Tree Star, Inc., Ashland, OR, USA). For gene expression array analyses, correlated ranked gene lists were generated based on Pearson's product moment correlation coefficient.
Relative P values and 95% confidence intervals were also calculated for the Pearson's correlations. GO analyses performed on the generated ranked gene lists displayed overall cluster enrichment score (log of geometric mean of EASE scores of all GEO terms included in the functional cluster) and both EASE score (modified Fisher exact test ; ref. 25) P values for GEO term enrichment and Bonferoni corrected P values to account for multiple testing. GSEA displayed the enrichment score and corresponding nominal P value representing the significance of enrichment of the single tested gene set (E2F1 up-regulated; ref. 19).

During G 1 /S transition, ␥-tubulin localizes to chromatin and interacts with E2F1
Immunofluorescence analysis using 3 different anti-␥-tubulin antibodies showed a constitutive localization of endogenous ␥-tubulin to the nucleus of NIH3T3 and U2OS cells (6)(7)(8). In addition, the centrosomic, nuclear, and cytosolic immunofluorescence-signal decreased with selective shRNA (Fig. 1A). Analysis of biochemical fractionations showed that part of the endogenous ␥-tubulin was found in the chromatin fraction (ref. 9 and Fig. 1B), and a densitometric analysis of the different fractions showed that 29.3 Ϯ 2.6 and 25.8 Ϯ 2.6% (nϭ3) of the total amount of endogenous ␥-tubulin was present in the chromatin of U2OS and NIH3T3, respectively. To determine variations in localization during the cell cycle, we investigated distribution of endogenous ␥-tubulin in synchronized NIH3T3 cells (3). This revealed accumulation in the chromatin fraction of cells in late G 1 and early S phase (6), in the latter reaching a maximum at 16 h ( Fig. 1C and Supplemental Fig. S1A). Translocation of ␥-tubulin to the chromatin coincided with a rise in E2F1 protein levels and an increase in hyperphosphorylated RB ( Fig. 1C and Supplemental Fig. S1A), events that mark the onset of cell division (1). Analysis of endogenous ␥-tubulin immunoprecipitates disclosed a cell cycle-dependent association of ␥-tubulin with endogenous E2F1, but not with cyclin E, Dp1, or RB (Fig. 1D). By comparison to other ␥-tubulin-interacting proteins, such as GCP2 (2), the E2F1-␥-tubulin complex appeared transiently at G 1 /S entry and in early S phase (Fig.  1D). Additional characterization showed that levels of E2F1-␥-tubulin complex decreased by shRNAi-induced reduction of either E2F1 or ␥-tubulin (Fig. 1E) and increased when U2OS cells were aphidicolin arrested (3,6) in early S phase (Fig. 1E). Next, we investigated the cellular localization of the E2F1-␥-tubulin by immunoprecipitating endogenous ␥-tubulin from fractions of U2OS and NIH3T3 cells. In comparison with the microtubule associated GCP2-␥-tubu-lin complex, E2F1-␥-tubulin occurred mainly in the chromatin fraction, suggesting that ␥-tubulin may modulate the nuclear transcriptional activity of E2F1 (Fig. 1F). Furthermore, E2F heterodimerization partner Dp1 was found associated with E2F1 immunoprecipitates but not with ␥-tubulin immunoprecipitates (Fig. 1D, E). These findings support a specific nuclear association between ␥-tubulin and E2F1 during early S phase.
To rule out nonspecific transcriptional variations that could cause the observed effect of ␥-tubulin on E2F activity, we studied variations in the protein levels of E2F. In the studied cells, the protein levels of endogenous E2F or ectopically expressed E2F varied, dependent on the ␥-tubulin protein levels. Unexpectedly, increased protein levels of E2Fs were detected when ␥-tubulin was coexpressed with E2F1, E2F1 ⌬193-284 , E2F2, and E2F3, but not E2F6 (Fig. 4B) or E2F1 ⌬193-359 (Fig. 4C), suggesting that ␥-tubulin protected E2F1, E2F1 ⌬193-284 , E2F2, and E2F3 from degradation. Accordingly, ␥-TUBULIN-shRNA-transfected cells showed , or C-␥tubGFP 334 -452 (C-␥TubGFP). Nuclei were detected using DAPI (blue). Scale bars ϭ 10 m. Bottom panels: cells (0.5ϫ10 6 ) were analyzed biochemically, as summarized in Fig. 1A, and examined by WB (nϭ3-6). C) U2OS cells expressing the various GFP-␥-tubulin constructs, as indicated, were separately immunoprecipitated with first anti-E2F1 (top panels), and then the same lysate was immunoprecipitated a second time with anti-GFP antibodies (bottom panels) and analyzed by WB. Part of the lysate used for the immunoprecipitations was run as loading control (total lys.). Cellulose membranes containing immunoprecipitated E2F1 were first analyzed by WB with an anti-GFP antibody and reprobed with an anti-E2F1. Arrowheads indicate the various GFPfused proteins. D) Purified full-length GST-␥-tubulin was incubated with His-E2F1, and the GST-␥-tubulin was subsequently retrieved by adsorption to glutathione-Sepharose 4B and examined by WB (nϭ3). Total amounts of loaded His-E2F1 were analyzed by WB (input). decreased E2F1, E2F2, and E2F3 levels (Fig. 4D), but the protective effect of ␥-tubulin on E2Fs was impaired in the presence of Dp1 (Fig. 3B and data not shown). Nonetheless, coexpression of Dp1 with E2Fs induced an increase in expression of E2Fs ( Fig. 4E and data not shown). Interestingly, the E2F ␥D (residues 193-359) encloses the DD (residues 195-284), which suggests that Dp1 and ␥-tubulin compete for the same E2F-interacting site and that the interaction of ␥-tubulin with the DD of E2F protects E2F from degradation. These results indicate the existence of two different E2F complexes: an active complex form of Dp1-E2F and a nonactive complex composed of ␥-tubulin-E2F. Fig. 4F); consequently, we analyzed the effects of ␥-tubulin on expression of an RBD-deficient E2F1 mutant (HA-E2F1 ⌬409 -426 ). Indeed, the absence of RBD did not influence the ␥-tubulin-induced increase in protein levels of HA-E2F1 ⌬409-426 ; instead, HA-E2F1 ⌬409 -426 was rapidly degraded (Fig. 4G). To ascertain that the effect on E2F levels was dependent on the C terminus of ␥-tubulin, we coexpressed HA-E2F1 or HA-E2F1 ⌬409 -426 with ␥-tubulin mutants and found that the ␥-tubulin mutants did not affect HA-E2F1 or HA-E2F1 ⌬409 -426 expression (Fig. 4H). Considering these findings, we concluded that ␥-tubulin's regulatory effect on E2Fs protein levels depends on the ␥-tubulin C terminus in a RB-binding-independent manner.

␥-Tubulin binds to DNA on the same DNA-binding motif as E2F
To determine whether ␥-tubulin, similar to Dp1 (1), binds to the E2F DNA binding site, we performed an electrophoretic mobility shift assay (EMSA; ref. 31). Surprisingly, GST-␥tub and GST-C-␥tub 334 -452 , but not GST-N-␥tub 1-333 , displayed binding to the radioactively labeled probe representing the E2F binding site, which was inhibited by an unlabeled competing wild-type E2F binding site (Fig. 5A). In addition, incubating His-E2F1 with GST-␥tub increased the DNA-binding activity of GST-␥tub relative to the amount of bound probe detected when the different components were incubated separately (Fig. 5B). To study ␥-tubulin's ability to bind to E2Fregulated promoters, a ChIP assay using ␥-tubulin antibodies was performed (24). Endogenous ␥-tubulin was present on the cyclin E promoter in aphidicolin-treated U2OS cells, and sequential ChIP assay (E2F ChIP followed by ␥-tubulin re-ChIP or vice versa) showed that endogenous E2F1 and E2F2, but not Dp1, colocalized with endogenous ␥-tubulin on cyclin E promoter (Fig.  5C). These results indicated that the C terminus of ␥-tubulin may interact directly with the ␥D of E2Fs and thereby negatively modulate E2F transcriptional activity. All the above-mentioned experiments suggested that ␥-tubulin and Dp1 compete for the same binding site on E2Fs. To confirm the hypothesis, we analyzed the Dp1 content on E2F1 immunoprecipitates from cells expressing variable protein levels of ␥-tubulin (Fig. 5D). We detected that lower ␥-tubulin levels were associated with higher levels of Dp1 in E2F1 immunoprecipitates (Fig. 5E). This result revealed that the mechanism by which ␥-tubulin moderates E2F activity is by competing out Dp1 in the E2F-Dp1 complex.

␥-Tubulin levels affect cell cycle progression and the expression of E2F-regulated genes
To further confirm the effect of ␥-tubulin on E2Fs, we altered ␥-tubulin levels in cells expressing HA-E2F1 in a synchronized cell population. Increased ␥-tubulin levels impaired the S-phase entry effect caused by HA-E2F1 by negatively regulating the expression of E2F transcriptional targets, such as cyclin E expression (Fig. 6A). Moreover, alterations of the ␥-tubulin levels during cell cycle in U2OS cells expressing GFP, ␥tubGFP, ␥TUBULIN-shRNA, and NLS-␥tubGFP mutants showed that a larger number of control cells expressing GFP or ␥tubGFP were in S phase compared to the cells with various ␥tubGFP mutants (Supplemental Fig. S2A). In a synchronized cell population, S-phase progression was delayed in NIH3T3 cells transfected with ␥Tubulin-shRNA or any of the NLS-␥tubGFP mutants, suggesting that ␥-tubulin levels and localization determine optimal cell cycle progression (Fig. 6B and Supplemental Fig. S2B).
Intriguingly, low expression or mutations of ␥-tubulin elevated E2F activity but delayed S-phase entry. To further elucidate the effect of reduced ␥-tubulin levels on E2F activity, an mRNA expression array was performed on control shRNA-and ␥-TUBULIN-shRNA-transfected cells, and the effect of ␥-TUBULIN reduction on an E2F1 target gene signature was examined (Table 1). GSEA revealed that on reduced mRNA expression levels of ␥-TUBULIN, a significant enrichment of E2F1 positively regulated target genes was found (enrichment score 0.34, nominal Pϭ0.026; Fig. 6C). It can also be noted that RB lay among the most positively enriched E2F1 targets (Table 1). In accordance with this, we found increased protein levels of RB in ␥TUBULIN-shRNAand ␥Tubulin-shRNA-transfected cells (Fig. 6B, D), which provides a potential explanation for the observed delay in S-phase entry (Fig. 6B). These findings further support the moderating effect of ␥-tubulin on E2F activities, as the increase in RB levels concurred with a decrease in levels of phosphorylated RB.

High expression of ␥-tubulin correlates with high expression of E2F1 and cell cycle-associated gene signatures in various tumor types
To identify underlying biological processes associated with ␥-tubulin expression in various tumor types, we made use of publically available expression array datasets, including 351 breast cancers, 121 lung cancers (NCBI GEO accession number GSE2109), and 290 colon cancers (NCBI GEO GSE14333; ref. 32). For each tumor type, all genes were ranked according to correlation to ␥-tubulin expression. As expected from our results, ␥-tubulin expression was significantly positively correlated with E2F1 expression in all threetumor types (Fig. 7). In addition, GO analysis was performed using the top 100 positively correlated genes to ␥-tubulin expression. In each tumor type, ␥-tubulincorrelated gene expression displayed highly significant enrichment of cell cycle-related processes (Supplemental Tables S1 and S2). The fact that high levels of ␥-tubulin coincide with high levels of cell cycle-regulated genes supports the hypothesis of ␥-tubulin acting as a cell cycle regulator and also suggests that ␥-tubulin may be involved in cell cycle regulation in multiple malignancies.

NLS-␥-tubulin mutants do not affect centrosome or ␣␤-tubulin dynamics and E2F1 does not associate with ␥-tubulin phosphorylated on Ser131
To establish that the observed effect of ␥tubGFP mutants on E2F activities and cell cycle was not caused by defective centrosomes or altered ␣␤-tubulin dynamics, we examined the effect of ␥tubGFP-R399A-K400A-R409A on centrosome and ␣␤-tubulin regulation. Ectopic expression of ␥tubGFP-R399A-K400A-K409A in U2OS cells did not influence the number of centrosomes or mitotic spindles formed or altered interphase microtubules or astral microtubule regrowth (Fig. 8A,  B). This finding suggests that the effects of ␥tubGFP mutants on cell cycle progression and E2F activity are not due to defective centrosomes or altered ␣␤-tubulin dynamics.
We have previously shown that ␥-tubulin is phosphorylated on Ser131 (pSer131), and pSer131 enhances microtubule polymerization in the centriole (3). Thus, in search for the mechanism that regulates nuclear translocation of ␥-tubulin, we examined the amount of . Extracts from synchronous NIH3T3 cells transfected with E2F1 and GFP or E2F1 and ␥tubGFP were prepared as in Fig. 1A and examined by WB using the indicated antibodies (nϭ4). Arrowheads indicate various GFP-fused proteins. B) Cell cycle distribution of synchronous control shRNA-or ␥-Tubulin-shRNA-transfected NIH3T3 cells. Cells were synchronized in G 0 as in A and released for different time periods, and DNA content of the samples was determined by flow cytometry. Percentage of S-phase cells is indicated in each panel (nϭ3). Synchronous control shRNA-or ␥-Tubulin-shRNA-transfected NIH3T3 cells were analyzed using the indicated antibodies (nϭ3). Total lysate was run in parallel as control for ␥-tubulin levels and as loading control (indicated). Asterisks indicate lanes with variation in protein loading (nϭ3). C) GSEA of E2F1-upregulated gene set performed on a ranked gene list of differentially expressed genes between ␥-TUBULIN-shRNA-transfected (nϭ4) and control-shRNA-transfected (nϭ5) cells. Genes were ranked in descending order with regard to level of expression in ␥-TUBULIN-shRNA-transfected cells. (enrichment score 0.34, nominal Pϭ0.026). D) U2OS cells expressing indicated constructs were used for immunoprecipitation of RB with anti-RB or control (C) antibody. Total lysate was run as loading control (bottom panels; nϭ3).

DISCUSSION
We describe an NLS that mediates ␥-tubulin translocation to the nucleus during S phase (6 -8). In the nucleus, ␥-tubulin governs E2F1, E2F2, and E2F3 transcriptional activities. We found that decreased ␥-tubulin levels or expression of NLS-␥-tubulin mutants increase E2F transcriptional activity, while elevated ␥-tubulin levels reduce  A) Tubulin in transfected cells was stained using anti-␥-tubulin (␥Tub; red) or anti-␣-tubulin antibody (red), as indicated; nuclei were detected with DAPI (blue). B) ␣-Tubulin (red) in U2OS cells stained immediately following cold treatment (0 min) or 2 min after adding warm medium. Scale bars ϭ 10 m. C, D) ␥-Tubulin phosphorylated on Ser131 does not associate with E2F1. Synchronous NIH3T3 cells (2.10 6 /time point) were lysed and divided into 2 samples. C) In one sample, ␥-tubulin was immunoprecipitated from extracts using ␥Tub, then detected by Western blotting (WB) using anti-E2F1 antibody, and finally stripped and reprobed with ␥Tub (bottom panel), as described in Fig. 1A. D) In the second sample, endogenous pSer131-␥-tubulin was immunoprecipitated with anti-pSer131-␥-tubulin antibody, subsequently developed by WB using anti-E2F1 antibody, and then stripped and reprobed with ␥Tub (bottom blot panel). Bottom panels illustrate DNA content of the cells detected by flow cytometry (percentage of S-phase cells indicated, nϭ3). E2F activity. Furthermore, the protein levels of recombinant E2F1, E2F2, and E2F3 vary depending on the ␥-tubulin and Dp1 expression. Low E2F protein levels were found in cells with reduced ␥-tubulin levels, despite the high measured E2F transcriptional activities. Increased expression of ␥-tubulin caused high protein levels of E2F, but the transcriptional activity of E2F was low. Also, we found that high expression levels of Dp1 increased the total levels of E2Fs; considering that low protein levels of ␥-tubulin decreased E2Fs expression, this finding suggest that the transient increase of E2Fs during G 1 and S phase could be caused by the dynamic formation of the Dp1-and ␥-tubulin-E2F complexes.
␥TUBULIN-shRNA induced an increase in E2F transcriptional activity and caused an E2F-dependent increase in the protein levels of RB. The RB signal transduction pathways can trigger a G 1 arrest (33,34); consequently, an increase in RB may be responsible for causing the observed S-phase entry delay in cells with reduced ␥-tubulin protein levels. In G 1 /S entry, ␥-tubulin binds to cyclin E promoter to ensure a transient expression of cyclin E, and, in doing so, ␥-tubulin coordinates the cell cycle. In addition, the chromatin-associated pool of ␥-tubulin was not bound to microtubules or GCP2-containing fractions, suggesting that ␥-tubulin has centrosome-independent functions. Altogether, these observations indicate that ␥-tubulin plays a role in the regulation of E2F transcriptional activity during the cell cycle.
Our finding of an E2F-mediated increase of RB in response to reduced ␥-tubulin levels implicates ␥-tubulin as a regulator in the G 1 /S phase transition. In line with this view, the presence or absence of centrosomes promotes either proliferation or a G 1 arrest (35). The mechanism by which centrosomes regulate the G 1 /S transition is unclear, but it remains possible that the triggering signal that translocates ␥-tubulin to the nucleus may originate at the centrosomes. Once centrosome duplication is initiated and cyclin E activity is no longer needed, the translocation of ␥-tubulin to the nucleus will inhibit the transcription of cyclin E, and in doing so, will prevent a cyclin E-dependent reduplication of the centrosomes (36). In this way, a timely regulation of cell cycle progression can be achieved.
Alteration in the RB-E2F signal transduction pathways are connected to cancer development in various tumors types, as uncontrolled E2F signaling may trigger cell division (1). Consequently, in these tumors, uncontrolled cell division coincides with expression of cell cycle-regulated genes, which should also include ␥-tubulin. To test this possibility, we examined ␥-tubulin-correlated gene expression in tumor material and found that ␥-tubulin displayed significant positive correlation to E2F1 expression, as well as significant enrichment of cell cycle-associated processes on GO analysis. These trends did not seem to be tumor type specific, as similar patterns were observed in multiple tumor types, including breast, lung, and colon cancers. This may suggest a role for ␥-tubulin in cell cycle regulation in not only normal cells but also in malignancies, however further investigations are required.
In summary, the mechanism that allows ␥-tubulin to moderate E2F transcriptional activity involves direct binding to E2Fs. This, in turn, moderates E2F transcriptional activity during S-phase entry. In the absence of ␥-tubulin, increased E2F activity elevates E2F-mediated expression of RB and delays S-phase entry (Fig. 9A). At the G 1 /S phase transition, RB releases E2Fs, which may induce E2F-␥-tubulin complex formation, ensuring a transient transcription of genes necessary for S-phase entry (Fig. 9B)  . ␥-Tubulin protein levels regulate E2F transcriptional activity. A) In the absence of ␥-tubulin, at G 1 /S phase transition, the released E2F-Dp1 complex transcribes RB. B) In the presence of ␥-tubulin, at G 1 /S transition, the released E2F-Dp1 complex transcribes gene products necessary for S-phase entry, such as cyclin E. As nuclear ␥-tubulin levels increase, the E2F-␥-tubulin complex forms and puts an end to the transcriptional activity of E2Fs.