Translationally controlled tumor protein: Biological Overview | References
Gene name - Translationally controlled tumor protein
Cytological map position - 86D8-86D8
Function - signaling
Keywords - control of growth, Rheb GEF, TSC signaling pathway
Symbol - Tctp
FlyBase ID: FBgn0037874
Genetic map position - 3R: 7,035,975..7,037,006 [+]
Classification - Translationally controlled tumour protein
Cellular location - cytoplasmic
|Recent literature||Le, T.P., Vuong, L.T., Kim, A.R., Hsu, Y.C. and Choi, K.W. (2016). 14-3-3 proteins regulate Tctp-Rheb interaction for organ growth in Drosophila. Nat Commun 7: 11501. PubMed ID: 27151460
14-3-3 family proteins regulate multiple signalling pathways. Understanding biological functions of 14-3-3 proteins has been limited by the functional redundancy of conserved isotypes. This study provides evidence that 14-3-3 proteins regulate two interacting components of Tor signalling in Drosophila, translationally controlled tumour protein (Tctp) and Rheb GTPase. Single knockdown of 14-3-3ɛ or 14-3-3ζ isoform does not show obvious defects in organ development but causes synergistic genetic interaction with Tctp and Rheb to impair tissue growth. 14-3-3 proteins physically interact with Tctp and Rheb. Knockdown of both 14-3-3 isoforms abolishes the binding between Tctp and Rheb, disrupting organ development. Depletion of 14-3-3s also reduces the level of phosphorylated S6 kinase, phosphorylated Thor/4E-BP and cyclin E (CycE). Growth defects from knockdown of 14-3-3 and Tctp are suppressed by CycE overexpression. This study suggests a novel mechanism of Tor regulation mediated by 14-3-3 interaction with Tctp and Rheb.
|Hong, S.T. and Choi, K.W. (2016). Antagonistic roles of Drosophila Tctp and Brahma in chromatin remodelling and stabilizing repeated sequences. Nat Commun 7: 12988. PubMed ID: 27687497
Genome stability is essential for all organisms. Translationally controlled tumour protein (TCTP) is a conserved protein associated with cancers. TCTP is involved in multiple intracellular functions, but its role in transcription and genome stability is poorly understood. This study demonstrates new functions of Drosophila TCTP (Tctp) in transcription and the stability of repeated sequences (rDNA and pericentromeric heterochromatin). Tctp binds Brahma (Brm) chromatin remodeler to negatively modulate its activity. Tctp mutants show abnormally high levels of transcription in a large set of genes and transposons. These defects are ameliorated by brm mutations. Furthermore, Tctp promotes the stability of repeated sequences by opposing the Brm function. Additional regulation of pericentromeric heterochromatin by Tctp is mediated by su(var)3-9 transcriptional regulation. Altogether, Tctp regulates transcription and the stability of repeated sequences by antagonizing excess Brm activity. This study provides insights into broader nuclear TCTP functions for the maintenance of genome stability.
Cellular growth and proliferation are coordinated during organogenesis. Misregulation of these processes leads to pathological conditions such as cancer. Tuberous sclerosis (TSC) is a benign tumour syndrome caused by mutations in either TSC1 or TSC2 tumour suppressor genes. Studies in Drosophila and other organisms have identified TSC signalling as a conserved pathway for growth control. Activation of the TSC pathway is mediated by Rheb (Ras homologue enriched in brain), a Ras superfamily GTPase. Rheb is a direct target of TSC2 (Gigas in Drosophila) and is negatively regulated by its GTPase-activating protein activity. However, molecules required for positive regulation of Rheb have not been identified. This study shows that a conserved protein, translationally controlled tumour protein (TCTP), is an essential new component of the TSC-Rheb pathway. Reducing Drosophila TCTP (dTCTP) levels reduces cell size, cell number and organ size, which mimics Drosophila Rheb (dRheb) mutant phenotypes. dTCTP is genetically epistatic to Tsc1 and dRheb, but acts upstream of dS6k, a downstream target of dRheb. dTCTP directly associates with dRheb and displays guanine nucleotide exchange activity with it in vivo and in vitro. Human TCTP (hTCTP) shows similar biochemical properties compared to dTCTP and can rescue dTCTP mutant phenotypes, suggesting that the function of TCTP in the TSC pathway is evolutionarily conserved. These studies identify TCTP as a direct regulator of Rheb and a potential therapeutic target for TSC disease (Hsu, 2007).
TCTP is a highly conserved protein upregulated in various tumours. Despite studies on the biochemical and structural properties of TCTP (Yarm, 2002; Liu, 2005; Yang, 2005; Thaw, 2001), the physiological significance of these findings has not been determined. Thus, the function of TCTP in vivo was studied using Drosophila as a model organism (Hsu, 2007).
First dTCTP expression was knocked down in developing flies by targeted expression of double-stranded RNA (dsRNA) for RNA interference (RNAi). Expression of dTCTP RNAi depleted endogenous dTCTP to nearly undetectable levels by different GAL4 drivers. Tissue-specific expression of dTCTP RNAi reduced the size of the eye, wing, notum, or a specific region in the wing pouch, corresponding to the expression domains of various GAL4 lines. The size reduction was caused by a decrease in both cell size and cell number, a typical phenotype for mutations in the insulin or TSC pathways. Ubiquitous expression of UAS-dTCTP RNAi by actin-GAL4 (act>dTCTPi) caused lethality around the third instar larval stage. A portion of these larvae was able to survive to the pupal stage with reduced body size, consistent with the organ size reduction. The lethality and phenotypes caused by dTCTP RNAi were rescued by co-expression of a dTCTP complementary DNA, indicating that these defects were due to a reduction of dTCTP levels. It is therefore concluded that dTCTP RNAi suppresses organ growth by affecting both cell size and number (Hsu, 2007).
The phenotypes of dTCTP loss-of-function mutants were further investigated because RNAi may represent a hypomorphic situation. dTCTPEy09182 is a hypomorphic allele of dTCTP resulting from a P-element insertion in its 5' untranslated region. Rare homozygous dTCTPEy09182 flies that escaped from lethality showed smaller body size compared with their heterozygous siblings. To create null alleles, imprecise excisions were generated from dTCTPEy09182. One imprecise excision line, dTCTPh59, showed a 1.1-kilobase deletion downstream of the insertion site that removes the entire dTCTP coding sequence. Western blot analysis showed no detectable dTCTP protein in dTCTPh59 early first instar larvae. Both dTCTPh59 homozygotes and dTCTPh59 heterozygotes for a deficiency chromosome uncovering the dTCTP region (dTCTPh59/Df(3R)M-Kx1) showed 100% lethality at the late first instar stage, indicating that this allele is a genetic null. Expression of dTCTP cDNA or a genomic DNA construct was able to rescue dTCTPh59 mutants, indicating that the lethality is due to deletion of the dTCTP gene (Hsu, 2007).
To examine the phenotypes of dTCTP null mutant cells, dTCTP mutant clones were generated using mitotic recombination. dTCTPh59 mutant clones showed growth disadvantage compared to their wild-type twin spots. The sizes of dTCTPh59 clones were similar to those of the twin spots 24 h after heat shock. However, the sizes of the twin spots were much larger than dTCTPh59 clones 48 h after heat shock, and most dTCTPh59 clones were eliminated by 60 h after heat shock. Similarly, using the EGUF/Hid technique (Stowers, 1999) to remove most wild-type cells in dTCTPh59 mosaic eyes resulted in either a no-eye or small-eye phenotype. Therefore, the null phenotypes were qualitatively comparable to the dTCTP RNAi phenotype, but more severe (Hsu, 2007).
The reduction in cell number caused by dTCTP RNAi and the behaviour of dTCTP mutant cells may result from a proliferation defect or abnormal cell death. These possibilities were tested using the MARCM (mosaic analysis with a repressible cell marker) technique. Similar to clones generated by traditional mitotic recombination, numerous small dTCTPh59 green-fluorescent-protein-expressing (GFP+) clones were observed at 24 h after heat shock. By 72 h after heat shock, very few GFP+ clones remained on the discs. In contrast, Cyclin E (CycE) overexpression, via the MARCM technique, within dTCTPh59 clones resulted in four times more dTCTPh59 cells at 72 h after heat shock. Similarly, CycE overexpression also suppressed the dTCTP RNAi phenotypes. Next whether the dTCTPh59 phenotypes can be attributed to abnormal cell death was tested. Expression of the P35 cell death inhibitor also significantly suppressed the dTCTPh59 phenotypes, leading to the presence of four times more GFP+ cells at 72 h after heat shock. These data indicate that loss of dTCTP causes defects in cell proliferation and triggers cell death (Hsu, 2007).
Insulin and TSC signalling are two parallel but interacting pathways for growth control. Inactivation of positive regulatory components, such as Insulin receptor (InR), dRheb and Tor, leads to a decrease in organ size by affecting cell size and cell number. In contrast, overexpression of InR and dRheb, as well as inactivation of negative regulatory components such as Tsc1 and Pten, causes tissue overgrowth. Given the similar phenotypes between dTCTP mutants and mutants in the insulin/TSC pathways, genetic epistasis experiments were performed to test whether dTCTP has a role in these two pathways. Overexpression of InR by patched (ptc)-GAL4 caused weak but consistent expansion of the distance between L3 and L4 wing veins. In contrast, co-expression of InR and dTCTP RNAi reduced the L3-L4 distance, resembling the dTCTP RNAi phenotype. Therefore, dTCTP is epistatic to (acts downstream of) InR (Hsu, 2007).
Next, the relationship between Tsc1 and dTCTP was examined. Mutations in Tsc1 or Tsc2 cause similar phenotypes because they function as a complex. Mosaic eyes and heads consisting primarily of Tsc1 mutant cells were much larger than wild type. Expression of dTCTP RNAi in Tsc1 mutant cells suppressed this overgrowth both in the eye and head. Furthermore, when the eye was composed of Tsc1 and dTCTP double mutant cells, the flies displayed a small or no-eye phenotype indistinguishable from the dTCTP single mutant phenotype, suggesting that dTCTP acts downstream or in parallel to Tsc1 (Hsu, 2007).
The relationship between dTCTP and dRheb was tested. Ectopic expression of dRheb using ptc-GAL4 resulted in a 15% increase in the L3-L4 distance compared with the ptc>GFP control. However, co-expression of dTCTP RNAi and dRheb in the ptc expression region showed the dTCTP RNAi phenotype, suggesting that dTCTP is epistatic to dRheb (Hsu, 2007).
Finally, whether activation of dS6k, a downstream effector of the insulin/TSC pathway, is dependent on dTCTP was tested. The level of dS6k activation was measured using a phospho-specific antibody (dS6k p-Thr 398). Extracts from act>dTCTPi larvae showed a significantly lower amount of activated dS6k compared with the controls, indicating that dTCTP is required for dS6k activation. Consistent with this, the eyeless (ey)>dTCTPi phenotype was dominantly enhanced by heterozygosity for a null mutation of dS6k (dS6kl-1). Removing a copy of dS6k caused an approximately 20% further reduction of eye size in ey>dTCTPi animals. Taken together, these data support a model wherein dTCTP functions either downstream or in parallel to Tsc and dRheb, but upstream of dS6k (Hsu, 2007).
Epistatic analysis suggests that dTCTP may be a new component in the TSC pathway. Because TCTP structurally resembles a small GTPase regulator, Mss4 (Thaw, 2001), it is proposed that dTCTP might directly associate with dRheb and positively regulate its activity. To test this, co-immunoprecipitation experiments were performed. Flag-tagged dTCTP immunoprecipitated together with Myc-tagged dRheb in 293T cell extracts, suggesting that dTCTP and dRheb form a complex in vivo. Furthermore, in vitro pull-down assays demonstrated direct binding of glutathione S-transferase (GST)-dTCTP to maltose binding protein (MBP)-dRheb. Notably, dTCTP showed preferential binding to nucleotide-free dRheb, a property shared among guanine nucleotide exchange factors (GEFs). To test whether dTCTP has GEF activity for dRheb, in vitro GDP release experiments were performed. MBP-dRheb alone showed weak intrinsic GDP dissociation. In contrast, addition of GST-dTCTP stimulated the GTP/GDP exchange on dRheb rapidly even when low amounts of dTCTP were used. The GEF-like activity is specific, as dTCTP did not accelerate the exchange reaction on dRas1, the closest GTPase to dRheb at sequence level. Moreover, a glutamic acid to valine mutation in the putative GTPase binding groove of dTCTP (dTCTPE12V) abolished this GEF activity, even at a high concentration. Because dTCTPE12V still retained binding activity to dRheb, this residue seems to have a critical function in catalytic reactions rather than binding between the two proteins. To determine whether E12 is critical for the function of dTCTP in vivo, genetic rescue experiments were performed. Whereas wild-type dTCTP was able to rescue fully the dTCTP RNAi phenotype, mutant dTCTPE12V failed to rescue the RNAi defects even though the mutant protein was expressed at a high level. Therefore, the conserved E12 residue of dTCTP is essential for its normal function in development (Hsu, 2007).
To test whether this in vitro GEF activity has a physiological relevance, the in vivo level of GTP bound to dRheb was determined in Drosophila S2 cells. S2 cells treated with dTCTP dsRNA or a control EGFP dsRNA were transfected with Myc-tagged dRheb. dTCTP-dsRNA-treated cells consistently showed a lower percentage of GTP-bound dRheb, suggesting that dTCTP is required for dRheb activation in vivo. These GEF-like properties displayed by dTCTP towards dRheb are particularly intriguing, as no Rheb GEFs have been reported. Further kinetic and structural analysis will help to elucidate whether dTCTP is a bona fide GEF enzyme for dRheb (Hsu, 2007).
Human TCTP (hTCTP) and dTCTP are roughly 50% identical in their protein sequence. dTCTP RNAi and mutant phenotypes can be rescued by expression of hTCTP. Furthermore, hTCTP interacts most strongly with the nucleotide-free hRheb and stimulates the GDP/GTP exchange of hRheb in vitro. These data suggest that the function of TCTP in the TSC pathway is likely to be conserved throughout evolution (Hsu, 2007).
dTCTP controls cell growth and proliferation by positively regulating dRheb activity. The data suggest that dTCTP may function as a GEF or a related regulatory factor to activate dRheb. Given the strong epistatic effect of dTCTP to dRheb, it is also possible that dTCTP may have additional roles in the TSC pathway, acting downstream of dRheb but upstream of S6k (Hsu, 2007).
TCTP has been implicated in a wide range of cancers. Nevertheless, overgrowth phenotypes as a result of dTCTP overexpression were not observed, suggesting that dTCTP is not sufficient to induce growth. Notably, reduction of TCTP levels is sufficient for suppression of malignancy in tumour reversion models. This study provides a possible explanation for this phenomenon. It will be intriguing to learn whether lowering the insulin/TSC signalling output can be a general mechanism for tumour reversion (Hsu, 2007).
Search PubMed for articles about Drosophila Tctp
Hsu, Y.-C., Chern, J. J., Cai, Y., Liu, M. and Choi, K.-W. (2007). Drosophila TCTP is essential for growth and proliferation through regulation of dRheb GTPase. Nature 445: 785-788. PubMed ID: 17301792
Liu, H., Peng, H.-W., Cheng, Y.-S., Yuan, H. S. and Yang-Yen, H.-F. (2005). Stabilization and enhancement of the antiapoptotic activity of Mcl-1 by TCTP. Mol. Cell. Biol. 25: 3117-3126. PubMed ID: 15798198
Stowers, R. S. and Schwarz, T. L. (1999). A genetic method for generating Drosophila eyes composed exclusively of mitotic clones of a single genotype. Genetics 152: 1631-1639. PubMed ID: 10430588
Thaw, P. et al. (2001). Structure of TCTP reveals unexpected relationship with guanine nucleotide-free chaperones. Nature Struct. Biol. 8: 701-704. PubMed ID: 11473261
Yang, Y. et al. (2005). An N-terminal region of translationally controlled tumor protein is required for its antiapoptotic activity. Oncogene 24: 4778-4788. PubMed ID: 15870695
Yarm, F. R. (2002). Plk phosphorylation regulates the microtubule-stabilizing protein TCTP. Mol. Cell. Biol. 22: 6209-6221. PubMed ID: 12167714
date revised: 7 March 2009
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