![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Center for Neuronal Survival, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada
Received 5 April 2004/ Returned for modification 12 May 2004/ Accepted 30 June 2004
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Gro/TLEs are transcriptional corepressors that lack DNA-binding activity of their own but are instead recruited to specific gene regulatory sequences via interaction with a number of different DNA-binding transcription factors. These include a family of related basic-helix-loop-helix proteins encoded by the hairy and Enhancer of split (Hes) genes of Drosophila (25) and their related vertebrate counterparts (13, 20, 22). A number of other transcription factors act as DNA-binding partners of Gro/TLEs, including Runt homology domain proteins (18, 19, 32), homedomain factors containing the engrailed homology region 1 motif (16, 21), paired domain proteins (4, 10), Tcf/Lef-related HMG box factors (18, 27), and winged helix domain proteins (35). As a result of these interactions, Gro/TLEs become recruited to selected target genes in context-dependent manners. Upon their recruitment to DNA, they are believed to mediate transcriptional repression by at least two mechanisms. One is thought to involve interactions with both histones and histone deacetylases (HDACs) resulting in the modification of the histone acetylation state (6, 8, 24). The other is hypothesized to involve inhibitory interactions with components of the basal transcriptional machinery (36).
The mechanisms that regulate Gro/TLE activities during cell differentiation are poorly defined. Gro/TLE1 becomes increasingly phosphorylated as a function of neuronal differentiation (14, 22). A change to a hyperphosphorylated state was also observed after the interaction of Gro/TLE with Hes1, one of its transcriptional cofactors during neuronal development (22). These observations suggested that changes in Gro/TLE1 phosphorylation that occur in response to cofactor binding might play roles in the regulation of the neural functions of Gro/TLE. Here we provide the first demonstration that protein kinase CK2 phosphorylates Gro/TLE1 at S239 in vivo. Phosphorylation of S239 is critical for cofactor-activated phosphorylation (CAP) that Gro/TLE1 undergoes in response to Hes1 binding. We also provide evidence that mutation of S239 into alanine decreases both the nuclear association and the transcription repression activity of Gro/TLE1. Finally, we demonstrate that Gro/TLE1 inhibits the differentiation of cortical neural progenitor cells into neurons and that phosphorylation of S239 is required for this function. Together, these results demonstrate that Gro/TLE1 is a physiological substrate of CK2 and provide the first characterization of events involved in the regulation of Gro/TLE activity during neuronal differentiation.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1.6-kb fragment encoding the N-terminal region. This fragment was subcloned into pCMV2-FLAG digested with EcoRV. The remaining portion of the Gro/TLE1 cDNA was then subcloned as a SmaI restriction fragment. For analysis of Gro/TLE1 point mutants, the plasmids pCMV2-FLAG-Gro/TLE1(S239A), pCMV2-FLAG-Gro/TLE1(S253A), pCMV2-FLAG-Gro/TLE1(S239A/S253A), pCMV2-FLAG-Gro/TLE1(S239E), pCMV2-FLAG-Gro/TLE1(S253E), and pCMV2-FLAG-Gro/TLE1(S239E/S253E) were obtained by generating the sequences containing the appropriate mutations by PCR-based strategies (information on oligonucleotide primers is available upon request), followed by subcloning into pBluescript SK plasmid and DNA sequencing. The verified point-mutated products were then subcloned into pCMV2-FLAG-Gro/TLE1 digested with BstEII and HpaI, replacing the corresponding wild-type sequence. Plasmids pCMV2-FLAG-Gro/TLE1(
285-335), pGEX2-Gro/TLE1-Q, pGEX2-Gro/TLE1-GP, pGEX2-Gro/TLE1-CcN, pGEX1-Gro/TLE1-SP, and pGEX3-Gro/TLE3-WDR were described (23). Constructs pGEX2-Gro/TLE1(CcN-S239A), pGEX2-Gro/TLE1(CcN-S253A) and pGEX2-Gro/TLE1(CcN-S239A/S253A) were generated by PCR amplification of the region of interest from pCMV2-FLAG-Gro/TLE1(S239A), pCMV2-FLAG-Gro/TLE1(S253A) or pCMV2-FLAG-Gro/TLE1(S239A/S253A), respectively, followed by subcloning into pGEX2. Plasmids pEBG-Hes1, pEBG-Hes1(WRPW), pEGFP, pCMV2-FLAG-Hes1, pCMV5-Runx2, and p6OSE2-Luc were described (12, 19, 20). Antibody production. An 11-amino-acid peptide containing residues 235 to 245 of human Gro/TLE1 with S239 converted to phosphoserine was synthesized (SHYDpSDGDKSD; Biosource), conjugated to maleimide-coupled KLH (Pierce), and injected into rabbits by using Freund adjuvant. Sera were passed over an affinity column of the immunizing phosphopeptide coupled to Sulfolink (Pierce), and retained antibodies were eluted and concentrated in a Centriplus-10 (Amicon).
In vitro kinase assays. Purification of fusion proteins of glutathione S-transferase (GST) and individual Gro/TLE domains from bacteria and in vitro phosphorylation assays with 0.5 U of purified CK2 (New England Biolabs)/µl were performed as described previously (22). After gel electrophoresis, proteins were transferred to nitrocellulose and exposed to film. After autoradiography, membranes were subjected to Western blotting with anti-GST antibodies. Alternatively, the products of kinase reactions were loaded onto SpinZyme phosphocellulose units (Pierce) and centrifuged at 3,000 x g for 30 s. The filters were then extensively washed with 75 mM phosphoric acid and dried. Bound radioactivity was measured in a scintillation counter.
Pharmacological inhibition of CK2. The selective CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) (28) was resuspended in dimethyl sulfoxide (DMSO). HEK293 cells were incubated in the presence of either DMSO alone or TBB at the concentrations indicated in the figure legends. Cells were then collected, and whole-cell lysates were prepared and subjected to Western blotting analysis.
Cell culture, transfection, protein-protein interaction assays, biochemical fractionation, and Western blotting. HeLa and HEK293 cells were cultured and, when appropriate, transfected by using the SuperFect reagent (Qiagen) (20, 22). Cell lysates were prepared and GST coprecipitation, immunoprecipitation, and Western blotting studies were performed as described previously (19, 22, 24). Antibodies used were anti-(pS239)Gro/TLE1 (1:2,000), panTLE (1:20 [24, 31]), anti-Gro/TLE1 (1:1,000 [14, 34]), anti-GST (1:1,000; Santa Cruz), anti-FLAG (1:10,000; Sigma), anti-HDAC2 (1:4,000; Zymed), antiphosphoserine (1:750; Abcam), and anti-histone H3 (1:1,000 [22, 23]). Postnuclear supernatant and chromatin-enriched fractions were prepared as described previously (22, 23). Immunoblots were scanned and analysis performed by using the NIH Image J program (rsb.info.nih.gov/ij/).
Indirect immunofluorescence. Staged mouse embryos were collected, fixed, sectioned, and subjected to immunohistochemistry as described previously (34). All animal procedures followed the guidelines of the Canadian Council for Animal Care. HEK293 and cortical neural progenitor cells were cultured on four-well chamber slides (Nalgene Nunc, Int.) as described previously (12), fixed, and permeabilized in 0.1% IGEPAL in HEPES-buffered saline containing 4% paraformaldehyde. Primary antibodies for immunofluorescence were either rabbit anti-(pS239)Gro/TLE1, rabbit anti-Gro/TLE1, rat panTLE, mouse anti-FLAG, mouse anti-Ki67 (1:25; BD Pharmingen), or mouse anti-NeuN (1:50; Chemicon). Detection was as described previously (12).
Transcription assays. HEK293 cells were transiently transfected with the reporter plasmid p6OSE2-Luc in the absence or presence of plasmids encoding either Runx2, FLAG-Gro/TLE1, FLAG-Gro/TLE1(S239A), or FLAG-Gro/TLE1(S239E) as described previously (19). In each case, 0.5 µg of pRSV-ß-galactosidase DNA was cotransfected to provide a means of normalizing the assays for transfection efficiency. Luciferase activity values were expressed as means ± the standard deviations.
Cortical neural progenitor cell culture, transfection, and analysis of neuronal differentiation. Primary neural progenitor cell cultures were established from dorsal telencephalic cortices dissected from embryonic day 13.0 (E13.0) mouse embryos as described previously (12). Cells were seeded into four-well chamber slides (Nalgene Nunc) at 4 x 105 cells/well, coated with 0.1% poly-D-lysine and 0.2% laminin (BD Biosciences), cultured in Neurobasal medium supplemented with 1% N2, 2% B27, 0.5 mM glutamine, 1% penicillin-streptomycin (Invitrogen), and 40 ng of FGF2 (Collaborative Research)/ml. After 48 h in vitro, cells were transfected by using plasmids encoding either green fluorescent protein (GFP) alone (0.2 µg/well), or both GFP (0.2 µg/well) and Gro/TLE1 (full-length or point mutants) (0.8 µg/well). The total amount of DNA was adjusted to 1.0 µg and mixed with 50 µl of OptiMEM medium (Invitrogen), followed by incubation for 5 min. An equal volume of OptiMEM medium was mixed separately with Lipofectamine 2000 reagent (Invitrogen; 2 µl/µg of DNA) and then combined with the DNA mixture and incubated for 20 min. Half the medium was removed and stored as a conditioned medium, and the DNA-Lipofectamine 2000 mix was then added dropwise to each well. After 24 h, the medium was removed and replaced with fresh medium that contained the conditioned medium. Approximately 2 days later, cells were fixed and subjected to double-label immunocytochemical analysis of the expression of GFP, Ki67, or NeuN. Digital image acquisition and analysis were performed with the Northern Eclipse software (Empix, Inc.). The results were expressed as mean values ± the standard deviation. For analysis of FLAG-Gro/TLE1 protein expression, cortical progenitor cells were cultured in six-well dishes and lysed 48 h after transfection, followed by immunoprecipitation and Western blotting with anti-FLAG antibodies.
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
|
We next determined whether CK2 was involved in the phosphorylation of S239 in vivo. HEK293 cells were cultured in the absence or presence of the selective CK2 inhibitor TBB, a cell-permeable compound that was shown to be specific for CK2 against a panel of more than 30 other protein kinases (28). Because disruption of CK2 activity is lethal in both embryos and cultured cells (3), we performed these assays under conditions of only partial CK2 inhibition to preserve cell viability (28, 29; data not shown). Lysates from cells treated with TBB or DMSO alone were subjected to Western blot analysis with anti-(pS239)Gro/TLE1. Exposure to TBB resulted in a significant decrease of anti-(pS239)Gro/TLE1 immunoreactivity (Fig. 3A and E, lanes 2 and 4) without decreasing the immunoreactivity detected with either the non-phospho-specific Gro/TLE1 antibody (Fig. 3B and E, lanes 2 and 4) or the panTLE antibody (Fig. 3C). Neither did TBB cause obvious changes in the immunoreactive profile decorated by an anti-phosphoserine antibody (Fig. 3D), in agreement with the demonstrated specificity of TBB for CK2 (28). Thus, TBB caused a specific decrease in the phosphorylation of S239 without affecting the expression levels of Gro/TLEs. These combined results show that Gro/TLE1 is phosphorylated in vivo by CK2 at S239.
|
|
Involvement of S239 in Hes1-activated hyperphosphorylation of Gro/TLE1. We next investigated whether phosphorylation of the CcN domain of Gro/TLE1 by CK2 was important for the CAP that Gro/TLE1 undergoes after interaction with its transcriptional partner Hes1 (22). Wild-type and mutated Gro/TLE1 proteins were expressed in HEK293 cells in the absence or presence of cotransfected Hes1. As previously shown (22), Hes1 induced a significant CAP of Gro/TLE1 resulting in the appearance of a hyperphosphorylated form of slower electrophoretic mobility (Fig. 5B, lanes 1 and 2). In contrast, only a modest gel retardation was observed when Hes1 was cotransfected with Gro/TLE1(S239A) (Fig. 5B, lanes 3 and 4). This mutated Gro/TLE1 protein interacted with Hes1 like its wild-type counterpart, indicating that reduced CAP of Gro/TLE1 was not caused by a perturbation of the Gro/TLE1:Hes1 interaction (Fig. 5E, lanes 7 and 8). As previously shown (20), this interaction was dependent on the C-terminal WRPW motif of Hes1 (Fig. 5E, lane 6). Thus, S239 is important for maximal Hes1-induced hyperphosphorylation of Gro/TLE1. We next examined both the single mutant Gro/TLE1(S253A) and the double mutant Gro/TLE1(S239A/S253A). The S253A mutation alone decreased, but did not block, the CAP of Gro/TLE1 mediated by Hes1 (Fig. 5B, lanes 5 and 6), whereas the double mutation completely inhibited it (Fig. 5B, lanes 7 and 8). Both Gro/TLE1(S253A) and Gro/TLE1(S239A/S253A) interacted with Hes1 like their wild-type counterpart (Fig. 5E, lanes 7 to 10). Hes1 expression had no effects on the electrophoretic mobility of the control protein HDAC2 (Fig. 5C). These combined results show that both S239 and S253 are important for the CAP of Gro/TLE1 induced by Hes1, implicating CK2-mediated phosphorylation of the CcN domain in these events.
|
285-335); Fig. 5A] blocked the Hes1-mediated CAP of Gro/TLE1 (Fig. 5H, lanes 3 and 4) without perturbing the Gro/TLE1: Hes1 interaction (Fig. 5I). Taken together, these observations strongly suggest that phosphorylation of S239 takes place independent of Hes1 binding but is important for additional phosphorylation events that are activated by Hes1 binding and result in the CAP of Gro/TLE1. Such events may directly target the region deleted in Gro/TLE1(285-335), which contains an abundance of serine and threonine residues. Alternatively, this region may be involved in conformational or protein-protein interaction mechanisms required for the Hes1-mediated CAP of Gro/TLE1. Importance of S239 for Gro/TLE1-mediated transcriptional repression. Based on these results and the previous demonstration that inhibition of CK2 activity reduces the transcription repression ability of Gro/TLE (22), we next tested whether S239 was important for Gro/TLE1-mediated transcriptional repression. HEK293 cells were transfected with a reporter plasmid containing the luciferase reporter gene under the control of tandem DNA-binding sites for the Runt-related protein Runx2. Previous studies have shown that Runx2 activates transcription from these sites and that Gro/TLE can repress this transactivation (32). In agreement with these results, we found that Runx2 induced a roughly 150-fold activation of reporter gene expression (designated as 100%) and wild-type Gro/TLE1 suppressed this activation (Fig. 6A, bars 1 to 4). In contrast, Gro/TLE1(S239A) did not repress Runx2-mediated transactivation (Fig. 6A, bars 5 and 6) when expressed at equivalent levels (Fig. 6B). Runx2 interacted equally with Gro/TLE1 and Gro/TLE1(S239A) (data not shown). Importantly, a point mutation that introduced a negatively charged glutamate residue at position 239 [Gro/TLE1(S239E)] did not disrupt the transcription repression ability of Gro/TLE1 (Fig. 6A, bars 7 and 8). Together, these results strongly suggest that phosphorylation of S239 provides local negative charges that are important for the transcription repression function of Gro/TLE1.
|
|
|
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Gro/TLE1 is phosphorylated by CK2 in vivo. Through in vitro kinase assays and the use of a phospho-specific antibody, we have demonstrated for the first time that CK2 phosphorylates Gro/TLE1 at S239. This finding is consistent with the evolutionary conservation of this site within the CcN motifs of Drosophila Gro (231SDQD) and human Gro/TLEs (Gro/TLE1, 239SDGD; Gro/TLE2, 228SDED; Gro/TLE3, 240SDGD; Gro/TLE4, 238SDGE). In vitro kinase assays have suggested further that CK2 can also phosphorylate Gro/TLE1 at a second sequence, 253SNED, located 25 residues C terminal to the NLS. A similar CK2 consensus sequence is conserved in the CcN motif of the C. elegans Gro/TLE family member UNC-37 (182TNDD), human Gro/TLE3 (253SNED) and Gro/TLE4 (252SNED), but not Gro/TLE2 or Drosophila Gro. Although it remains to be determined whether the 253SNED site is phosphorylated by CK2 in vivo, the observation that UNC-37 contains a conserved TNDD, but not SDXD/E, sequence C terminal to its NLS, suggests that S/TNE/DD motifs within the CcN domain of certain Gro/TLEs are also targets of CK2.
The conservation of at least one CK2 target site within the CcN domain of different Gro/TLE family members suggests that CK2-mediated phosphorylation of this region is an important event in the functions of all Gro/TLE proteins. Because CK2 is a ubiquitous and constitutively active protein kinase (reference 28 and references therein), it is likely that phosphorylation of Gro/TLE1 by CK2 is a constitutive event. In agreement with this, we have found that Gro/TLE1 becomes phosphorylated at S239 rapidly after its translation. Moreover, Gro/TLE1 is phosphorylated at S239 in virtually all cells examined. In particular, our results suggest that Gro/TLE1 is phosphorylated at S239 in both mitotic neural progenitor cells and postmitotic neurons in the developing cerebral cortex. These combined observations suggest that phosphorylation of S239 is not correlated with specific functions of Gro/TLE1 in either proliferating or differentiated cells but may instead represent a general regulatory event underlying its activity.
Phosphorylation of S239 of Gro/TLE1 is important for nuclear interaction and transcriptional repression. Insight into the role of CK2-mediated phosphorylation of Gro/TLE1 is provided by studies of other proteins, including SV40 T-antigen, nucleoplasmin, and IFI 16, that also contain CcN motifs that are phosphorylated at their resident CK2 sites (1; reference 11 and references therein). Where examined, CK2 phosphorylation promotes the nuclear association of these proteins. Although it was originally hypothesized that CK2-mediated phosphorylation of the CcN motif increased the affinity of the NLS for the importin heterodimer, recent studies have shown that the region containing the CK2 phosphorylation site of the CcN motif of SV40 T-antigen does not make contacts with importins and phosphorylation does not increase the affinity of recognition by the NLS-recognizing importin heterodimer (11). Similarly, although CK2-mediated phosphorylation of the CcN motif of IFI 16 promotes its nuclear accumulation, it appears to do so independently of recognition by importins but rather through facilitating binding to nuclear components (1). In agreement with these observations, the present findings have shown that mutation of S239 into alanine does not prevent the nuclear localization of Gro/TLE1 but instead decreases its nuclear retention during subcellular fractionation. This observation is consistent with the previous demonstration that pharmacological inhibition of CK2 also decreases the nuclear retention of Gro/TLE1 (22). These combined results strongly suggest that CK2-mediated phosphorylation of the CcN domain of Gro/TLE1 is not required for nuclear translocation but instead promotes the nuclear interaction of Gro/TLE1.
Our studies have suggested at least one mechanism by which phosphorylation of the CcN domain contributes to the nuclear binding of Gro/TLE1. We have shown that both pharmacological inhibition of CK2 activity (22) and mutation of S239 into alanine result not only in a weakened nuclear interaction but also a reduced Hes1-mediated CAP of Gro/TLE1. These findings strongly suggest that the nuclear association of Gro/TLE1 is promoted by a hierarchical sequence of phosphorylation events in which Gro/TLE1 first becomes phosphorylated by CK2 within its CcN domain. This primary phosphorylation is not required for either nuclear translocation or interaction with Hes1 but is important for both the secondary phosphorylation of Gro/TLE1 that occurs during CAP and its nuclear retention. We propose that the constitutive, CK2-mediated phosphorylation of the CcN domain is required for subsequent, cofactor-regulated phosphorylation events that either directly or indirectly strengthen the interaction of Gro/TLE1 with yet-to-be-defined transcriptional or structural factors that stabilize its association with nuclei.
A corollary to this model is that inhibition of CK2 phosphorylation should reduce the ability of Gro/TLE1 to mediate transcriptional repression. In agreement with this, we have demonstrated that both pharmacological inhibition of CK2 activity (22) and mutation of S239 (the present study) weaken the ability of Gro/TLE to repress transcription. Moreover, mutation of S239 impairs the biological activity of Gro/TLE1 during neuronal differentiation, an effect that is consistent with a loss or reduction of transcription repression function (see below for further discussion). These combined observations suggest that a combination of constitutive and regulated posttranslational modifications establish a phosphorylation state that promotes intranuclear interactions that render Gro/TLE1 competent to mediate transcriptional repression.
Phosphorylation of S239 is important for Gro/TLE1 activity during cortical neurogenesis. The possible involvement of a hierarchy of phosphorylation reactions in the regulation of Gro/TLE1 activity is consistent with the observation that this protein becomes increasingly phosphorylated as a function of neuronal differentiation in primary cortical neural progenitors or P19 cells (14, 22). We have found that S239 is phosphorylated in both undifferentiated neural progenitors and differentiated neurons, suggesting that it is unlikely that these phosphorylation changes are due to a developmentally regulated phosphorylation of S239. However, we have demonstrated that mutation of S239 suppresses the ability of Gro/TLE1 to inhibit the transition of cortical neural progenitor cells into neurons. Gro/TLE1 is coexpressed with, and acts as a transcriptional corepressor for, different DNA-binding factors that inhibit and/or delay cortical neuronal differentiation and also promote the CAP of Gro/TLE1, including Hes1 and brain factor 1 (22, 30, 35). We therefore propose that S239 of Gro/TLE1 needs to be phosphorylated by CK2 in order to allow the developmentally regulated CAP of Gro/TLE1 that occurs during neurogenesis as a result of its interaction with such DNA-binding cofactors. In turn, the modification of Gro/TLE1 caused by the regulated CAP promotes its nuclear binding and transcription repression activity, which are required for its antineurogenic function. In this scenario, CK2 phosphorylation of Gro/TLE1 is a critical event for the regulation of its activity during neuronal development. Given the widespread expression of CK2, it is possible that similar mechanisms also modulate Gro/TLE activity outside of the nervous system.
|
ACKNOWLEDGMENTS |
|---|
This study was supported by grants from the Canadian Institutes of Health Research (CIHR) to S.S. H.N.N. is a CIHR Postdoctoral Fellow. S.S. is a Senior Scholar of the Fonds de la Recherche en Sante du Quebec.
|
FOOTNOTES |
|---|
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2. Brinkmeier, M. L., M. A. Potok, K. B. Cha, T. Gridley, S. Stifani, J. Meeldijk, H. Clevers, and S. A. Camper. 2003. TCF and Groucho-related genes influence pituitary growth and development. Mol. Endocrinol. 17:2152-2161.
3. Buchou, T., M. Vernet, O. Blond, H. H. Jensen, H. Pointu, B. B. Olsen, C. Cochet, O. G. Isinger, and B. Boldyreff. 2003. Disruption of the regulatory ß subunit of protein kinase CK2 in mice leads to a cell-autonomous defect and early embryonic lethality. Mol. Cell. Biol. 23:908-915.
4. Cai, Y., P. D. Brophy, I. Levitan, S. Stifani, and G. R. Dressler. 2003. Groucho suppresses Pax2 transactivation by inhibition of JNK-mediated phosphorylation. EMBO J. 22:5522-5529.[CrossRef][Medline]
5. Carvalho, L. R., K. S. Woods, B. B. Mendonca, N. Marcal, A. L. Zamparini, S. Stifani, J. M. Brickman, I. V. J. Arnhold, and M. Dattani. 2003. A homozygous mutation in HESX1 is associated with evolving hypopituitarism due to impaired repressor-corepressor interaction. J. Clin. Investig. 112:1192-1201.[CrossRef][Medline]
6. Chen, G., J. Fernandez, S. Mische, and A. J. Courey. 1999. A functional interaction between the histone deacetylase rpd3 and the corepressor Groucho in Drosophila development. Genes Dev. 13:2218-2230.
7. Chen, G., and A. J. Courey. 2000. Groucho/TLE family proteins and transcriptional repression. Gene 249:1-16.[CrossRef][Medline]
8. Courey, A. J., and S. Jia. 2001. Transcriptional repression: the long and the short of it. Genes Dev. 15:2786-2796.
9. Dasen, J. S., J. P. Barbera, T. S. Herman, S. O. Connell, L. Olson, B. Ju, J. Tolkuhn, S. H. Back, D. W. Rose, and M. G. Rosenfeld. 2001. Temporal regulation of a paired-like homeodomain repressor/TLE corepressor complex and a related activator is required for pituitary organogenesis. Genes Dev. 15:3193-3207.
10. Eberhard, D., G. Jimenez, B. Heavy, and M. Busslinger. 2000. Transcriptional repression by Pax5 (BSAP) through interaction with corepressors of the Groucho family. EMBO J. 19:2292-2303.[CrossRef][Medline]
11. Fontes, M. R., T. Teh, G. Toth, A. John, I. Pavo, D. A. Jans, and B. Kobe. 2003. Role of flanking sequences and phosphorylation in the recognition of the simian-virus-40 large T-antigen nuclear localization sequences by importin-alpha. Biochem. J. 375:339-349.[CrossRef][Medline]
12. Gratton, M. O., E. Torban, S. Belanger-Jasmin, F. Theriault, M. S. German, and S. Stifani. 2003. Hes6 promotes cortical neurogenesis and inhibits Hes1 transcription repression activity by multiple mechanisms. Mol. Cell. Biol. 23:6922-6935.
13. Grbavec, D., R. Lo, Y. Liu, and S. Stifani. 1998. Transducin-like Enhancer of split 2, a mammalian homologue of Drosophila Groucho, acts as a transcriptional repressor, interacts with Hairy/Enhancer of split proteins, and is expressed during neuronal development. Eur. J. Biochem. 258:339-349.[Medline]
14. Husain, J., R. Lo, D. Grbavec, and S. Stifani. 1996. Affinity for the nuclear compartment and expression during cell differentiation implicate phosphorylated Groucho/TLE1 forms of higher molecular mass in nuclear functions. Biochem. J. 317:523-531.
15. Javed, A., B. Guo, S. Hiebert, J. Y. Choi, J. Green, S. C. Zhao, M. A. Osborne, S. Stifani, J. L. Stein, J. B. Lian, A. J. van Wijnen, and G. S. Stein. 2000. Groucho/TLE/R-esp proteins associate with the nuclear matrix and repress RUNX (CBF
/AML/PEBP2) dependent activation of tissue-specific gene transcription. J. Cell Sci. 113:2221-2231.[Abstract]
16. Jimenez, G., Z. Paroush, and D. Ish-Horowicz. 1997. Groucho acts as a corepressor for a subset of negative regulators, including Hairy and Engrailed. Genes Dev. 11:3072-3082.
17. Kobayashi, M., K. Nishikawa, T. Suzuki, and M. Yamamoto. 2001. The homeobox protein Six3 interacts with the Groucho corepressor and acts as a transcriptional repressor in eye and forebrain formation. Dev. Biol. 232:315-326.[CrossRef][Medline]
18. Levanon, D., R. E. Goldstein, Y. Bernstein, H. Tang, D. Goldenberg, S. Stifani, Z. Paroush, and Y. Groner. 1998. Transcriptional repression by AML1 and LEF-1 is mediated by the TLE/Groucho corepressors. Proc. Natl. Acad. Sci. USA 95:11590-11595.
19. McLarren, K. W., R. Lo, D. Grbavec, K. Thirunavukkarasu, G. Karsenty, and S. Stifani. 2000. The mammalian basic helix loop helix protein HES-1 binds to and modulates the transactivating function of the Runt-related factor Cbfa1. J. Biol. Chem. 275:530-538.
20. McLarren, K. W., F. Theriault, and S. Stifani. 2001. Association with the nuclear matrix and interaction with Groucho and RUNX proteins regulate the transcription repression ability of the basic helix loop helix factor Hes1. J. Biol. Chem. 276:1578-1584.
21. Muhr, J., E. Andersson, M. Persson, T. M. Jessell, and J. Ericson. 2001. Groucho-mediated transcriptional repression establishes progenitor cell pattern and neuronal fate in the ventral neural tube. Cell 104:861-873.[CrossRef][Medline]
22. Nuthall, H. N., J. Husain, K. W. McLarren, and S. Stifani. 2002. Role for Hes1-induced phosphorylation in Groucho-mediated transcriptional repression. Mol. Cell. Biol. 22:389-399.
23. Nuthall, H. N., K. Joachim, A. Palaparti, and S. Stifani. 2002. A role for cell cycle-regulated phosphorylation in Groucho-mediated transcriptional repression. J. Biol. Chem. 277:51049-51057.
24. Palaparti, A., A. Baratz, and S. Stifani. 1997. The Groucho/Transducin-like Enhancer of split transcriptional repressors interact with the genetically defined amino-terminal silencing domain of histone H3. J. Biol. Chem. 272:26604-26610.
25. Paroush, Z., R. L. Finley, T. Kidd, S. M. Wainwright, P. W. Ingham, R. Brent, and D. Ish-Horowicz. 1994. Groucho is required for Drosophila neurogenesis, segmentation, and sex determination and interacts directly with hairy-related bHLH proteins. Cell 79:805-815.[CrossRef][Medline]
26. Pflugrad, A., J. Y. Meir, T. M. Barnes, and D. M. Miller III. 1997. The Groucho-like transcription factor UNC-37 functions with the neural specificity gene unc-4 to govern motor neuron identity in C. elegans. Development 124:1699-1709.[Abstract]
27. Roose, J., M. Molenaar, J. Peterson, J. Hurenkamp, H. Brantjies, P. Moerer, M. van de Wetering, O. Destree, and H. Clevers. 1998. The Xenopus Wnt effector XTcf-3 interacts with Groucho-related transcriptional repressors. Nature 395:608-612.[CrossRef][Medline]
28. Sarno, S., H. Reddy, F. Meggio, M. Ruzzene, S. P. Davies, A. Donella-Deana, D. Shugar, and L. A. Pinna. 2001. Selectivity of 4,5,6,7-tetrabromobenzotriazole, an ATP site-directed inhibitor of protein kinase CK2 (casein kinase-2'). FEBS Lett. 496:44-48.[CrossRef][Medline]
29. Sarno, S., S. Moro, F. Meggio, G. Zagotto, D. Dal Ben, P. Ghisellini, R. Battistutta, G. Zanotti, and L. A. Pinna. 2002. Toward the rational design of protein kinase casein kinase-2 inhibitors. Pharmacol. Ther. 93:159-168.[CrossRef][Medline]
30. Sonderegger, C. K., and P. K. Vogt. 2003. Binding of the corepressor TLE1 to Qin enhances Qin-mediated transformation of chicken embryo fibroblasts. Oncogene 22:1749-1757.[CrossRef][Medline]
31. Stifani, S., C. M. Blaumueller, N. J. Redhead, R. E. Hill, and S. Artavanis-Tsakonas. 1992. Human homologs of a Drosophila Enhancer of split gene product define a novel family of nuclear proteins. Nat. Genet. 2:119-127.[CrossRef][Medline]
32. Thirunavukkarasu, K., M. Mahajan, K. W. McLarren, S. Stifani, and G. Karsenty. 1998. Two domains unique to osteoblast-specific transcription factor Osf2/Cbf1 contribute to its transactivating function and its inability to heterodimerize with Cbfß. Mol. Cell. Biol. 18:4197-4208.
33. Yao, J., Y. Liu, J. Husain, R. Lo, A. Palaparti, J. Henderson, and S. Stifani. 1998. Combinatorial expression patterns of individual TLE proteins during cell determination and differentiation suggest non-redundant functions for mammalian homologs of Drosophila Groucho. Dev. Growth Differ. 40:133-146.[CrossRef][Medline]
34. Yao, J., Y. Liu, R. Lo, I. Tretjakoff, A. Peterson, and S. Stifani. 2000. Disrupted development of the cerebral hemispheres in transgenic mice expressing the mammalian Groucho homologue Transducin-like Enhancer of split 1 in postmitotic neurons. Mech. Dev. 93:105-115.[CrossRef][Medline]
35. Yao, J., E. Lai, and S. Stifani. 2001. The winged-helix protein brain factor 1 interacts with Groucho and Hes proteins to repress transcription. Mol. Cell. Biol. 21:1962-1972.
36. Zhang, H., and S. W. Emmons. 2002. Caenorhabditis elegans unc-37/groucho interacts genetically with components of the transcriptional mediator complex. Genetics 160:799-803.
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| J. Bacteriol. | J. Virol. | Eukaryot. Cell |
|---|
| Microbiol. Mol. Biol. Rev. | Clin. Vaccine Immunol. | All ASM Journals |
|---|
, P < 0.05.
