Cell cycle-dependent regulation of the forkhead transcription factor FOXK2 by CDK·cyclin complexes.
ABSTRACT: Several mammalian forkhead transcription factors have been shown to impact on cell cycle regulation and are themselves linked to cell cycle control systems. Here we have investigated the little studied mammalian forkhead transcription factor FOXK2 and demonstrate that it is subject to control by cell cycle-regulated protein kinases. FOXK2 exhibits a periodic rise in its phosphorylation levels during the cell cycle, with hyperphosphorylation occurring in mitotic cells. Hyperphosphorylation occurs in a cyclin-dependent kinase (CDK)·cyclin-dependent manner with CDK1·cyclin B as the major kinase complex, although CDK2 and cyclin A also appear to be important. We have mapped two CDK phosphorylation sites, serines 368 and 423, which play a role in defining FOXK2 function through regulating its stability and its activity as a transcriptional repressor protein. These two CDK sites appear vital for FOXK2 function because expression of a mutant lacking these sites cannot be tolerated and causes apoptosis.
Project description:A major target of insulin signaling is the FoxO family of Forkhead transcription factors, which translocate from the nucleus to the cytoplasm following insulin-stimulated phosphorylation. Here we show that the Forkhead transcription factors FoxK1 and FoxK2 are also downstream targets of insulin action, but that following insulin stimulation, they translocate from the cytoplasm to nucleus, reciprocal to the translocation of FoxO1. FoxK1/FoxK2 translocation to the nucleus is dependent on the Akt-mTOR pathway, while its localization to the cytoplasm in the basal state is dependent on GSK3. Knockdown of FoxK1 and FoxK2 in liver cells results in upregulation of genes related to apoptosis and down-regulation of genes involved in cell cycle and lipid metabolism. This is associated with decreased cell proliferation and altered mitochondrial fatty acid metabolism. Thus, FoxK1/K2 are reciprocally regulated to FoxO1 following insulin stimulation and play a critical role in the control of apoptosis, metabolism and mitochondrial function.
Project description:The forkhead transcription factor FOXK2 plays a critical role in suppressing tumorigenesis and mediating cytotoxic drug action in breast cancer. However, the mechanism by which the biological function of FOXK2 is regulated remains poorly understood. Here, we investigated the role of SUMOylation in modulating FOXK2-mediated drug sensitivity. We identified SUMOylation consensus motifs within the FOXK2 sequence and constructed two SUMOylation-defective double mutants by converting lysine 527 and 633 to arginines and glutamic acid 529 and 635 to alanines, respectively. We found that both the FOXK2 SUMOylation-deficient (K527/633?R) and (E529/635?A) mutants were ineffective in mediating the cytotoxic function of paclitaxel when compared to the wild-type (WT) FOXK2. When overexpressed, unlike the wild-type (WT) FOXK2, the K527/633?R mutant had little effect on the sensitivity of MCF-7 and MDA-MB-231 cells to paclitaxel, as examined by cell viability and clonogenic assays. Our results also showed that MCF-7 cells overexpressing the K527/633?R mutant form of FOXK2 or the empty expression vector have lower protein and mRNA levels of its tumour suppressive transcriptional target FOXO3 compared to the wild-type FOXK2. Consistently, ChIP assays revealed that unlike wild-type FOXK2, the SUMOylation-defective (K527/633?R) mutant is unable to bind to the FOXO3 promoter, despite expressing comparable levels of protein and having the same subcellular localization as the wild-type FOXK2 in MCF-7 cells. Interestingly, expression of neither the wild-type nor the K527/633?R mutant FOXK2 had any effect on the proliferation and paclitaxel sensitivity of the MCF-7 TaxR paclitaxel-resistant cells. In agreement, both the wild-type and the (K527/633?R) mutant FOXK2 failed to bind to the endogenous FOXO3 promoter in these cells. Collectively, our results suggest that SUMOylation positively regulates FOXK2 transcriptional activity and has a role in mediating the cytotoxic response to paclitaxel through the tumour suppressor FOXO3.
Project description:The transcriptional control circuitry in eukaryotic cells is complex and is orchestrated by combinatorially acting transcription factors. Forkhead transcription factors often function in concert with heterotypic transcription factors to specify distinct transcriptional programs. Here, we demonstrate that FOXK2 participates in combinatorial transcriptional control with the AP-1 transcription factor. FOXK2 binding regions are widespread throughout the genome and are often coassociated with AP-1 binding motifs. FOXK2 acts to promote AP-1-dependent gene expression changes in response to activation of the AP-1 pathway. In this context, FOXK2 is required for the efficient recruitment of AP-1 to chromatin. Thus, we have uncovered an important new molecular mechanism that controls AP-1-dependent gene expression.
Project description:There are numerous forkhead transcription factors in mammalian cells but we know little about the molecular functions of the majority of these. FOXK2 is a ubiquitously expressed family member suggesting an important function across multiple cell types. Here, we show that FOXK2 binds to the SIN3A and PR-DUB complexes. The PR-DUB complex contains the important tumour suppressor protein, the deubiquitinase BAP1. FOXK2 recruits BAP1 to DNA, promotes local histone deubiquitination and causes changes in target gene activity. Our results therefore provide an important link between BAP1 and the transcription factor FOXK2 and demonstrate how BAP1 can be recruited to specific regulatory loci.
Project description:Estrogen receptors (ERs) are critical regulators of breast cancer development. Identification of molecules that regulate the function of ERs may facilitate the development of more effective breast cancer treatment strategies. In this study, we showed that the forkhead transcription factor FOXK2 interacted with ER?, and inhibited ER?-regulated transcriptional activities by enhancing the ubiquitin-mediated degradation of ER?. This process involved the interaction between FOXK2 and BRCA1/BARD1, the E3 ubiquitin ligase of ER?. FOXK2 interacted with BARD1 and acted as a scaffold protein for BRCA1/BARD1 and ER?, leading to enhanced degradation of ER?, which eventually accounted for its decreased transcriptional activity. Consistent with these observations, overexpression of FOXK2 inhibited the transcriptional activity of ER?, decreased the transcription of ER? target genes, and suppressed the proliferation of ER?-positive breast cancer cells. In contract, knockdown of FOXK2 in MCF-7 cells promoted cell proliferation. However, when ER? was also knocked down, knockdown of FOXK2 had no effect on cell proliferation. These findings suggested that FOXK2 might act as a negative regulator of ER?, and its association with both ER? and BRCA1/BARD1 could lead to the down-regulation of ER? transcriptional activity, effectively regulating the function of ER?.
Project description:Activation of the DNA-damage checkpoint culminates in the inhibition of cyclin-dependent kinase (Cdk) complexes to prevent cell-cycle progression. We have shown recently that Cdk activity is required for activation of the Forkhead transcription factor FoxM1, an important regulator of gene expression in the G2 phase of the cell cycle. Here, we show that FoxM1 is transcriptionally active during a DNA-damage-induced G2 arrest and is essential for checkpoint recovery. Paradoxically, Cdk activity, although reduced after checkpoint activation, is required to maintain FoxM1-dependent transcription during the arrest and for expression of pro-mitotic targets such as cyclin A, cyclin B and Plk1. Indeed, we find that cells need to retain sufficient levels of Cdk activity during the DNA-damage response to maintain cellular competence to recover from a DNA-damaging insult.
Project description:Clear-cell renal cell carcinoma (ccRCC) is one of the most common urological malignant neoplasms. In addition, ccRCC is a highly aggressive cancer with a concomitant poor prognosis. Forkhead box K2 (FOXK2) has been reported to involve in many molecular mechanisms.However, little is known about the role FOXK2 plays in clear-cell renal cell carcinoma. Our previous study showed that FOXK2 mRNA and protein expression were decreased in human ccRCC tissues and suppressed the proliferation of ccRCC cells both in vitro and in vivo. We used microarrays (Affymetrix HTA2.0 Array) to detail the differentially expressed genes after overexpression of FOXK2, and aim to find potential one or more target gene/genes of FOXK2 Overall design: We conducted the genome-wide transcriptome profiling of human ccRCC cell line 769-P after transfection with pLV-EGFP-FOXK2 (overexpression of FOXK2) and plv-EGFP empty vector (control)
Project description:Chromatin immunoprecipitation of FOXK2 (tagged with Flag and His tags) in U2OS cells detected by SOLiD sequencing. ***Correction March 2014: The sample “FOXK2_Dox_treated” has been renamed, it was originally named “FOXK2_rep2”. A new sample “FOXK2_rep2” has been added, with new files. It has come to our attention that one of the FOXK2 ChIP-seq replicates 'FOXK2_rep2' that we used in our paper recent paper (Ji, Z., Donaldson, I.J., Liu, J., Hayes, A., Zeef, L.A.H. and Sharrocks, A.D. (2012) The forkhead transcription factor FOXK2 promotes AP-1-mediated transcriptional regulation. Mol. Cell. Biol. 32, 385-398. doi:10.1128/MCB.05504-11) was incorrect. The replicate was actually treated with doxorubicin prior to ChIP-seq analysis resulting in the loss of many FOXK2 binding events.***
Project description:Cells escape the need for mitogens at a restriction point several hours before entering S phase. The restriction point has been proposed to result from CDK4/6 initiating partial Rb phosphorylation to trigger a bistable switch whereby cyclin E-CDK2 and Rb mutually reinforce each other to induce Rb hyperphosphorylation. Here, using single-cell analysis, we unexpectedly found that cyclin E/A-CDK activity can only maintain Rb hyperphosphorylation starting at the onset of S phase and that CDK4/6 activity, but not cyclin E/A-CDK activity, is required to hyperphosphorylate Rb throughout G1 phase. Mitogen removal in G1 results in a gradual loss of CDK4/6 activity with a high likelihood of cells sustaining Rb hyperphosphorylation until S phase, at which point cyclin E/A-CDK activity takes over. Thus, it is short-term memory, or transient hysteresis, in CDK4/6 activity following mitogen removal that sustains Rb hyperphosphorylation, demonstrating a probabilistic rather than an irreversible molecular mechanism underlying the restriction point.
Project description:The forkhead transcription factor FoxM1 controls expression of a large number of genes that are specifically expressed during the G(2) phase of the cell cycle. Throughout most of the cell cycle, FoxM1 activity is restrained by an autoinhibitory mechanism, involving a repressor domain present in the N-terminal part of the protein. Activation of FoxM1 in G(2) is achieved by Cyclin A/Cyclin-dependent kinase (Cdk)-mediated phosphorylation, which alleviates autoinhibition by the N-terminal repressor domain. Here, we show that FoxM1 interacts with B55?, a regulatory subunit of protein phosphatase 2A (PP2A). B55? binds the catalytic subunit of PP2A, and this promotes dephosphorylation and inactivation of FoxM1. Indeed, we find that overexpression of B55? results in decreased FoxM1 activity. Inversely, depletion of B55? results in premature activation of FoxM1. The activation of FoxM1 that is observed upon depletion of B55? is fully dependent on Cyclin A/Cdk-mediated phosphorylation of FoxM1. Taken together, these data demonstrate that B55? acts to antagonize Cyclin A/Cdk-dependent activation of FoxM1, to ensure that FoxM1 activity is restricted to the G(2) phase of the cell cycle.