Arjan B. Brenkman

FOXO4 transcriptional activity is regulated by monoubiquitination and USP7/HAUSP

FOXO (Forkhead box O) transcription factors are important regulators of cellular metabolism, cell-cycle progression and cell death. FOXO activity is regulated by multiple post-translational modifications, including phosphorylation, acetylation and polyubiquitination. Here, we show that FOXO becomes monoubiquitinated in response to increased cellular oxidative stress, resulting in its re-localization to the nucleus and an increase in its transcriptional activity. Deubiquitination of FOXO requires the deubiquitinating enzyme USP7/HAUSP (herpesvirus-associated ubiquitin-specific protease), which interacts with and deubiquitinates FOXO in response to oxidative stress. Oxidative stress-induced ubiquitination and deubiquitination by USP7 do not influence FOXO protein half-life. However, USP7 does negatively regulate FOXO transcriptional activity towards endogenous promoters. Our results demonstrate a novel mechanism of FOXO regulation and indicate that USP7 has an important role in regulating FOXO-mediated stress responses.

Regulation of Treg functionality by acetylation-mediated Foxp3 protein stabilization

Regulatory T cells (Tregs) are a specific subset of lymphocytes that are critical for the maintenance of self-tolerance. Expression levels of the transcription factor Foxp3 have been causally associated with Treg differentiation and function. Recent studies show that Foxp3 can also be transiently expressed in effector T cells; however, stable Foxp3 expression is required for development of a functional Treg suppressor phenotype. Here, we demonstrate that Foxp3 is acetylated, and this can be reciprocally regulated by the histone acetyltransferase p300 and the histone deacetylase SIRT1. Hyperacetylation of Foxp3 prevented polyubiquitination and proteasomal degradation, therefore dramatically increasing stable Foxp3 protein levels. Moreover, using mouse splenocytes, human peripheral blood mononuclear cells, T cell clones, and skin-derived T cells, we demonstrate that treatment with histone deacetylase inhibitors resulted in significantly increased numbers of functional Treg cells. Taken together, our data demonstrate that modulation of the acetylation state of Foxp3 provides a novel molecular mechanism for assuring rapid temporal control of Foxp3 levels in T cells, thereby regulating Treg numbers and functionality. Manipulating Foxp3 acetylation levels could therefore provide a new therapeutic strategy to control inappropriate (auto)immune responses.

Mps1 Phosphorylates Borealin to Control Aurora B Activity and Chromosome Alignment

Maintenance of chromosomal stability relies on coordination between various processes that are critical for proper chromosome segregation in mitosis. Here we show that monopolar spindle 1 (Mps1) kinase, which is essential for the mitotic checkpoint, also controls correction of improper chromosome attachments. We report that Borealin/DasraB, a member of the complex that regulates the Aurora B kinase, is directly phosphorylated by Mps1 on residues that are crucial for Aurora B activity and chromosome alignment. As a result, cells lacking Mps1 kinase activity fail to efficiently align chromosomes due to impaired Aurora B function at centromeres, leaving improper attachments uncorrected. Strikingly, Borealin/DasraB bearing phosphomimetic mutations restores Aurora B activity and alignment in Mps1-depleted cells. Mps1 thus coordinates attachment error correction and checkpoint signaling, two crucial responses to unproductive chromosome attachments.

Redox-sensitive cysteines bridge p300/CBP-mediated acetylation and FoxO4 activity

Cellular damage invoked by reactive oxygen species plays a key role in the pathobiology of cancer and aging. Forkhead box class O (FoxO) transcription factors are involved in various cellular processes including cell cycle regulation, apoptosis and resistance to reactive oxygen species, and studies in animal models have shown that these transcription factors are of vital importance in tumor suppression, stem cell maintenance and lifespan extension. Here we report that the activity of FoxO in human cells is directly regulated by the cellular redox state through a unique mechanism in signal transduction. We show that reactive oxygen species induce the formation of cysteine-thiol disulfide-dependent complexes of FoxO and the p300/CBP acetyltransferase, and that modulation of FoxO biological activity by p300/CBP-mediated acetylation is fully dependent on the formation of this redox-dependent complex. These findings directly link cellular redox status to the activity of the longevity protein FoxO.

Mdm2 induces mono-ubiquitination of FOXO4.

Background The Forkhead box O (FOXO) class of transcription factors are involved in the regulation of several cellular responses including cell cycle progression and apoptosis. Furthermore, in model organisms FOXOs act as tumor suppressors and affect aging. Previously, we noted that FOXOs and p53 are remarkably similar within their spectrum of regulatory proteins [1]. For example, the de-ubiquitinating enzyme USP7 removes ubiquitin from both FOXO and p53. However, Skp2 has been identified as E3 ligase for FOXO1, whereas Mdm2 is the prime E3 ligase for p53. Principal Findings/Methodology Here we provide evidence that Mdm2 acts as an E3 ligase for FOXO as well. In vitro incubation of Mdm2 and FOXO results in ATP-dependent (multi)mono-ubiquitination of FOXO similar to p53. Furthermore, in vivo co-expression of Mdm2 and FOXO induces FOXO mono-ubiquitination and consistent with this result, siRNA-mediated depletion of Mdm2 inhibits mono-ubiquitination of FOXO induced by hydrogen peroxide. Regulation of FOXO ubiquitination by Mdm2 is likely to be direct since Mdm2 and FOXO co-immunoprecipitate. In addition, Mdm2-mediated ubiquitination regulates FOXO transcriptional activity. Conclusions/Significance These data identify Mdm2 as a novel E3 ligase for FOXOs and extend the analogous mode of regulation between FOXO and p53.

Chromosomal instability by inefficient Mps1 auto-activation due to a weakened mitotic checkpoint and lagging chromosomes.

Background Chromosomal instability (CIN), a feature widely shared by cells from solid tumors, is caused by occasional chromosome missegregations during cell division. Two of the causes of CIN are weakened mitotic checkpoint signaling and persistent merotelic attachments that result in lagging chromosomes during anaphase. Principal Findings Here we identify an autophosphorylation event on Mps1 that is required to prevent these two causes of CIN. Mps1 is phosphorylated in mitotic cells on at least 7 residues, 4 of which by autophosphorylation. One of these, T676, resides in the activation loop of the kinase domain and a mutant that cannot be phosphorylated on T676 is less active than wild-type Mps1 but is not kinase-dead. Strikingly, cells in which endogenous Mps1 was replaced with this mutant are viable but missegregate chromosomes frequently. Anaphase is initiated in the presence of misaligned and lagging chromosomes, indicative of a weakened checkpoint and persistent merotelic attachments, respectively. Conclusions/Significance We propose that full activity of Mps1 is essential for maintaining chromosomal stability by allowing resolution of merotelic attachments and to ensure that single kinetochores achieve the strength of checkpoint signaling sufficient to prevent premature anaphase onset and chromosomal instability. To our knowledge, phosphorylation of T676 on Mps1 is the first post-translational modification in human cells of which the absence causes checkpoint weakening and CIN without affecting cell viability.

The Peptidyl-Isomerase Pin1 Regulates p27kip1 Expression through Inhibition of Forkhead Box O Tumor Suppressors

The Forkhead box O (FOXO) protein family is an evolutionarily conserved subclass of transcription factors recently identified as bona fide tumor suppressors. Preventing the accumulation of cellular damage due to oxidative stress is thought to underlie its tumor-suppressive role. Oxidative stress, in turn, also feedback controls FOXO4 function. Regulation of this process, however, is poorly understood but may be relevant to the ability of FOXO to control tumor suppression. Here, we characterize novel FOXO4 phosphorylation sites after increased cellular oxidative stress and identify the isomerase Pin1, a protein frequently found to be overexpressed in cancer, as a critical regulator of p27(kip1) through FOXO4 inhibition. We show that Pin1 requires these phosphorylation events to act negatively on FOXO4 transcriptional activity. Consistent with this, oxidative stress induces binding of Pin1 to FOXO, thereby attenuating its monoubiquitination, a yet uncharacterized mode of substrate modulation by Pin1. We have previously shown that monoubiquitination is involved in controlling nuclear translocation in response to cellular stress, and indeed, Pin1 prevents nuclear FOXO4 accumulation. Interestingly, Pin1 acts on FOXO through stimulation of the activity of the deubiquitinating enzyme HAUSP/USP7. Ultimately, this results in decreased transcriptional activity towards target genes, including the cell cycle arrest gene p27(kip1). Notably, in a primary human breast cancer panel, low p27(kip1) levels inversely correlated with Pin1 expression. Thus, Pin1 is identified as a novel negative FOXO regulator, interconnecting FOXO phosphorylation and monoubiquitination in response to cellular stress to regulate p27(kip1).

Regulation of histone H3K4 tri-methylation and PAF complex recruitment by the Ccr4-Not complex

Efficient transcription is linked to modification of chromatin. For instance, tri-methylation of lysine 4 on histone H3 (H3K4) strongly correlates with transcriptional activity and is regulated by the Bur1/2 kinase complex. We found that the evolutionarily conserved Ccr4-Not complex is involved in establishing H3K4 tri-methylation in Saccharomyces cerevisiae. We observed synthetic lethal interactions of Ccr4-Not components with BUR1 and BUR2. Further analysis indicated that the genes encoding the Not-proteins are essential for efficient regulation of H3K4me3, but not H3K4me1/2, H3K36me2 or H3K79me2/3 levels. Moreover, regulation of H3K4me3 levels by NOT4 is independent of defects in RNA polymerase II loading. We found NOT4 to be important for ubiquitylation of histone H2B via recruitment of the PAF complex, but not for recruitment or activation of the Bur1/2 complex. These results suggest a mechanism in which the Ccr4-Not complex functions parallel to or downstream of the Bur1/2 kinase to facilitate H3K4me3 via PAF complex recruitment.

AGC kinases regulate phosphorylation and activation of eukaryotic translation initiation factor 4B

Eukaryotic translation initiation factor 4B (eIF4B) plays a critical role during the initiation of protein synthesis and its activity can be regulated by multiple phosphorylation events. In a search for novel protein kinase B (PKB/c-akt) substrates, we identified eIF4B as a potential target. Using an in vitro kinase assay, we found that PKB can directly phosphorylate eIF4B on serine 422 (ser422). Activation of a conditional PKB mutant, interleukin-3 (IL-3) or insulin stimulation resulted in PKB-dependent phosphorylation of this residue in vivo. This was prevented by pretreatment of cells with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 or pharmacological inhibition of PKB. Pretreatment of cells with rapamycin, inhibiting mTOR or U0126 to inhibit MEK, had little effect on eIF4B ser422 phosphorylation. In contrast, following amino-acid refeeding, eIF4B ser422 phosphorylation was found to be mammalian target of rapamycin (mTOR)-dependent. We further identified eIF4B ser406 as a novel mitogen-regulated phosphorylation site. Insulin-induced phosphorylation of eIF4B ser406 was dependent on both MEK and mTOR activity. Utilizing a novel translational control luciferase assay, we could further demonstrate that phosphorylation of ser406 or ser422 is essential for optimal translational activity of eIF4B. These data provide novel insights into complex multikinase regulation of eIF4B phosphorylation and reveal an important mechanism by which PKB can regulate translation, potentially critical for the transforming capacity of this AGC kinase family member.

Activation of forkhead box O transcription factors by oncogenic BRAF promotes p21cip1-dependent senescence.

Oncogene-induced senescence (OIS) is a potent tumor-suppressive mechanism that is thought to come at the cost of aging. The Forkhead box O (FOXO) transcription factors are regulators of life span and tumor suppression. However, whether and how FOXOs function in OIS have been unclear. Here, we show a role for FOXO4 in mediating senescence by the human BRAF(V600E) oncogene, which arises commonly in melanoma. BRAF(V600E) signaling through mitogen-activated protein kinase/extracellular signal-regulated kinase kinase resulted in increased reactive oxygen species levels and c-Jun NH(2) terminal kinase-mediated activation of FOXO4 via its phosphorylation on Thr(223), Ser(226), Thr(447), and Thr(451). BRAF(V600E)-induced FOXO4 phosphorylation resulted in p21(cip1)-mediated cell senescence independent of p16(ink4a) or p27(kip1). Importantly, melanocyte-specific activation of BRAF(V600E) in vivo resulted in the formation of skin nevi expressing Thr(223)/Ser(226)-phosphorylated FOXO4 and elevated p21(cip1). Together, these findings support a model in which FOXOs mediate a trade-off between cancer and aging.