Regulation of Chk1 kinase by autoinhibition and ATR-mediated phosphorylation

The checkpoint kinase Chk1 undergoes ATR-mediated phosphorylation and activation in response to unreplicated DNA, but the precise mechanism of Chk1 activation is not known. In this study, we have analyzed the domain structure of Xenopus Chk1 and explored the mechanism of its activation by ATR-mediated phosphorylation. We show that the C-terminal region of Xenopus Chk1 contains an autoinhibitory region (AIR), which largely overlaps with a bipartite, unusually long ( approximately 85-amino acid) nuclear localization signal. When coexpressed in oocytes or embryos, the AIR can interact with and inhibit the kinase domain of Chk1, but not full-length Chk1, suggesting an autoinhibitory intramolecular interaction in the Chk1 molecule. If linked with the preceding ATR phosphorylation domain that has either phospho-mimic mutation or genuine phosphorylation, however, the AIR can no longer interact with or inhibit the kinase domain, suggesting a conformational change of the AIR by ATR-mediated phosphorylation. Even in full-length Chk1, such phospho-mimic mutation can interfere with the autoinhibitory intramolecular interaction, but only if this interaction is somewhat weakened by an additional mutation in the AIR. These results provide significant insights into the mechanism of Chk1 activation at the DNA replication checkpoint.     

ATR-mediated checkpoint pathways regulate phosphorylation and activation of human Chk1

Chk1 is an evolutionarily conserved protein kinase that regulates cell cycle progression in response to checkpoint activation. In this study, we demonstrated that agents that block DNA replication or cause certain forms of DNA damage induce the phosphorylation of human Chk1. The phosphorylated form of Chk1 possessed higher intrinsic protein kinase activity and eluted more quickly on gel filtration columns. Serines 317 and 345 were identified as sites of phosphorylation in vivo, and ATR (the ATM- and Rad3-related protein kinase) phosphorylated both of these sites in vitro. Furthermore, phosphorylation of Chk1 on serines 317 and 345 in vivo was ATR dependent. Mutants of Chk1 containing alanine in place of serines 317 and 345 were poorly activated in response to replication blocks or genotoxic stress in vivo, were poorly phosphorylated by ATR in vitro, and were not found in faster-eluting fractions by gel filtration. These findings demonstrate that the activation of Chk1 in response to replication blocks and certain forms of genotoxic stress involves phosphorylation of serines 317 and 345. In addition, this study implicates ATR as a direct upstream activator of Chk1 in human cells.     

CHK2-dependent phosphorylation of BRCA1 in hypoxia

Hypoxia induces a diverse spectrum of changes in the expression and activity of numerous DNA repair factors within the tumor microenvironment. In particular, we and others have shown that hypoxia induces phosphorylation and activation of the checkpoint kinase, CHK2, in an ATM-dependent manner. One downstream target of CHK2, the BRCA1 protein, plays a critical role in both DNA repair and cell cycle checkpoint regulation in mammalian cells. Here we report that BRCA1 is specifically phosphorylated on Serine 988 in response to hypoxic stress, and phosphorylation at this site is dependent on CHK2 expression. These findings enhance our understanding of ATM-CHK2 pathway activation in hypoxia, and they identify a novel role for BRCA1 in the response to hypoxic stress.     

Direct requirement for Xmus101 in ATR-mediated phosphorylation of Claspin bound Chk1 during checkpoint signaling

TopBP1-like proteins, which include Xenopus laevis Xmus101, are required for DNA replication and have been linked to replication checkpoint control. A direct role for TopBP1/Mus101 in checkpoint control has been difficult to prove, however, because of the requirement for replication in generating the DNA structures that activate the checkpoint. Checkpoint activation occurs in X. laevis egg extracts upon addition of an oligonucleotide duplex (AT70). We show that AT70 bypasses the requirement for replication in checkpoint activation. We take advantage of this replication-independent checkpoint system to determine the role of Xmus101 in the checkpoint. We find that Xmus101 is essential for AT70-mediated checkpoint signaling and that it functions to promote phosphorylation of Claspin bound Chk1 by the ataxia-telangiectasia and Rad-3-related (ATR) protein kinase. We also identify a separation-of-function mutant of Xmus101. In extracts expressing this mutant, replication of sperm chromatin occurs normally; however, the checkpoint response to stalled replication forks fails. These data demonstrate that Xmus101 functions directly during signal relay from ATR to Chk1.     

p53 C-terminal phosphorylation by CHK1 and CHK2 participates in the regulation of DNA-damage-induced C-terminal acetylation

The tumor suppressor protein p53 mediates stress-induced growth arrest or apoptosis and plays a major role in safeguarding genome integrity. In response to DNA damage, p53 can be modified at multiple sites by phosphorylation and acetylation. We report on the characterization of p53 C-terminal phosphorylation by CHK1 and CHK2, two serine/threonine (Ser/Thr) protein kinases, previously implicated in the phosphorylation of the p53 N terminus. Using tryptic phosphopeptide mapping, we have identified six additional CHK1 and CHK2 sites residing in the final 100 amino acids of p53. Phosphorylation of at least three of these sites, Ser366, Ser378, and Thr387, was induced by DNA damage, and the induction at Ser366 and Thr387 was abrogated by small interfering RNA targeting chk1 and chk2. Furthermore, mutation of these phosphorylation sites has a different impact on p53 C-terminal acetylation and on the activation of p53-targeted promoters. Our results demonstrate a possible interplay between p53 C-terminal phosphorylation and acetylation, and they provide an additional mechanism for the control of the activity of p53 by CHK1 and CHK2.     

An essential role for hGle1 nucleocytoplasmic shuttling in mRNA export

Gle1 is required for mRNA export in yeast and human cells. Here, we report that two human Gle1 (hGle1) isoforms are expressed in HeLa cells (hGle1A and B). The two encoded proteins are identical except for their COOH-terminal regions. hGle1A ends with a unique four-amino acid segment, whereas hGle1B has a COOH-terminal 43-amino acid span. Only hGle1B, the more abundant isoform, localizes to the nuclear envelope (NE) and pore complex. To test whether hGle1 is a dynamic shuttling transport factor, we microinjected HeLa cells with recombinant hGle1 and conducted photobleaching studies of live HeLa cells expressing EGFP-hGle1. Both strategies show that hGle1 shuttles between the nucleus and cytoplasm. An internal 39-amino acid domain is necessary and sufficient for mediating nucleocytoplasmic transport. Using a cell-permeable peptide strategy, we document a role for hGle1 shuttling in mRNA export. An hGle1 shuttling domain (SD) peptide impairs the export of both total poly(A)+ RNA and the specific dihydrofolate reductase mRNA. Coincidentally, SD peptide-treated cells show decreased endogenous hGle1 localization at the NE and reduced nucleocytoplasmic shuttling of microinjected, recombinant hGle1. These findings pinpoint the first functional motif in hGle1 and link hGle1 to the dynamic mRNA export mechanism.     

Delta-catenin-induced dendritic morphogenesis. An essential role of p190RhoGEF interaction through Akt1-mediated phosphorylation

Delta-catenin was first identified through its interaction with Presenilin-1 and has been implicated in the regulation of dendrogenesis and cognitive function. However, the molecular mechanisms by which delta-catenin promotes dendritic morphogenesis were unclear. In this study, we demonstrated delta-catenin interaction with p190RhoGEF, and the importance of Akt1-mediated phosphorylation at Thr-454 residue of delta-catenin in this interaction. We have also found that delta-catenin overexpression decreased the binding between p190RhoGEF and RhoA, and significantly lowered the levels of GTP-RhoA but not those of GTP-Rac1 and -Cdc42. Delta-catenin T454A, a defective form in p190RhoGEF binding, did not decrease the binding between p190RhoGEF and RhoA. Delta-catenin T454A also did not lower GTP-RhoA levels and failed to induce dendrite-like process formation in NIH 3T3 fibroblasts. Furthermore, delta-catenin T454A significantly reduced the length and number of mature mushroom shaped spines in primary hippocampal neurons. These results highlight signaling events in the regulation of delta-catenin-induced dendrogenesis and spine morphogenesis.     

An essential role for PICK1 in NMDA receptor-dependent bidirectional synaptic plasticity

PICK1 is a calcium-sensing, PDZ domain-containing protein that interacts with GluR2 and GluR3 AMPA receptor (AMPAR) subunits and regulates their trafficking. Although PICK1 has been principally implicated in long-term depression (LTD), PICK1 overexpression in CA1 pyramidal neurons causes a CaMK- and PKC-dependent potentiation of AMPAR-mediated transmission and an increase in synaptic GluR2-lacking AMPARs, mechanisms associated with NMDA receptor (NMDAR)-dependent long-term potentiation (LTP). Here, we directly tested whether PICK1 participates in both hippocampal NMDAR-dependent LTP and LTD. We show that the PICK1 potentiation of AMPAR-mediated transmission is NMDAR dependent and fully occludes LTP. Conversely, blockade of PICK1 PDZ interactions or lack of PICK1 prevents LTP. These observations demonstrate an important role for PICK1 in LTP. In addition, deletion of PICK1 or blockade of PICK1 PDZ binding prevented NMDAR-dependent LTD. Thus, PICK1 plays a critical role in bidirectional NMDAR-dependent long-term synaptic plasticity in the hippocampus.     

An essential role for Dicer in adipocyte differentiation

Dicer is a cellular enzyme required for the processing of pre‐miRNA molecules into mature miRNA, and Dicer and miRNA biogenesis have been found to play important roles in a variety of physiologic processes. Recently, reports of alterations in miRNA expression levels in cultured pre‐adipogenic cell lines during differentiation and findings of differences between the miRNA expression signatures of white and brown adipose have suggested that miRNA molecules might regulate adipocyte differentiation and the formation of adipose tissue. However, direct evidence that miRNAs regulate adipogenesis is lacking. To determine if Dicer and mature miRNA govern adipocyte differentiation, we utilized primary cells isolated from mice bearing Dicer‐conditional alleles to study adipogenesis in the presence or absence of miRNA biogenesis. Our results reveal that Dicer is required for adipogenic differentiation of mouse embryonic fibroblasts and primary cultures of pre‐adipocytes. Furthermore, the requirement for Dicer in adipocyte differentiation is not due to miRNA‐mediated alterations in cell proliferation, as deletion of the Ink4a locus and the prevention of premature cellular senescence normally induced in primary cells upon Dicer ablation fails to rescue adipogenic differentiation in fibroblasts and pre‐adipocytes. J. Cell. Biochem. 110: 812–816, 2010. © 2010 Wiley‐Liss, Inc.     

An essential role for Akt1 in dendritic cell function and tumor immunotherapy

Current dendritic cell (DC) vaccine preparations involving ex vivo differentiation and maturation produce short-lived, transiently active DCs that may curtail T-cell responses in vivo. We demonstrate that Akt1, downregulation of which decreases DC lifespan, is critical for proinflammatory signal-mediated DC survival and maturation. Lipopolysaccharide or CD40 signaling stabilizes Akt1, promoting both activation and Bcl-2-dependent survival of DCs. Expression of a potent allele encoding a lipid raft-targeted Akt1, M(F)-DeltaAkt, is sufficient for maturation and survival of murine bone marrow-derived DCs in vivo. M(F)-DeltaAkt-transduced DCs enhanced T-cell proliferation, activation and long-term memory responses, enabling eradication of large pre-established lymphomas and aggressive B16 melanomas. Human myeloid DCs expressing constitutively active M(F)-DeltahAkt also survived significantly longer and promoted antigen-specific T-cell responses. Thus, Akt1 is a critical regulator of DC lifespan, and its manipulation in DCs can improve the clinical efficacy of DC-based tumor vaccines.     

MCL-1 inhibits BAX in the absence of MCL-1/BAX Interaction

The BCL-2 family of proteins plays a major role in the control of apoptosis as the primary regulator of mitochondrial permeability. The pro-apoptotic BCL-2 homologues BAX and BAK are activated following the induction of apoptosis and induce cytochrome c release from mitochondria. A second class of BCL-2 homologues, the BH3-only proteins, is required for the activation of BAX and BAK. The activity of both BAX/BAK and BH3-only proteins is opposed by anti-apoptotic BCL-2 homologues such as BCL-2 and MCL-1. Here we show that anti-apoptotic MCL-1 inhibits the function of BAX downstream of its initial activation and translocation to mitochondria. Although MCL-1 interacted with BAK and inhibited its activation, the activity of MCL-1 against BAX was independent of an interaction between the two proteins. However, the anti-apoptotic function of MCL-1 required the presence of BAX. These results suggest that the pro-survival activity of MCL-1 proceeds via inhibition of BAX function at mitochondria, downstream of its activation and translocation to this organelle.     

MCL-1 is essential for survival but dispensable for metabolic fitness of FOXP3+ regulatory T cells

FOXP3⁺ regulatory T (Treg) cells are essential for maintaining immunological tolerance. Given their importance in immune-related diseases, cancer and obesity, there is increasing interest in targeting the Treg cell compartment therapeutically. New pharmacological inhibitors that specifically target the prosurvival protein MCL-1 may provide this opportunity, as Treg cells are particularly reliant upon this protein. However, there are two distinct isoforms of MCL-1; one located at the outer mitochondrial membrane (OMM) that is required to antagonize apoptosis, and another at the inner mitochondrial membrane (IMM) that is reported to maintain IMM structure and metabolism via ATP production during oxidative phosphorylation. We set out to elucidate the relative importance of these distinct biological functions of MCL-1 in Treg cells to assess whether MCL-1 inhibition might impact upon the metabolism of cells able to resist apoptosis. Conditional deletion of Mcl1 in FOXP3⁺ Treg cells resulted in a lethal multiorgan autoimmunity due to the depletion of the Treg cell compartment. This striking phenotype was completely rescued by concomitant deletion of the apoptotic effector proteins BAK and BAX, indicating that apoptosis plays a pivotal role in the homeostasis of Treg cells. Notably, MCL-1-deficient Treg cells rescued from apoptosis displayed normal metabolic capacity. Moreover, pharmacological inhibition of MCL-1 in Treg cells resistant to apoptosis did not perturb their metabolic function. We conclude that Treg cells require MCL-1 only to antagonize apoptosis and not for metabolism. Therefore, MCL-1 inhibition could be used to manipulate Treg cell survival for clinical benefit without affecting the metabolic fitness of cells resisting apoptosis.Subject terms: Disease genetics, Immune cell death     

Translational repression of MCL-1 couples stress-induced eIF2 alpha phosphorylation to mitochondrial apoptosis initiation

The integrated stress response (ISR) integrates a broad range of environmental and endogenous stress signals to the phosphorylation of the alpha-subunit of eukaryotic translation initiation factor 2 (eIF2 alpha). Although intense or prolonged activation of this pathway is known to induce apoptosis, the molecular mechanisms coupling stress-induced eIF2 alpha phosphorylation to the cell death machinery have remained incompletely understood. In this study, we characterized apoptosis initiation in response to classical activators of the ISR (tunicamycin, UVC, elevated osmotic pressure, arsenite). We found that all applied stress stimuli activated a mitochondrial pathway of apoptosis initiation. Rapid and selective down-regulation of the anti-apoptotic BCL-2 family protein MCL-1 preceded the activation of BAX, BAK, and caspases. Stabilization of MCL-1 blocked apoptosis initiation, while cells with reduced MCL-1 protein content were strongly sensitized to stress-induced apoptosis. Stress-induced elimination of MCL-1 occurred with unchanged protein turnover and independently of MCL-1 mRNA levels. In contrast, stress-induced phosphorylation of eIF2 alpha at Ser(51) was both essential and sufficient for the down-regulation of MCL-1 protein in stressed cells. These findings indicate that stress-induced phosphorylation of eIF2 alpha is directly coupled to mitochondrial apoptosis regulation via translational repression of MCL-1. Down-regulation of MCL-1 enables but not enforces apoptosis initiation in stressed cells.     

An essential role for Prox1 in the induction of the lymphatic endothelial cell phenotype

The process of angiogenesis has been well documented, but little is known about the biology of lymphatic endothelial cells and the molecular mechanisms controlling lymphangiogenesis. The homeobox gene Prox1 is expressed in a subpopulation of endothelial cells that, after budding from veins, gives rise to the mammalian lymphatic system. In Prox1(-)(/-) embryos, this budding becomes arrested at around embryonic day (E)11.5, resulting in embryos without lymphatic vasculature. Unlike the endothelial cells that bud off in E11.5 wild-type embryos, those of Prox1-null embryos did not co-express any lymphatic markers such as VEGFR-3, LYVE-1 or SLC. Instead, the mutant cells appeared to have a blood vascular phenotype, as determined by their expression of laminin and CD34. These results suggest that Prox1 activity is required for both maintenance of the budding of the venous endothelial cells and differentiation toward the lymphatic phenotype. On the basis of our findings, we propose that a blood vascular phenotype is the default fate of budding embryonic venous endothelial cells; upon expression of Prox1, these budding cells adopt a lymphatic vasculature phenotype.     

An essential role for Pin1 in Xenopus laevis embryonic development revealed by specific inhibitors

The peptidyl prolyl cis/trans isomerase (PPIase) Pin1 plays an important role in phosphorylation-dependent events of the cell cycle. This function is linked to its display of two phosphothreonine/phosphoserine-proline binding motifs, one within the type IV WW domain and a second within the parvulin-like catalytic domain. By microinjection of the compound Ac-Phe-D-Thr(PO3H2)-Pip-Nal-Gln-NH2, which inhibits Xenopus laevis Pin1 with a Ki value of 19.4+/-1.5 nM, into the animal pole of X. laevis embryos at the two-cell stage, the impact of Pin1 PPIase activity on cell cycle progression and embryonic development could be analysed, independent of WW domain-mediated phosphoprotein binding. Injected embryos showed a dramatically decreased survival rate at late stages of development that could only be partially compensated by co-injection with mRNAs of enzymatically active Pin1 variants, demonstrating that the phosphorylation-specific PPIase activity of Pin1 is essential for cell division and development in X. laevis.