Double-knockout Mouse Embryonic Fibroblasts Research Articles

Functional characterization of the CRY2 circadian clock component variant p.Ser420Phe revealed a new degradation pathway for CRY2

Cryptochromes (CRYs) are essential components of the circadian clock, playing a pivotal role as transcriptional repressors. Despite their significance, the precise mechanisms underlying CRYs' involvement in the circadian clock remain incompletely understood. In this study, we identified a rare CRY2 variant, p.Ser420Phe, from the 1000 Genomes Project and Ensembl database that is located in the functionally important coiled-coil-like helix (CC−helix) region. Functional characterization of this variant at the cellular level revealed that p.Ser420Phe CRY2 had reduced repression activity on CLOCK:BMAL1−driven transcription due to its reduced affinity to the core clock protein PER2 and defective translocation into the nucleus. Intriguingly, the CRY2 variant exhibited an unexpected resistance to degradation via the canonical proteasomal pathway, primarily due to the loss of interactions with E3 ligases (FBXL3 and FBXL21), which suggests Ser-420 of CRY2 is required for the interaction with E3 ligases. Further studies revealed that wild type and CRY2 variant are degraded by the lysosomal-mediated degradation pathway, a mechanism not previously associated with CRY2. Surprisingly, our complementation study with Cry1−/−Cry2−/− double knockout mouse embryonic fibroblast cells indicated that CRY2 variant caused 7 hours shorter circadian period length in contrast to the observed prolonged period length in CRY2−/− cell lines. In summary, this study reveals a hitherto unknown degradation pathway for CRY2, shedding new light on the regulation of circadian rhythm period length.

Functionally confirmed compound heterozygous ADAM17 missense loss-of-function variants cause neonatal inflammatory skin and bowel disease 1

A disintegrin and metalloprotease 17 (ADAM17) is the major sheddase that processes more than 80 substrates, including tumour necrosis factor-α (TNFα). The homozygous genetic deficiency of ADAM17 causing a complete loss of ADAM17 expression was reported to be linked to neonatal inflammatory skin and bowel disease 1 (NISBD1). Here we report for the first time, a family with NISBD1 caused by functionally confirmed compound heterozygous missense variants of ADAM17, namely c.1699T>C (p.Cys567Arg) and c.1799G>A (p.Cys600Tyr). Both variants were detected in two siblings with clinical features of NISBD1, such as erythroderma with exudate in whole body, recurrent skin infection and sepsis and prolonged diarrhoea. In a cell-based assay using Adam10/17 double-knockout mouse embryonic fibroblasts (Adam10/17−/− mEFs) exogenously expressing each of these mutants, phorbol 12-myristate 13-acetate-stimulated shedding was strongly reduced compared with wild-type ADAM17. Thus, in vitro functional assays demonstrated that both missense variants cause the loss-of-function of ADAM17, resulting in the development of NISBD1. Our study further expands the spectrum of genetic pathology underlying ADAM17 in NISBD1 and establishes functional assay systems for its missense variants.

S6K1 Is Indispensible for Stress-Induced Microtubule Acetylation and Autophagic Flux.

Autophagy is a specific macromolecule and organelle degradation process. The target macromolecule or organelle is first enclosed in an autophagosome, and then delivered along acetylated microtubules to the lysosome. Autophagy is triggered by stress and largely contributes to cell survival. We have previously shown that S6K1 kinase is essential for autophagic flux under stress conditions. Here, we aimed to elucidate the underlying mechanism of S6K1 involvement in autophagy. We stimulated autophagy in S6K1/2 double-knockout mouse embryonic fibroblasts by exposing them to different stress conditions. Transient gene overexpression or silencing, immunoblotting, immunofluorescence, flow cytometry, and ratiometric fluorescence analyses revealed that the perturbation of autophagic flux in S6K1-deficient cells did not stem from impaired lysosomal function. Instead, the absence of S6K1 abolished stress-induced tubulin acetylation and disrupted the acetylated microtubule network, in turn impairing the autophagosome-lysosome fusion. S6K1 overexpression restored tubulin acetylation and autophagic flux in stressed S6K1/2-deficient cells. Similar effect of S6K1 status was observed in prostate cancer cells. Furthermore, overexpression of an acetylation-mimicking, but not acetylation-resistant, tubulin variant effectively restored autophagic flux in stressed S6K1/2-deficient cells. Collectively, S6K1 controls tubulin acetylation, hence contributing to the autophagic flux induced by different stress conditions and in different cells.

The Arg-293 of Cryptochrome1 is responsible for the allosteric regulation of CLOCK-CRY1 binding in circadian rhythm

Mammalian circadian clocks are driven by transcription/translation feedback loops composed of positive transcriptional activators (BMAL1 and CLOCK) and negative repressors (CRYPTOCHROMEs (CRYs) and PERIODs (PERs)). CRYs, in complex with PERs, bind to the BMAL1/CLOCK complex and repress E-box-driven transcription of clock-associated genes. There are two individual CRYs, with CRY1 exhibiting higher affinity to the BMAL1/CLOCK complex than CRY2. It is known that this differential binding is regulated by a dynamic serine-rich loop adjacent to the secondary pocket of both CRYs, but the underlying features controlling loop dynamics are not known. Here we report that allosteric regulation of the serine-rich loop is mediated by Arg-293 of CRY1, identified as a rare CRY1 SNP in the Ensembl and 1000 Genomes databases. The p.Arg293His CRY1 variant caused a shortened circadian period in a Cry1-/-Cry2-/- double knockout mouse embryonic fibroblast cell line. Moreover, the variant displayed reduced repressor activity on BMAL1/CLOCK driven transcription, which is explained by reduced affinity to BMAL1/CLOCK in the absence of PER2 compared with CRY1. Molecular dynamics simulations revealed that the p.Arg293His CRY1 variant altered a communication pathway between Arg-293 and the serine loop by reducing its dynamicity. Collectively, this study provides direct evidence that allosterism in CRY1 is critical for the regulation of circadian rhythm.

Mitochondrial residence of the apoptosis inducer BAX is more important than BAX oligomerization in promoting membrane permeabilization

Permeabilization of the mitochondrial outer membrane is a key step in the intrinsic apoptosis pathway, triggered by the release of mitochondrial intermembrane space proteins into the cytoplasm. The BCL-2-associated X apoptosis regulator (BAX) protein critically contributes to this process by forming pores in the mitochondrial outer membrane. However, the relative roles of the mitochondrial residence of BAX and its oligomerization in promoting membrane permeabilization are unclear. To this end, using both cell-free and cellular experimental systems, including membrane permeabilization, size-exclusion chromatography-based oligomer, and retrotranslocation assays, along with confocal microscopy analysis, here we studied two BAX C-terminal variants, T182I and G179P. Neither variant formed large oligomers when activated in liposomes. Nevertheless, the G179P variant could permeabilize liposome membranes, suggesting that large BAX oligomers are not essential for the permeabilization. However, when G179P was transduced into BAX/BCL2 agonist killer (BAK) double-knockout mouse embryonic fibroblasts, its location was solely cytoplasmic, and it then failed to mediate cell death. In contrast, T182I was inefficient in both liposome insertion and permeabilization. Yet, when transduced into cells, BAXT182I resided predominantly on mitochondria, because of its slow retrotranslocation and mediated apoptosis as efficiently as WT BAX. We conclude that BAX's mitochondrial residence in vivo, regulated by both targeting and retrotranslocation, is more significant for its pro-apoptotic activity than its ability to insert and to form higher-order oligomers in model membranes. We propose that this finding should be taken into account when developing drugs that modulate BAX activity.

ULK1 Phosphorylates and Regulates Mineralocorticoid Receptor

Mineralocorticoid receptor (MR) signaling regulates both renal Na-Cl reabsorption and K+ excretion. Wepreviously demonstrated that phosphorylation of S843 in the MR ligand-binding domain in renal intercalated cells is involved in the balance of these activities by regulating ligand binding and signaling. However, the kinase that phosphorylates MRS843 is unknown. Using a high-throughput screen assay of 197 kinases, we found that ULK1 is the principal kinase that is responsible for the phosphorylation of MRS843. The results were confirmed by invitro kinase assay, mass spectrometry, and siRNA knockdown experiments. Notably, phosphorylation at MRS843 was markedly reduced in ULK1/2 double knockout mouse embryonic fibroblasts. Upstream, we show that ULK1 activity is inhibited by phosphorylation induced by angiotensin II via mTOR in cell culture and invivo. These findings implicate mTOR and ULK1 as regulators of MR activity in intercalated cells, a pathway that is critical for maintaining electrolyte homeostasis.

Abstract 3324: FATE1 promotes mitochondrial hyperfusion and supports maintenance of mitochondrial networks following apoptosis stimulation

Abstract FATE1 (fetal and adult testis expressed 1), also known as the cancer-testis antigen BJ-HCC-2, is expressed in testis and tumor tissues. FATE1 was recently described as a major survival factor in tumor cells of various origins, by mediating the degradation of the pro-apoptotic BH3-only protein Bik (Maxfield et al., in Nat Commun 2015 Nov 16;6:8840) and through ER-mitochondrial uncoupling (Doghman-Bouguerra et al., in EMBO Rep 2016 Sep;17(9):1264-80). Interestingly, FATE1 shares high sequence homology with the mitochondrial Drp1 receptor MFF (mitochondrial fission factor). We thus investigated a possible role of FATE1 impact on mitochondrial morphology following the stimulation of apoptosis in cancer cells. We found that, similar to MFF, FATE1 is localized to outer mitochondrial membranes and, unlike MFF, additionally localized to the endoplasmic reticulum. Importantly, in contrast to MFF, FATE1 overexpression does not recruit Drp1 to mitochondria, and instead promotes hyperfusion of mitochondrial networks. Co-immunoprecipitation experiments and reconstitution of Mfn2 or Mfn1 in double knockout mouse embryonic fibroblasts indicate a role for the mitochondrial fusion protein Mfn2, but not Mfn1. As FATE1 overexpressing cancer cells were more resistant to mitochondrial fragmentation in response to TNF and valinomycin treatments, we propose FATE1 as a novel regulator of mitochondrial morphology changes occurring during apoptosis, with possible implications in FATE1-mediated resistance of cancer cells to chemotherapy. Citation Format: Anne Hamacher-Brady, Verena Lang, Nathan R. Brady. FATE1 promotes mitochondrial hyperfusion and supports maintenance of mitochondrial networks following apoptosis stimulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3324. doi:10.1158/1538-7445.AM2017-3324

P53 suppresses ribonucleotide reductase via inhibiting mTORC1.

Balanced deoxyribonucleotides pools are essential for cell survival and genome stability. Ribonucleotide reductase is the rate-limiting enzyme for the production of deoxyribonucleotides. We report here that p53 suppresses ribonucleotide reductase subunit 1 (RRM1) and 2 (RRM2) via inhibiting mammalian target of rapamycin complex 1 (mTORC1). In vitro, cancer cell lines and mouse embryonic fibroblast cells were treated with different concentrations of pharmacological inhibitors for different times. In vivo, rhabdomyosarcoma Rh30 cell tumor-bearing mice were treated with rapamycin or AZD8055. Protein levels and phosphorylation status were assessed by immunoblotting and mRNA levels were determined by real time RT-PCR. Pharmacological inhibition of mTORC1 with rapamycin, mTOR kinase with AZD8055 or protein kinase B with MK2206 resulted in decrease of RRM1 and RRM2 in Rh30 cells both in vitro and in mouse tumor xenografts. Moreover, eukaryotic translational initiation factor 4E-binding proteins 1 and 2 double knockout mouse embryonic fibroblast cells demonstrated an elevation of RRM1 and RRM2. Furthermore, down-regulation of mTOR-protein kinase B signaling or cyclin dependent kinase 4 led to decrease of RRM1 and RRM2 mRNAs. In addition, TP53 mutant cancer cells had elevation of RRM1 and RRM2, which was reduced by rapamycin. Importantly, human double minute 2 inhibitor nutlin-3 decreased RRM1 and RRM2 in TP53 wild type rhabdomyosarcoma Rh18 but not in TP53 mutated Rh30 cells. Our data demonstrated that mTOR enhances the cap-dependent protein translation and gene transcription of RRM1 and RRM2. Our findings might provide an additional mechanism by which p53 maintains genome stability.

Tet1 and Tet2 Protect DNA Methylation Canyons against Hypermethylation.

DNA methylation is a dynamic epigenetic modification with an important role in cell fate specification and reprogramming. The Ten eleven translocation (Tet) family of enzymes converts 5-methylcytosine to 5-hydroxymethylcytosine, which promotes passive DNA demethylation and functions as an intermediate in an active DNA demethylation process. Tet1/Tet2 double-knockout mice are characterized by developmental defects and epigenetic instability, suggesting a requirement for Tet-mediated DNA demethylation for the proper regulation of gene expression during differentiation. Here, we used whole-genome bisulfite and transcriptome sequencing to characterize the underlying mechanisms. Our results uncover the hypermethylation of DNA methylation canyons as the genomic key feature of Tet1/Tet2 double-knockout mouse embryonic fibroblasts. Canyon hypermethylation coincided with disturbed regulation of associated genes, suggesting a mechanistic explanation for the observed Tet-dependent differentiation defects. Based on these results, we propose an important regulatory role of Tet-dependent DNA demethylation for the maintenance of DNA methylation canyons, which prevents invasive DNA methylation and allows functional regulation of canyon-associated genes.

Molecular evidence of Zn chelation of the procaspase activating compound B-PAC-1 in B cell lymphoma.

The resistance of apoptosis in cancer cells is pivotal for their survival and is typically ruled by mutations or dysregulation of core apoptotic cascade. Mantle cell lymphoma (MCL) is a non-Hodgkin's B-cell malignancy expressing higher anti-apoptotic proteins providing survival advantage. B-PAC-1, a procaspase activating compound, induces apoptosis by sequestering Zn bound to procaspase-3, but the amino acids holding Zn in Caspase-3 is not known. Here we show that reintroduction of WT caspase-3 or 7 in Caspase3–7 double knock-out (DKO) mouse embryonic fibroblasts (MEF) promoted B-PAC-1 to induce apoptosis (27–43%), but not in DKO MEFs or MEFs expressing respective Casp3–7 catalytic mutants (12–13%). Using caspase-6 and -9 exosite analysis, we identified and mutated predicted Zn-ligands in caspase-3 (H108A, C148S and E272A) and overexpressed into DKO MEFs. Mutants carrying E272A abrogated Zn-reversal of apoptosis induced by B-PAC-1 via higher XIAP and smac expressions but not in H108A or C148S mutants. Co-immunoprecipitation analysis revealed stronger XIAP-caspase-3 interaction suggesting a novel mechanism of impulsive apoptosis resistance by disrupting predicted Zn-ligands in caspase-3. B-PAC-1 sponsored apoptosis in MCL cell lines (30–73%) via caspase-3 and PARP cleavages accompanied by loss of Mcl-1 and IAPs including XIAP while Zn substantially abrogated B-PAC-1-driven apoptosis (18–36%). In contrary, Zn is dispensable to inhibit staurosporin, bendamustine, ABT199 or MK206-induced apoptosis. Consistent to cell lines, B-PAC-1 stimulated cell death in primary B-lymphoma cells via caspase-3 cleavage with decline in both Mcl-1 and XIAP. This study underscores the first genetic evidence that B-PAC-1 driven apoptosis is mediated via Zn chelation.

Cbl-family ubiquitin ligases and their recruitment of CIN85 are largely dispensable for epidermal growth factor receptor endocytosis

Members of the casitas B-lineage lymphoma (Cbl) family (Cbl, Cbl-b and Cbl-c) of ubiquitin ligases serve as negative regulators of receptor tyrosine kinases (RTKs). An essential role of Cbl-family protein-dependent ubiquitination for efficient ligand-induced lysosomal targeting and degradation is now well-accepted. However, a more proximal role of Cbl and Cbl-b as adapters for CIN85-endophilin recruitment to mediate ligand-induced initial internalization of RTKs is supported by some studies but refuted by others. Overexpression and/or incomplete depletion of Cbl proteins in these studies is likely to have contributed to this dichotomy. To address the role of endogenous Cbl and Cbl-b in the internalization step of RTK endocytic traffic, we established Cbl/Cbl-b double-knockout (DKO) mouse embryonic fibroblasts (MEFs) and demonstrated that these cells lack the expression of both Cbl-family members as well as endophilin A, while they express CIN85. We show that ligand-induced ubiquitination of EGFR, as a prototype RTK, was abolished in DKO MEFs, and EGFR degradation was delayed. These traits were reversed by ectopic human Cbl expression. EGFR endocytosis, assessed using the internalization of 125I-labeled or fluorescent EGF, or of EGFR itself, was largely retained in Cbl/Cbl-b DKO compared to wild type MEFs. EGFR internalization was also largely intact in Cbl/Cbl-b depleted MCF-10A human mammary epithelial cell line. Inducible shRNA-mediated knockdown of CIN85 in wild type or Cbl/Cbl-b DKO MEFs had no impact on EGFR internalization. Our findings, establish that, at physiological expression levels, Cbl, Cbl-b and CIN85 are largely dispensable for EGFR internalization. Our results support the model that Cbl–CIN85–endophilin complex is not required for efficient internalization of EGFR, a prototype RTK.

MicroRNA 17-92 cluster mediates ETS1 and ETS2-dependent RAS-oncogenic transformation.

The ETS-family transcription factors Ets1 and Ets2 are evolutionarily conserved effectors of the RAS/ERK signaling pathway, but their function in Ras cellular transformation and biology remains unclear. Taking advantage of Ets1 and Ets2 mouse models to generate Ets1/Ets2 double knockout mouse embryonic fibroblasts, we demonstrate that deletion of both Ets1 and Ets2 was necessary to inhibit HrasG12V induced transformation both in vitro and in vivo. HrasG12V expression in mouse embryonic fibroblasts increased ETS1 and ETS2 expression and binding to cis-regulatory elements on the c-Myc proximal promoter, and consequently induced a robust increase in MYC expression. The expression of the oncogenic microRNA 17-92 cluster was increased in HrasG12V transformed cells, but was significantly reduced when ETS1 and ETS2 were absent. MYC and ETS1 or ETS2 collaborated to increase expression of the oncogenic microRNA 17-92 cluster in HrasG12V transformed cells. Enforced expression of exogenous MYC or microRNA 17-92 rescued HrasG12V transformation in Ets1/Ets2-null cells, revealing a direct function for MYC and microRNA 17-92 in ETS1/ETS2-dependent HrasG12V transformation.

An Interaction between Bcl-xL and the Voltage-dependent Anion Channel (VDAC) Promotes Mitochondrial Ca2+ Uptake

The role of the antiapoptotic protein Bcl-xL in regulating mitochondrial Ca(2+) ([Ca(2+)]mito) handling was examined in wild-type (WT) and Bcl-xL knock-out (Bcl-xL-KO) mouse embryonic fibroblast cells. Inositol 1,4,5-trisphosphate-generating agonist evoked cytosolic Ca(2+) transients that produced a larger [Ca(2+)]mito uptake in WT cells compared with Bcl-xL-KO. In permeabilized cells, stepping external [Ca(2+)] from 0 to 3 μm also produced a larger [Ca(2+)]mito uptake in WT; moreover, the [Ca(2+)]mito uptake capacity of Bcl-xL-KO cells was restored by re-expression of mitochondrially targeted Bcl-xL. Bcl-xL enhancement of [Ca(2+)]mito uptake persisted after dissipation of the mitochondrial membrane potential but was absent in mitoplasts lacking an outer mitochondrial membrane. The outer membrane-localized voltage-dependent anion channel (VDAC) is a known Ca(2+) permeability pathway that directly interacts with Bcl-xL. Bcl-xL interacted with VDAC1 and -3 isoforms, and peptides based on the VDAC sequence disrupted Bcl-xL binding. Peptides reduced [Ca(2+)]mito uptake in WT but were without effect in Bcl-xL-KO cells. In addition, peptides reduced [Ca(2+)]mito uptake in VDAC1 and VDAC3 knock-out but not VDAC1 and -3 double knock-out mouse embryonic fibroblast cells, confirming that Bcl-xL interacts functionally with VDAC1 and -3 but not VDAC2. Thus, an interaction between Bcl-xL and VDAC promotes matrix Ca(2+) accumulation by increasing Ca(2+) transfer across the outer mitochondrial membrane.

Abstract LB-149: Sustained activity of the mTOR pathway is required for DNA damage-induced up-regulation of RRM2 in cancer cells.

Abstract Balanced deoxyribonucleotides (dNTPs) are essential for the accurate and efficient replication and repair DNA synthesis. Increased or imbalanced dNTP pool leads to a mutator phenotype, while decreased dNTP level impairs DNA replication and repair, and hence cell survival. Ribonucleotide reductase (RNR) is the rate-limiting enzyme for the production of dNTPs from ribonucleotides and is under tight control during cell cycle and in response to stress. Increase of RNR is required for yeast cells to survive DNA damage and replication stress. However, the regulation and function of RNR in response to DNA damage remains unclear and controversial in mammalian cells. We report here that the mTOR pathway controls both RRM1 and RRM2 but not p53R2. mTOR signaling is required for mitomycin C (MMC) induced up-regulation of RRM2 in rhabdomyosarcoma Rh30 cells. Pharmacological inhibition of mTOR kinase with AZD8055 or mTORC1 inhibitor rapamycin resulted in the decrease of both RRM1 and RRM2, but not p53R2. 4E-BP1 and 4E-BP2 double knockout mouse embryonic fibroblast cells (4E-BP1/2-/- MEF) showed increased basal expression of RRM1 and RRM2, indicating that mTORC1 controls RRM1 and RRM2 via cap-dependent protein translation. In addition, pharmacological inhibition of either mTORC2-Akt signaling or CDK4/6 led to decrease of the mRNAs of RRM1 and RRM2, accompanied with the suppression of pRb-S780 signaling, suggesting that mTORC2-Akt signaling controls the gene transcription of RRM1 and RRM2 via regulating G1 cyclin-dependent kinases (CDK4/6). Both rapamycin and AZD8055 impaired the induction of endogenous RRM2 by MMC, but not ectopically expressed RRM2, which is not controlled by mTOR signaling. Our data demonstrate that mTOR controls both the protein translation and gene transcription of RRM1 and RRM2. DNA damage induced up-regulation of RRM2 depends on sustained mTOR signaling, suggesting that control of RNR by mTOR signaling may be one of the mechanisms by which cell growth-promoting signaling pathways enhance the survival and genome instability of cancer cells under constant DNA replication and damage induced stress. Supported by UPHS award CA77776. Citation Format: Changxian Shen, Peter J. Houghton. Sustained activity of the mTOR pathway is required for DNA damage-induced up-regulation of RRM2 in cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-149. doi:10.1158/1538-7445.AM2013-LB-149

Non-canonical function of Bax in stress-induced nuclear protein redistribution.

Bax and Bak (Bax/Bak) are essential pro-apoptotic proteins of the Bcl-2 family that trigger mitochondrial outer membrane permeabilization (MOMP) in a Bcl-2/Bcl-xL-inhibitable manner. We recently discovered a new stress-related function for Bax/Bak-regulation of nuclear protein redistribution (NPR) from the nucleus to cytoplasm. This effect was independent of Bax/Bak N-terminus exposure and not inhibited by Bcl-xL over-expression. Here, we studied the molecular mechanism governing this novel non-canonical response. Wild-type (WT) and mutant versions of Bax were re-expressed in Bax/Bak double-knockout mouse embryonic fibroblasts and their ability to promote NPR, apoptotic events, and changes in lamin A mobility was examined. Our results show that, in this system, Bax expression was sufficient to restore NPR such as in WT cells undergoing apoptosis. This activity of Bax was uncoupled from cytochrome c release from the mitochondria (indicative of MOMP) and required its membrane localization, α helices 5/6, and the Bcl-2 homology 3 (BH3) domain. Moreover, enrichment of Bax in the nuclear envelope by the so-called Klarsicht/ANC-1/Syne-1 homology domain effectively triggered NPR as in WT Bax, but without inducing MOMP or cell death. Bax-induced NPR was associated with impairment in lamin A mobility, implying a connection between these two nuclear envelope-associated events. Overall, the results indicate a new MOMP-independent, stress-induced Bax function on the nuclear envelope.

Repression of transcription of presenilin‐1 inhibits γ‐secretase independent ER Ca2+ leak that is impaired by FAD mutations

Genetic deletion or mutations of presenilin genes (PS1/PS2) cause familial Alzheimer's disease and calcium (Ca²⁺) signaling abnormalities. PS1/PS2 act as endoplasmic reticulum (ER) Ca²⁺ leak channels that facilitate passive Ca²⁺ leak across ER membrane. Studies with PS1/PS2 double knockout (PS1/PS2-DKO) mouse embryonic fibroblasts showed that PS1/PS2 were responsible for 80% of passive Ca²⁺ leak from the lumen of endoplasmic reticulum to cytosol. Transient transfection of the wild type PS1 expression construct increased cytoplasmic Ca²⁺ as a result of Ca²⁺ leak across ER membrane whereas the FADPS1 (PS1-M146V) mutation construct alone or in combination with the wild type PS1 expression construct abrogated Ca²⁺ leak in SK-N-SH cells. Inhibition of basal c-jun-NH2-terminal kinase (JNK) activity by JNK inhibitor SP600125 repressed PS1 transcription and PS1 protein expression by augmenting p53 protein level in SK-N-SH cells (Lee and Das 2008). In this report we also showed that repression of PS1 transcription by JNK inhibitor SP600125 inhibited passive Ca²⁺ leak across ER membrane which could be rescued by expressing PS1 wild type and not by expressing FADPS1 (PS1-M146V) under a SP600125 non-responsive promoter. Treatment of SK-N-SH cells with SP600125 also triggered InsP3R-mediated Ca²⁺ release from the ER by addition of 500 nM bradykinin, an agonist of InsP3 receptor (InsP3R1) without changing the expression of InsP3R1. This data confirms that SP600125 increases the Ca²⁺ store in the ER by inhibiting PS1-mediated Ca²⁺ leak across ER membrane. p53, ZNF237 and Chromodomain helicase DNA-binding protein 3 which are repressors of PS1 transcription, also reduced Ca²⁺ leak across ER membrane in SK-N-SH cells but γ-secretase inhibitor or dominant negative γ-secretase-specific PS1 mutant (PS1-D257A) had no significant effect. Therefore, p53, ZNF237, and Chromodomain helicase DNA-binding protein 3 inhibit the function ER Ca²⁺ leak channels to regulate both ER and cytoplasmic Ca²⁺ levels and may potentially control Ca²⁺-signaling function of PS1.

A Single Mutation in Arrestin-2 Prevents ERK1/2 Activation by Reducing c-Raf1 Binding

Arrestins regulate the signaling and trafficking of G protein-coupled receptors (GPCRs). GPCR complexes with both nonvisual arrestins channel signaling to G protein-independent pathways, one of which is the activation of extracellular signal regulated kinase 1/2 (ERK1/2). Here we used alanine-scanning mutagenesis of residues on the nonreceptor-binding surface conserved between arrestin-2 and arrestin-3. We show that an Arg307Ala mutation significantly reduced arrestin-2 binding to c-Raf1, whereas the binding of the mutant to active phosphorylated receptor and downstream kinases MEK1 and ERK2 was not affected. In contrast to wild-type arrestin-2, the Arg307Ala mutant failed to rescue arrestin-dependent ERK1/2 activation via β2-adrenergic receptor in arrestin-2/3 double knockout mouse embryonic fibroblasts. Thus, Arg307 plays a specific role in arrestin-2 binding to c-Raf1 and is indispensable in the productive scaffolding of c-Raf1-MEK1-ERK1/2 signaling cascade. Arg307Ala mutation specifically eliminates arrestin-2 signaling through ERK, which makes arrestin-2-Arg307Ala the first signaling-biased arrestin mutant constructed. In the crystal structure the side chain of homologous arrestin-3 residue Lys308 points in a different direction. Alanine substitution of Lys308 does not significantly affect c-Raf1 binding to arrestin-3 and its ability to promote ERK1/2 activation, suggesting that the two nonvisual arrestins perform the same function via distinct molecular mechanisms.

Role of guanine‐binding protein inhibitory alpha in activation of the Akt/mTORC1 pathway by growth factors

The Akt/mTOR pathway is essential for cell growth, migration and survival mediated by growth factors. Although it has been known that growth factor receptors called receptor tyrosine kinases (RTKs) and adaptor proteins including Grb2, Gabs, IRSs as well as FRSs are critical for activation of this pathway, it is still not well understood how these adaptor proteins are activated. We have found that the heterotrimeric guanine‐nucleotide binding protein (G protein) alpha inhibitory (Gia) is critical for mediating this activation. In Gi1a and Gi3a double knockout mouse embryonic fibroblasts (Gia1/3 DKO MEFs), the activation of the Akt/mTORC pathway was largely impaired by epidermal growth factor (EGF), EGF family members, fibroblast growth factors, and low doses of platelet derived growth factor (PDGF). In contrast, Gia1 and Gi3a double deficiency had no profound defect in activation of this pathway by insulin and insulin‐like growth factors. Mechanistic study results indicated that Gia proteins are important for the association of adaptor proteins and PI3K regulatory subunit p85. Further analysis revealed that loss of Gia proteins impaired MEFs migration and proliferation in response to growth factors. In addition, Gia proteins are important for cancer cell growth and migration. Overall, our results suggest that Gia proteins are new players in growth factor signaling and the pathogenesis process of cancer.

BH3 Domains other than Bim and Bid Can Directly Activate Bax/Bak

Bcl-2 family proteins regulate a critical step in apoptosis referred to as mitochondrial outer membrane permeabilization (MOMP). Members of a subgroup of the Bcl-2 family, known as the BH3-only proteins, activate pro-apoptotic effectors (Bax and Bak) to initiate MOMP. They do so by neutralizing pro-survival Bcl-2 proteins and/or directly activating Bax/Bak. Bim and Bid are reported to be direct activators; however, here we show that BH3 peptides other than Bim and Bid exhibited various degrees of direct activation of the effector Bax or Bak, including Bmf and Noxa BH3s. In the absence of potent direct activators, such as Bim and Bid, we unmasked novel direct activator BH3 ligands capable of inducing effector-mediated cytochrome c release and liposome permeabilization, even when both Bcl-xL- and Mcl-1-type anti-apoptotic proteins were inhibited. The ability of these weaker direct activator BH3 peptides to cause MOMP correlated with that of the corresponding full-length proteins to induce apoptosis in the absence of Bim and Bid. We propose that, in certain contexts, direct activation by BH3-only proteins other than Bim and Bid may significantly contribute to MOMP and apoptosis.

Intrinsic Cooperation between p16INK4a and p21Waf1/Cip1 in the Onset of Cellular Senescence and Tumor Suppression In vivo

Although the p16(INK4a) and p21Waf1/Cip1 cyclin-dependent kinase (CDK) inhibitors are known to play key roles in cellular senescence in vitro, their roles in senescence remain rather poorly understood in vivo. This situation is partly due to the possibility of compensatory effect(s) between p16INK4a and p21Waf1/Cip1 or to the upregulation of functionally related CDK inhibitors. To directly address the cooperative roles of p16INK4a and p21Waf1/Cip1 in senescence in vivo, we generated a mouse line simply lacking both p16INK4a and p21Waf1/Cip1 genes [double-knockout (DKO)]. Mouse embryonic fibroblasts (MEF) derived from DKO mice displayed no evidence of cellular senescence when cultured serially in vitro. Moreover, DKO MEFs readily escaped Ras-induced senescence and overrode contact inhibition in culture. This was not the case in MEFs lacking either p16INK4a or p21Waf1/Cip1, indicating that p16(INK4a) and p21Waf1/Cip1 play cooperative roles in cellular senescence and contact inhibition in vitro. Notably, we found the DKO mice to be extremely susceptible to 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate-induced skin carcinogenesis that involves oncogenic mutation of the H-ras gene. Mechanistic investigations suggested that the high incidence of cancer in DKO mice likely reflected a cooperative effect of increased benign skin tumor formation caused by p21Waf1/Cip1 loss, with increased malignant conversion of benign skin tumors caused by p16(INK4a) loss. Our findings establish an intrinsic cooperation between p16INK4a and p21Waf1/Cip1 in the onset of cellular senescence and tumor suppression in vivo.