Showing papers in "Genes & Development in 1999"

CDK inhibitors: positive and negative regulators of G1-phase progression

Abstract: Mitogen-dependent progression through the first gap phase (G1) and initiation of DNA synthesis (S phase) during the mammalian cell division cycle are cooperatively regulated by several classes of cyclin-dependent kinases (CDKs) whose activities are in turn constrained by CDK inhibitors (CKIs). CKIs that govern these events have been assigned to one of two families based on their structures and CDK targets. The first class includes the INK4 proteins (inhibitors of CDK4), so named for their ability to specifically inhibit the catalytic subunits of CDK4 and CDK6. Four such proteins [p16 (Serrano et al. 1993), p15 (Hannon and Beach 1994), p18 (Guan et al. 1994; Hirai et al. 1995), and p19 (Chan et al. 1995; Hirai et al. 1995)] are composed of multiple ankyrin repeats and bind only to CDK4 and CDK6 but not to other CDKs or to D-type cyclins. The INK4 proteins can be contrasted with more broadly acting inhibitors of the Cip/Kip family whose actions affect the activities of cyclin D-, E-, and A-dependent kinases. The latter class includes p21 (Gu et al. 1993; Harper et al. 1993; El-Deiry et al. 1993; Xiong et al. 1993a; Dulic et al. 1994; Noda et al. 1994), p27 (Polyak et al. 1994a,b; Toyoshima and Hunter 1994), and p57 (Lee et al. 1995; Matsuoka et al. 1995), all of which contain characteristic motifs within their amino-terminal moieties that enable them to bind both to cyclin and CDK subunits (Chen et al. 1995, 1996; Nakanishi et al. 1995; Warbrick et al. 1995; Lin et al. 1996; Russo et al. 1996). Based largely on in vitro experiments and in vivo overexpression studies, CKIs of the Cip/Kip family were initially thought to interfere with the activities of cyclin D-, E-, and A-dependent kinases. More recent work has altered this view and revealed that although the Cip/Kip proteins are potent inhibitors of cyclin Eand A-dependent CDK2, they act as positive regulators of cyclin Ddependent kinases. This challenges previous assumptions about how the G1/S transition of the mammalian cell cycle is governed, helps explain some enigmatic features of cell cycle control that also involve the functions of the retinoblastoma protein (Rb) and the INK4 proteins, and changes our thinking about how either p16 loss or overexpression of cyclin D-dependent kinases contribute to cancer. Here we focus on the biochemical interactions that occur between CKIs and cyclin Dand E-dependent kinases in cultured mammalian cells, emphasizing the manner by which different positive and negative regulators of the cell division cycle cooperate to govern the G1-to-S transition. To gain a more comprehensive understanding of the biology of CDK inhibitors, readers are encouraged to refer to a rapidly emerging but already extensive literature (for review, see Elledge and Harper 1994; Sherr and Roberts 1995; Chellappan et al. 1998; Hengst and Reed 1998a; Kiyokawa and Koff 1998; Nakayama 1998; Ruas and Peters 1998).

Cellular survival: a play in three Akts

Abstract: The programmed cell death that occurs as part of normal mammalian development was first observed nearly a century ago (Collin 1906). It has since been established that approximately half of all neurons in the neuroaxis and >99.9% of the total number of cells generated during the course of a human lifetime go on to die through a process of apoptosis (for review, see Datta and Greenberg 1998; Vaux and Korsmeyer 1999). The induction of developmental cell death is a highly regulated process and can be suppressed by a variety of extracellular stimuli. The purification in the 1950s of the nerve growth factor (NGF), which promotes the survival of sympathetic neurons, set the stage for the discovery that peptide trophic factors promote the survival of a wide variety of cell types in vitro and in vivo (Levi-Montalcini 1987). The profound biological consequences of growth factor (GF) suppression of apoptosis are exemplified by the critical role of target-derived neurotrophins in the survival of neurons and the maintenance of functional neuronal circuits. (Pettmann and Henderson 1998). Recently, the ability of trophic factors to promote survival have been attributed, at least in part, to the phosphatidylinositide 38-OH kinase (PI3K)/c-Akt kinase cascade. Several targets of the PI3K/c-Akt signaling pathway have been recently identified that may underlie the ability of this regulatory cascade to promote survival. These substrates include two components of the intrinsic cell death machinery, BAD and caspase 9, transcription factors of the forkhead family, and a kinase, IKK, that regulates the NF-kB transcription factor. This article reviews the mechanisms by which survival factors regulate the PI3K/c-Akt cascade, the evidence that activation of the PI3K/c-Akt pathway promotes cell survival, and the current spectrum of c-Akt targets and their roles in mediating c-Akt-dependent cell survival.

BCL-2 family members and the mitochondria in apoptosis

Abstract: A variety of physiological death signals, as well as pathological cellular insults, trigger the genetically programmed pathway of apoptosis (Vaux and Korsmeyer 1999). Apoptosis manifests in two major execution programs downstream of the death signal: the caspase pathway and organelle dysfunction, of which mitochondrial dysfunction is the best characterized (for reviews, see Green and Reed 1998; Thornberry and Lazebnik 1998). As the BCL-2 family members reside upstream of irreversible cellular damage and focus much of their efforts at the level of mitochondria, they play a pivotal role in deciding whether a cell will live or die (Fig. 1). The founder of this family, the BCL-2 proto-oncogene, was discovered at the chromosomal breakpoint of t(14;18) bearing human B-cell lymphomas. The BCL-2 family of proteins has expanded significantly and includes both proas well as anti-apoptotic molecules. Indeed, the ratio between these two subsets helps determine, in part, the susceptibility of cells to a death signal (Oltvai et al. 1993) (Fig. 1). An additional characteristic of the members of this family is their frequent ability to form homoas well as heterodimers, suggesting neutralizing competition between these proteins. A further characteristic of probable functional significance is their ability to become integral membrane proteins. BCL-2 family members possess up to four conserved BCL-2 homology (BH) domains designated BH1, BH2, BH3, and BH4, which correspond to a-helical segments (Adams and Cory 1998; Kelekar and Thompson 1998; Reed 1998) (Fig. 2). Many of the anti-apoptotic members display sequence conservation in all four domains. The pro-apoptotic molecules frequently display less sequence conservation of the first a-helical segment, BH4. Deletion and mutagenesis studies argue that the amphipathic a-helical BH3 domain serves as a critical death domain in the pro-apoptotic members. This concept is supported by an emerging subset of “BH3-domain-only” members who display sequence homology only within the BH3 domain and to date are all pro-apoptotic. However, the three-dimensional structure of at least one BH3-domainonly molecule, BID, demonstrates a very similar overall a-helical content to the anti-apoptotic molecule BCL-XL (Chou et al. 1999; McDonnell et al. 1999). Many BCL-2 family members also contain a carboxy-terminal hydrophobic domain, which in the case of BCL-2 is essential for its targeting to membranes such as the mitochondrial outer membrane (Nguyen et al. 1993).

Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal Neh2 domain

Abstract: Transcription factor Nrf2 is essential for the antioxidant responsive element (ARE)-mediated induction of phase II detoxifying and oxidative stress enzyme genes. Detailed analysis of differential Nrf2 activity displayed in transfected cell lines ultimately led to the identification of a new protein, which we named Keap1, that suppresses Nrf2 transcriptional activity by specific binding to its evolutionarily conserved amino-terminal regulatory domain. The closest homolog of Keap1 is a Drosophila actin-binding protein called Kelch, implying that Keap1 might be a Nrf2 cytoplasmic effector. We then showed that electrophilic agents antagonize Keap1 inhibition of Nrf2 activity in vivo, allowing Nrf2 to traverse from the cytoplasm to the nucleus and potentiate the ARE response. We postulate that Keap1 and Nrf2 constitute a crucial cellular sensor for oxidative stress, and together mediate a key step in the signaling pathway that leads to transcriptional activation by this novel Nrf2 nuclear shuttling mechanism. The activation of Nrf2 leads in turn to the induction of phase II enzyme and antioxidative stress genes in response to electrophiles and reactive oxygen species.

IAP family proteins—suppressors of apoptosis

Abstract: Apoptosis is a physiological cell suicide program that is critical for the development and maintenance of healthy tissues. Dysregulation of cell death pathways occur in cancer, autoimmune and immunodeficiency diseases, reperfusion injury after ischemic episodes, and in neurodegenerative disorders. Thus, proteins involved in apoptosis regulation are of intense biological interest and many are attractive therapeutic targets. This review discusses the Inhibitor of Apoptosis (IAP) family of proteins. First discovered in baculoviruses, IAPs were shown to be involved in suppressing the host cell death response to viral infection. Interestingly, ectopic expression of some baculoviral IAPs blocks apoptosis in mammalian cells, suggesting conservation of the cell death program among diverse species and commonalities in the mechanism used by the IAPs to inhibit apoptosis. Although the mechanism used by the IAPs to suppress cell death remains debated, several studies have provided insights into the biochemical functions of these intriguing proteins. Moreover, a variety of reports have suggested an important role for the IAPs in some human diseases.

Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls

Abstract: All eukaryotic cells have an extensive membranous labyrinth network of branching tubules and flattened sacs called the endoplasmic reticulum (ER). Approximately one-third of all cellular proteins are translocated into the lumen of the ER where post-translational modification, folding, and oligomerization occurs. The ER provides a unique oxidizing compartment for the folding of membrane and secretory proteins that are destined to the cell surface, as well as for proteins destined to other intracellular organelles, such as lysosomes and the Golgi compartment. Numerous cellular proteins reside within the ER through a mechanism that requires their continuous vesicle-mediated retrieval from post-ER compartments within the early secretory pathway. These ERresident proteins are chaperones and catalysts of protein folding that form a matrix on which newly synthesized proteins attain their final conformation. The ER is also the site of synthesis of cellular lipids and sterols. In addition, the ER is the major signal-transducing organelle within the cell that continuously responds to environmental cues to release calcium. The ER is exquisitely sensitive to alterations in homeostasis, where, upon a variety of different stimuli, signals are transduced from the ER to the cytoplasm and the nucleus to eventually result in adaptation for survival or induction of apoptosis. The immediate response occurs at the translational apparatus, whereas changes in gene expression promote long-term adaptation or apoptotic cell death. Recent evidence supports findings that these signaling pathways influence pathogenesis associated with viral infection and genetic disease. The purpose of this review is to summarize what is presently known about the diversity of molecular signaling mechanisms that coordinate the complex ER stress response at the translational and transcriptional level in yeast and in higher eukaryotic cells.

The SIR2/3/4 complex and SIR2 alone promote longevity in Saccharomyces cerevisiae by two different mechanisms

Abstract: The SIR genes are determinants of life span in yeast mother cells. Here we show that life span regulation by the Sir proteins is independent of their role in nonhomologous end joining. The short life span of a sir3 or sir4 mutant is due to the simultaneous expression of a and a mating-type information, which indirectly causes an increase in rDNA recombination and likely increases the production of extrachromosomal rDNA circles. The short life span of a sir2 mutant also reveals a direct failure to repress recombination generated by the Fob1p-mediated replication block in the rDNA. Sir2p is a limiting component in promoting yeast longevity, and increasing the gene dosage extends the life span in wild-type cells. A possible role of the conserved SIR2 in mammalian aging is discussed.

Indian hedgehog signaling regulates proliferation and differentiation of chondrocytes and is essential for bone formation

Abstract: The mechanisms that control cell proliferation and cell differentiation during morphogenesis of the endochondral skeleton of vertebrates are poorly understood. Indian hedgehog (Ihh) signaling from prehypertrophic chondrocytes has been implicated in the control of chondrocyte maturation by way of feedback control of a second secreted factor parathyroid hormone-related peptide (PTHrP) at the articular surfaces. Analysis of an Ihh null mutant suggests a more extensive role for Ihh in skeletal development. Mutants display markedly reduced chondrocyte proliferation, maturation of chondrocytes at inappropriate position, and a failure of osteoblast development in endochondral bones. Together, the results suggest a model in which Ihh coordinates diverse aspects of skeletal morphogenesis through PTHrP-dependent and independent processes.

RANK is essential for osteoclast and lymph node development.

Abstract: The physiological role of the TNF receptor (TNFR) family member, RANK, was investigated by generating RANK-deficient mice. RANK(-/-) mice were characterized by profound osteopetrosis resulting from an apparent block in osteoclast differentiation. RANK expression was not required for the commitment, differentiation, and functional maturation of macrophages and dendritic cells from their myeloid precursors but provided a necessary and specific signal for the differentiation of myeloid-derived osteoclasts. RANK(-/-) mice also exhibited a marked deficiency of B cells in the spleen. RANK(-/-) mice retained mucosal-associated lymphoid tissues including Peyer's patches but completely lacked all other peripheral lymph nodes, highlighting an additional major role for RANK in lymph node formation. These experiments reveal that RANK provides critical signals necessary for lymph node organogenesis and osteoclast differentiation.

Bmp4 is required for the generation of primordial germ cells in the mouse embryo.

Abstract: Before gastrulation, the mouse embryo consists of three distinct cell lineages which were established in the blastocyst during the peri-implantation period, that is, epiblast, extraembryonic endoderm, and trophectoderm. The epiblast, from which the entire fetus will form, as well as the extraembryonic mesoderm and amnion ectoderm, is a cup-shaped epithelium apposed on its open end to the extraembryonic ectoderm, a trophectoderm derivative. Both epiblast and extraembryonic ectoderm are covered by visceral endoderm, which is part of the extraembryonic endoderm lineage (Hogan et al. 1994). The primordial germ cells (PGCs) of the mouse embryo are derived from part of the population of epiblast cells that will give rise mainly to the extraembryonic mesoderm. Precursors of the PGCs are located before gastrulation in the extreme proximal region of the epiblast adjacent to the extraembryonic ectoderm, and have descendants not only in the germ line, but also in extraembryonic structures, that is, the allantois, blood islands, and yolk sac mesoderm, as well as both layers of the amnion. At embryonic day (E) 6.0, these precursors lie scattered in a ring that extends up to three cell diameters from the junction with the extraembryonic ectoderm (Lawson and Hage 1994). Early in gastrulation, they converge toward the primitive streak in the posterior of the embryo and translocate through it. Allocation to the germ cell lineage is thought to occur in ∼45 cells around E7.2, after the precursors have passed through the streak and have come to reside in the extraembryonic mesoderm (Lawson and Hage 1994). This is about the time when the putative PGCs can first be identified morphologically in a cluster posterior to the primitive streak in a position that will later become the base of the allantois (Ginsburg et al. 1990). PGCs stain strongly in a characteristic pattern for alkaline phosphatase (AP) activity (Chiquoine 1954), which by this stage is due to tissue nonspecific AP (Hahnel et al. 1990; MacGregor et al. 1995). The PGCs continue to express AP during their proliferation in the developing hindgut and migration into the genital ridges (for review, see Buehr 1997). Transplantation studies have shown that genetically marked distal epiblast cells from pre- and early-primitive streak-stage embryos, which would normally contribute to neuroectoderm and never to the PGCs, can give rise to PGCs and extraembryonic mesoderm when grafted to the proximal epiblast (Tam and Zhou 1996). These results raise the possibility that PGC precursors are induced by extracellular factors and/or cell interactions present locally at the junction between the extraembryonic ectoderm and epiblast. Candidate genes encoding putative germ cell precursor inducing factors are predicted to be expressed in the mouse embryo before and during gastrulation. One such factor is Bone Morphogenetic Protein 4 (Bmp4), a member of the TGFβ superfamily of intercellular signaling proteins (Hogan 1996; Waldrip et al. 1998). Most mouse embryos homozygous for a null mutation in Bmp4 die around gastrulation (∼E6.5) (Winnier et al. 1995). On some genetic backgrounds, however, a proportion of the mutant embryos survive until the early somite stage and show severe defects, particularly in the extraembryonic mesoderm (Winnier et al. 1995). In this paper, we exploit this late phenotype to show that PGC formation absolutely requires Bmp4 signaling. In addition, the size of the founding population of PGCs is significantly reduced in heterozygous mutant embryos. By using a Bmp4–lacZ reporter allele, we have definitively localized Bmp4 expression before gastrulation in the extraembryonic ectoderm and in mid- to late- primitive streak stage embryos in the extraembryonic mesoderm. Thus, Bmp4 is expressed at the right time and in the right place to play a role both in the quantitative induction of PGC precursors in the proximal epiblast and in their allocation to the germ cell lineage in the extraembryonic mesoderm. Furthermore, by analyzing genetic chimeras, we have clearly established a role for Bmp4 in the induction of PGC precursors and demonstrate for the first time that a secreted signal from the extraembryonic ectoderm is required for the normal development of the epiblast.

TRAF6 deficiency results in osteopetrosis and defective interleukin-1, CD40, and LPS signaling

Abstract: Bone resorption and remodeling is an intricately controlled, physiological process that requires the function of osteoclasts. The processes governing both the differentiation and activation of osteoclasts involve signals induced by osteoprotegerin ligand (OPGL), a member of tumor necrosis factor (TNF) superfamily, and its cognate receptor RANK. The molecular mechanisms of the intracellular signal transduction remain to be elucidated. Here we report that mice deficient in TNF receptor-associated factor 6 (TRAF6) are osteopetrotic with defects in bone remodeling and tooth eruption due to impaired osteoclast function. Using in vitro assays, we demonstrate that TRAF6 is crucial not only in IL-1 and CD40 signaling but also, surprisingly, in LPS signaling. Furthermore, like TRAF2 and TRAF3, TRAF6 is essential for perinatal and postnatal survival. These findings establish unexpectedly diverse and critical roles for TRAF6 in perinatal and postnatal survival, bone metabolism, LPS, and cytokine signaling.

XRCC3 promotes homology-directed repair of DNA damage in mammalian cells

Abstract: Homology-directed repair of DNA damage has recently emerged as a major mechanism for the maintenance of genomic integrity in mammalian cells. The highly conserved strand transferase, Rad51, is expected to be critical for this process. XRCC3 possesses a limited sequence similarity to Rad51 and interacts with it. Using a novel fluorescence-based assay, we demonstrate here that error-free homology-directed repair of DNA double-strand breaks is decreased 25-fold in an XRCC3-deficient hamster cell line and can be restored to wild-type levels through XRCC3 expression. These results establish that XRCC3-mediated homologous recombination can reverse DNA damage that would otherwise be mutagenic or lethal.

Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism

Abstract: The multisubunit eukaryotic translation initiation factor (eIF) 4F recruits 40S ribosomal subunits to the 5′ end of mRNA. The eIF4F subunit eIF4E interacts directly with the mRNA 5′ cap structure. Assembly of the eIF4F complex is inhibited by a family of repressor polypeptides, the eIF4E-binding proteins (4E-BPs). Binding of the 4E-BPs to eIF4E is regulated by phosphorylation: Hypophosphorylated 4E-BP isoforms interact strongly with eIF4E, whereas hyperphosphorylated isoforms do not. 4E-BP1 is hypophosphorylated in quiescent cells, but is hyperphosphorylated on multiple sites following exposure to a variety of extracellular stimuli. The PI3-kinase/Akt pathway and the kinase FRAP/mTOR signal to 4E-BP1. FRAP/mTOR has been reported to phosphorylate 4E-BP1 directly in vitro. However, it is not known if FRAP/mTOR is responsible for the phosphorylation of all 4E-BP1 sites, nor which sites must be phosphorylated to release 4E-BP1 from eIF4E. To address these questions, a recombinant FRAP/mTOR protein and a FRAP/mTOR immunoprecipitate were utilized in in vitro kinase assays to phosphorylate 4E-BP1. Phosphopeptide mapping of the in vitro-labeled protein yielded two 4E-BP1 phosphopeptides that comigrated with phosphopeptides produced in vivo. Mass spectrometry analysis indicated that these peptides contain phosphorylated Thr-37 and Thr-46. Thr-37 and Thr-46 are efficiently phosphorylated in vitro by FRAP/mTOR when 4E-BP1 is bound to eIF4E. However, phosphorylation at these sites was not associated with a loss of eIF4E binding. Phosphorylated Thr-37 and Thr-46 are detected in all phosphorylated in vivo 4E-BP1 isoforms, including those that interact with eIF4E. Finally, mutational analysis demonstrated that phosphorylation of Thr-37/Thr-46 is required for subsequent phosphorylation of several carboxy-terminal serum-sensitive sites. Taken together, our results suggest that 4E-BP1 phosphorylation by FRAP/mTOR on Thr-37 and Thr-46 is a priming event for subsequent phosphorylation of the carboxy-terminal serum-sensitive sites.

Targeted mRNA degradation by double-stranded RNA in vitro

Abstract: Double-stranded RNA (dsRNA) directs gene-specific, post-transcriptional silencing in many organisms, including vertebrates, and has provided a new tool for studying gene function. The biochemical mechanisms underlying this dsRNA interference (RNAi) are unknown. Here we report the development of a cell-free system from syncytial blastoderm Drosophila embryos that recapitulates many of the features of RNAi. The interference observed in this reaction is sequence specific, is promoted by dsRNA but not single-stranded RNA, functions by specific mRNA degradation, and requires a minimum length of dsRNA. Furthermore, preincubation of dsRNA potentiates its activity. These results demonstrate that RNAi can be mediated by sequence-specific processes in soluble reactions.

Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation

Abstract: ATP-dependent nucleosome remodeling and core histone acetylation and deacetylation represent mechanisms to alter nucleosome structure. NuRD is a multisubunit complex containing nucleosome remodeling and histone deacetylase activities. The histone deacetylases HDAC1 and HDAC2 and the histone binding proteins RbAp48 and RbAp46 form a core complex shared between NuRD and Sin3-histone deacetylase complexes. The histone deacetylase activity of the core complex is severely compromised. A novel polypeptide highly related to the metastasis-associated protein 1, MTA2, and the methyl-CpG-binding domain-containing protein, MBD3, were found to be subunits of the NuRD complex. MTA2 modulates the enzymatic activity of the histone deacetylase core complex. MBD3 mediates the association of MTA2 with the core histone deacetylase complex. MBD3 does not directly bind methylated DNA but is highly related to MBD2, a polypeptide that binds to methylated DNA and has been reported to possess demethylase activity. MBD2 interacts with the NuRD complex and directs the complex to methylated DNA. NuRD may provide a means of gene silencing by DNA methylation.

Molecular mechanism of nucleotide excision repair

Abstract: From its very beginning, life has faced the fundamental problem that the form in which genetic information is stored is not chemically inert. DNA integrity is challenged by the damaging effect of numerous chemical and physical agents, compromizing its function. To protect this Achilles heel, an intricate network of DNA repair systems has evolved early in evolution. One of these is nucleotide excision repair (NER), a highly versatile and sophisticated DNA damage removal pathway that counteracts the deleterious effects of a multitude of DNA lesions, including major types of damage induced by environmental sources. The most relevant lesions subject to NER are cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts (6-4PPs), two major kinds of injury produced by the shortwave UV component of sunlight. In addition, numerous bulky chemical adducts are eliminated by this process. Within the divergent spectrum of NER lesions, significant distortion of the DNA helix appears to be a common denominator. Defects in NER underlie the extreme photosensitivity and predisposition to skin cancer observed with the prototype repair syndrome xeroderma pigmentosum (XP). Seven XP complementation groups have been identified, representing distinct repair genes XPA–G (discussed in detail below). In the last decade, all key NER factors have been cloned and the core of the ‘cut-and-paste’ reaction has been reconstituted in vitro from purified components. Recently, XPC (complexed to hHR23B) has been identified as a DNA-damage sensor and repair-recruitment factor. The general transcription factor complex TFIIH, containing the XPB and XPD helicases, mediates strand separation at the site of the lesion. XPA verifies the damage in an open DNA conformation and is crucial in the assembly of the remainder of the repair machinery. Replication protein A (RPA) stabilizes the opened DNA complex and is involved in positioning the XPG and ERCC1–XPF endonucleases responsible for the DNA incisions around the lesion. After removal of the damagecontaining oligonucleotide, typically 24–32 nucleotides in length, general replication factors fill in the remaining gap and close it. Two modes of NER can be distinguished: repair of lesions over the entire genome, referred to as global genome NER (GG–NER), and repair of transcription-blocking lesions present in transcribed DNA strands, hence called transcription-coupled NER (TC–NER). Most XP groups harbor defects in a common component of both NER subpathways. GG–NER is dependent on the activity of all factors mentioned above, including the GG– NER-specific complex XPC–hHR23B. The rate of repair for GG–NER strongly depends on the type of lesion. For instance, 6-4PPs are removed much faster from the genome than CPDs, probably because of differences in affinity of the damage sensor XPC–hHR23B. In addition, the location (accessibility) of a lesion influences the removal rate in vivo. In TC–NER, damage is detected by the elongating RNA polymerase II complex when it encounters a lesion. Interestingly, a distinct disorder, Cockayne syndrome (CS), is associated with a specific defect in transcription-coupled repair. The identification of two complementation groups (CS-A and CS-B) shows that at least two gene products are specifically needed for fast and efficient repair of transcribed strands. Phenotypically, CS is a very pleiotropic condition characterized by photosensitivity as well as severe neurological, developmental, and premature aging features. Most of these symptoms are not seen even with totally NERdeficient XP patients. The additional symptoms of CS suggest that transcription-coupled repair and/or the CS proteins have functions beyond NER. Also, non-NERspecific lesions (such as oxidative damage) that stall transcription elongation appear to be removed in a transcription-coupled fashion, linking a blocked polymerase to multiple repair pathways. Intriguingly, some XP-B, XP-D, and XP-G patients display CS features combined with XP manifestations. Yet other XP-B and XP-D individuals suffer from the CS-like brittle-hair syndrome trichothiodystrophy (TTD). This clinical conundrum points to additional roles of these NER factors as well. A recent mouse model for TTD has linked mutations in the XPD subunit of the dual functional TFIIH complex with deficiencies in basal transcription underlying at least some of the TTD manifestations. Thus, NER defects are associated with a surprisingly wide clinical heterogeneity due to additional functions of the NER factors involved. 1Corresponding author. E-MAIL Hoeijmakers@gen.fgg.eur.nl; FAX 31 10 408 9468.

A role for ATR in the DNA damage-induced phosphorylation of p53

Abstract: Phosphorylation at Ser-15 may be a critical event in the up-regulation and functional activation of p53 during cellular stress. In this report we provide evidence that the ATM–Rad3-related protein ATR regulates phosphorylation of Ser-15 in DNA-damaged cells. Overexpression of catalytically inactive ATR (ATRki) in human fibroblasts inhibited Ser-15 phosphorylation in response to γ-irradiation and UV light. In γ-irradiated cells, ATRki expression selectively interfered with late-phase Ser-15 phosphorylation, whereas ATRki blocked UV-induced Ser-15 phosphorylation in a time-independent manner. ATR phosphorylated p53 at Ser-15 and Ser-37 in vitro, suggesting that p53 is a target for phosphorylation by ATR in DNA-damaged cells.

Multiple docking sites on substrate proteins form a modular system that mediates recognition by ERK MAP kinase

Abstract: MAP kinases phosphorylate specific groups of substrate proteins. Here we show that the amino acid sequence FXFP is an evolutionarily conserved docking site that mediates ERK MAP kinase binding to substrates in multiple protein families. FXFP and the D box, a different docking site, form a modular recognition system, as they can function independently or in combination. FXFP is specific for ERK, whereas the D box mediates binding to ERK and JNK MAP kinase, suggesting that the partially overlapping substrate specificities of ERK and JNK result from recognition of shared and unique docking sites. These findings enabled us to predict new ERK substrates and design peptide inhibitors of ERK that functioned in vitro and in vivo.

Roles of ephrinB ligands and EphB receptors in cardiovascular development: demarcation of arterial/venous domains, vascular morphogenesis, and sprouting angiogenesis

Abstract: Eph receptor tyrosine kinases and their cell-surface-bound ligands, the ephrins, regulate axon guidance and bundling in the developing brain, control cell migration and adhesion, and help patterning the embryo. Here we report that two ephrinB ligands and three EphB receptors are expressed in and regulate the formation of the vascular network. Mice lacking ephrinB2 and a proportion of double mutants deficient in EphB2 and EphB3 receptor signaling die in utero before embryonic day 11.5 (E11.5) because of defects in the remodeling of the embryonic vascular system. Our phenotypic analysis suggests complex interactions and multiple functions of Eph receptors and ephrins in the embryonic vasculature. Interaction between ephrinB2 on arteries and its EphB receptors on veins suggests a role in defining boundaries between arterial and venous domains. Expression of ephrinB1 by arterial and venous endothelial cells and EphB3 by veins and some arteries indicates that endothelial cell-to-cell interactions between ephrins and Eph receptors are not restricted to the border between arteries and veins. Furthermore, expression of ephrinB2 and EphB2 in mesenchyme adjacent to vessels and vascular defects in ephB2/ephB3 double mutants indicate a requirement for ephrin-Eph signaling between endothelial cells and surrounding mesenchymal cells. Finally, ephrinB ligands induce capillary sprouting in vitro with a similar efficiency as angiopoietin-1 (Ang1) and vascular endothelial growth factor (VEGF), demonstrating a stimulatory role of ephrins in the remodeling of the developing vascular system.

A mechanism of repression of TGFbeta/ Smad signaling by oncogenic Ras.

Abstract: TGFβ can override the proliferative effects of EGF and other Ras-activating mitogens in normal epithelial cells. However, epithelial cells harboring oncogenic Ras mutations often show a loss of TGFβ antimitogenic responses. Here we report that oncogenic Ras inhibits TGFβ signaling in mammary and lung epithelial cells by negatively regulating the TGFβ mediators Smad2 and Smad3. Oncogenically activated Ras inhibits the TGFβ-induced nuclear accumulation of Smad2 and Smad3 and Smad-dependent transcription. Ras acting via Erk MAP kinases causes phosphorylation of Smad2 and Smad3 at specific sites in the region linking the DNA-binding domain and the transcriptional activation domain. These sites are separate from the TGFβ receptor phosphorylation sites that activate Smad nuclear translocation. Mutation of these MAP kinase sites in Smad3 yields a Ras-resistant form that can rescue the growth inhibitory response to TGFβ in Ras-transformed cells. EGF, which is weaker than oncogenic mutations at activating Ras, induces a less extensive phosphorylation and cytoplasmic retention of Smad2 and Smad3. Our results suggest a mechanism for the counterbalanced regulation of Smad2/Smad3 by TGFβ and Ras signals in normal cells, and for the silencing of antimitogenic TGFβ functions by hyperactive Ras in cancer cells.

Inflammatory mast cells up-regulate angiogenesis during squamous epithelial carcinogenesis

Abstract: Expression of HPV16 early region genes in basal keratinocytes of transgenic mice elicits a multistage pathway to squamous carcinoma. We report that infiltration by mast cells and activation of the matrix metalloproteinase MMP-9/gelatinase B coincides with the angiogenic switch in premalignant lesions. Mast cells infiltrate hyperplasias, dysplasias, and invasive fronts of carcinomas, but not the core of solid tumors, where they degranulate in close apposition to capillaries and epithelial basement membranes, releasing mast-cell-specific serine proteases MCP-4 (chymase) and MCP-6 (tryptase). MCP-6 is shown to be a mitogen for dermal fibroblasts that proliferate in the reactive stroma, whereas MCP-4 can activate progelatinase B and induce hyperplastic skin to become angiogenic in an in vitro bioassay. Notably, premalignant angiogenesis is abated in a mast-cell-deficient (KITW/KITWWv) HPV16 transgenic mouse. The data indicate that neoplastic progression in this model involves exploitation of an inflammatory response to tissue abnormality. Thus, regulation of angiogenesis during squamous carcinogenesis is biphasic: In hyperplasias, dysplasias, and invading cancer fronts, inflammatory mast cells are conscripted to reorganize stromal architecture and hyperactivate angiogenesis; within the cancer core, upregulation of angiogenesis factors in tumor cells apparently renders them self-sufficient at sustaining neovascularization.

The DNA-dependent protein kinase

Abstract: The DNA-dependent protein kinase (DNA–PK) is anuclear serine/threonine protein kinase that is activatedupon association with DNA. Biochemical and geneticdata have revealed DNA–PK to be composed of a largecatalytic subunit, termed DNA–PKcs, and a regulatoryfactor termed Ku. In recent years, mammalian DNA–PKhas been shown to be a crucial component of both theDNA double-strand break (DSB) repair machinery andthe

The SCFβ-TRCP–ubiquitin ligase complex associates specifically with phosphorylated destruction motifs in IκBα and β-catenin and stimulates IκBα ubiquitination in vitro

Abstract: Ubiquitin-mediated proteolysis has a central role in controlling the intracellular levels of several important regulatory molecules such as cyclins, CKIs, p53, and IkappaBalpha. Many diverse proinflammatory signals lead to the specific phosphorylation and subsequent ubiquitin-mediated destruction of the NF-kappaB inhibitor protein IkappaBalpha. Substrate specificity in ubiquitination reactions is, in large part, mediated by the specific association of the E3-ubiquitin ligases with their substrates. One class of E3 ligases is defined by the recently described SCF complexes, the archetype of which was first described in budding yeast and contains Skp1, Cdc53, and the F-box protein Cdc4. These complexes recognize their substrates through modular F-box proteins in a phosphorylation-dependent manner. Here we describe a biochemical dissection of a novel mammalian SCF complex, SCFbeta-TRCP, that specifically recognizes a 19-amino-acid destruction motif in IkappaBalpha (residues 21-41) in a phosphorylation-dependent manner. This SCF complex also recognizes a conserved destruction motif in beta-catenin, a protein with levels also regulated by phosphorylation-dependent ubiquitination. Endogenous IkappaBalpha-ubiquitin ligase activity cofractionates with SCFbeta-TRCP. Furthermore, recombinant SCFbeta-TRCP assembled in mammalian cells contains phospho-IkappaBalpha-specific ubiquitin ligase activity. Our results suggest that an SCFbeta-TRCP complex functions in multiple transcriptional programs by activating the NF-kappaB pathway and inhibiting the beta-catenin pathway.

Growth factors acting via endothelial cell-specific receptor tyrosine kinases: VEGFs, Angiopoietins, and ephrins in vascular development

Abstract: The term ‘vasculogenesis’ refers to the earliest stages of vascular development, during which vascular endothelial cell precursors undergo differentiation, expansion, and coalescence to form a network of primitive tubules (Risau 1997). This initial lattice, consisting purely of endothelial cells that have formed rather homogenously sized interconnected vessels, has been referred to as the primary capillary plexus. The primary plexus is then remodeled by a process referred to as angiogenesis (Risau 1997), which involves the sprouting, branching, and differential growth of blood vessels to form the more mature appearing vascular patterns seen in the adult organism. This latter phase of vascular development also involves the sprouting and penetration of vessels into previously avascular regions of the embryo, and also the differential recruitment of associated supporting cells, such as smooth muscle cells and pericytes, as well as fibroblasts, to different segments of the vasculature (Folkman and D’Amore 1996; Lindahl et al. 1997). The adult vascular network is comprised of large arteries, internally lined by endothelial cells and well ensheathed by smooth muscle cells, that progressively branch into smaller and smaller vessels, terminating in precapillary arterioles that then give rise to capillaries. Capillaries are comprised almost entirely of endothelial cells that are only occasionally coated by a smooth muscle cell-like pericyte. Capillaries then feed into postcapillary venules that progressively associate into larger and larger venous structures; venous structures are fully enveloped by smooth muscle cells, though not to the same degree as arterial structures. The development of a functioning vascular network requires a remarkable degree of coordination between different cell types undergoing complex changes, and is exquisitely dependent upon signals exchanged between these cell types. Vascular endothelial growth factor (VEGF-A) provided the first example of a growth factor specific for the vascular endothelium, and VEGF-A has since been shown to be a critical regulator of endothelial cell development. Not surprisingly, the specificity of VEGF-A for the vascular endothelium results from the restricted distribution of VEGF-A receptors to these cells. The need to regulate the multitude of cellular interactions involved during vascular development suggested that VEGF-A might not be alone as an endothelial cell-specific growth factor. Indeed, there has been a recent explosion in the number of growth factors that specifically act on the vascular endothelium. This explosion involves the VEGF family, which now totals at least five members. In addition, an entirely unrelated family of growth factors, known as the Angiopoietins, recently has been identified as acting via endothelial cell-specific receptors known as the Ties. Most recently, particular members of the very large ephrin family have been identified as having unique roles on endothelium, and at least in some cases appear to act via a receptor that is not only largely restricted to the vascular endothelium but to the endothelium lining venous as opposed to arterial vessels. As we describe in detail below, a variety of studies highlighted by the analyses of knockout mice (as summarized in Table 1) have implicated the VEGFs, the Angiopoietins, and the ephrins as critical players in particular aspects of vascular development.

Disruption of the ARF–Mdm2–p53 tumor suppressor pathway in Myc-induced lymphomagenesis

Abstract: Transgenic mice expressing the c-Myc oncogene driven by the immunoglobulin heavy chain enhancer (Emu) develop B-cell lymphoma and exhibit a mean survival time of approximately 6 months. The protracted latent period before the onset of frank disease likely reflects the ability of c-Myc to induce a p53-dependent apoptotic program that initially protects animals against tumor formation but is disabled when overtly malignant cells emerge. In cultured primary mouse embryo fibroblasts, c-Myc activates the p19(ARF)-Mdm2-p53 tumor suppressor pathway, enhancing p53-dependent apoptosis but ultimately selecting for surviving immortalized cells that have sustained either p53 mutation or biallelic ARF deletion. Here we report that p53 and ARF also potentiate Myc-induced apoptosis in primary pre-B-cell cultures, and that spontaneous inactivation of the ARF-Mdm2-p53 pathway occurs frequently in tumors arising in Emu-myc transgenic mice. Many Emu-myc lymphomas sustained either p53 (28%) or ARF (24%) loss of function, whereas Mdm2 levels were elevated in others. Its overexpression in some tumors lacking p53 function raises the possibility that Mdm2 can contribute to lymphomagenesis by interacting with other targets. Emu-myc transgenic mice hemizygous for ARF displayed accelerated disease (11-week mean survival), and 80% of these tumors lost the wild-type ARF allele. All ARF-null Emu-myc mice died of lymphoma within a few weeks of birth. About half of the tumors arising in ARF hemizygous or ARF nullizygous Emu-myc transgenic mice also overexpressed Mdm2. Therefore, Myc activation strongly selects for spontaneous inactivation of the ARF-Mdm2-p53 pathway in vivo, cancelling its protective checkpoint function and accelerating progression to malignancy.

Cleavage of the death domain kinase RIP by caspase-8 prompts TNF-induced apoptosis.

Abstract: Although the molecular mechanisms of TNF signaling have been largely elucidated, the principle that regulates the balance of life and death is still unknown. We report here that the death domain kinase RIP, a key component of the TNF signaling complex, was cleaved by Caspase-8 in TNF-induced apoptosis. The cleavage site was mapped to the aspartic acid at position 324 of RIP. We demonstrated that the cleavage of RIP resulted in the blockage of TNF-induced NF-κB activation. RIPc, one of the cleavage products, enhanced interaction between TRADD and FADD/MORT1 and increased cells' sensitivity to TNF. Most importantly, the Caspase-8 resistant RIP mutants protected cells against TNF-induced apopotosis. These results suggest that cleavage of RIP is an important process in TNF-induced apoptosis. Further more, RIP cleavage was also detected in other death receptor-mediated apoptosis. Therefore, our study provides a potential mechanism to convert cells from life to death in death receptor-mediated apoptosis.

A Cbfa1-dependent genetic pathway controls bone formation beyond embryonic development

Abstract: The molecular mechanisms controlling bone extracellular matrix (ECM) deposition by differentiated osteoblasts in postnatal life, called hereafter bone formation, are unknown. This contrasts with the growing knowledge about the genetic control of osteoblast differentiation during embryonic development. Cbfa1, a transcriptional activator of osteoblast differentiation during embryonic development, is also expressed in differentiated osteoblasts postnatally. The perinatal lethality occurring in Cbfa1-deficient mice has prevented so far the study of its function after birth. To determine if Cbfa1 plays a role during bone formation we generated transgenic mice overexpressing Cbfa1 DNA-binding domain (ΔCbfa1) in differentiated osteoblasts only postnatally. ΔCbfa1 has a higher affinity for DNA than Cbfa1 itself, has no transcriptional activity on its own, and can act in a dominant-negative manner in DNA cotransfection assays. ΔCbfa1-expressing mice have a normal skeleton at birth but develop an osteopenic phenotype thereafter. Dynamic histomorphometric studies show that this phenotype is caused by a major decrease in the bone formation rate in the face of a normal number of osteoblasts thus indicating that once osteoblasts are differentiated Cbfa1 regulates their function. Molecular analyses reveal that the expression of the genes expressed in osteoblasts and encoding bone ECM proteins is nearly abolished in transgenic mice, and ex vivo assays demonstrated that ΔCbfa1-expressing osteoblasts were less active than wild-type osteoblasts. We also show that Cbfa1 regulates positively the activity of its own promoter, which has the highest affinity Cbfa1-binding sites characterized. This study demonstrates that beyond its differentiation function Cbfa1 is the first transcriptional activator of bone formation identified to date and illustrates that developmentally important genes control physiological processes postnatally.

ATP-dependent remodeling and acetylation as regulators of chromatin fluidity.

Abstract: It has become widely accepted that modification of nucleosome structure is an important regulatory mechanism. The hypothesis that the acetylation of histones is involved in regulation was first formed over thirty years ago by Allfrey and colleagues (Allfrey et al. 1964). Subsequent genetic studies suggested that complexes that utilize ATP hydrolysis to alter chromatin structure might also play a regulatory role. In the past 5 years, numerous ATP-dependent remodeling complexes, acetyltransferases, and acetyltransferase complexes have been isolated and characterized. With the identification of these complexes, it is now possible to examine how these complexes modulate gene expression, and how the action of these complexes can be coordinated. The two major classes of chromatin modifying complexes that have been characterized differ in whether or not they use covalent modification to alter chromatin structure (recent reviews include Felsenfeld et al. 1996; Hartzog and Winston 1997; Tsukiyama and Wu 1997; Gregory and Horz 1998; Imhof and Wolffe 1998; Kadonaga 1998; Kuo and Allis 1998; Mizzen and Allis 1998; Pollard and Peterson 1998; Varga-Weisz and Becker 1998; Workman and Kingston 1998). The first class consists of histone acetyltransferase (HAT) and deacetylase complexes, which, respectively, add and remove acetyl groups from the amino termini of the four core histones; increased acetylation is usually (but not always) associated with activation of gene expression, whereas decreased acetylation is associated with repression of gene expression. The second class consists of ATP-dependent chromatin remodeling complexes, which alter chromatin structure by changing the location or conformation of the nucleosome. These structural changes are accomplished without covalent modification, and can be involved in either activation or repression. In addition to these two major classes of complexes, there are also other recently identified complexes such as FACT, DRIP/ARC, and SPT4/SPT5 that help the transcription machinery contend with nucleosome structure (Hartzog et al. 1998; LeRoy et al. 1998; Orphanides et al. 1998; Wada et al. 1998; Naar et al. 1999; Rachez et al. 1999). The mode of action of these other complexes is not yet understood. This review focuses on ATP-dependent remodeling complexes and how these complexes interact with acetylation complexes to regulate gene expression.

Whose end is destruction: cell division and the anaphase-promoting complex.

Abstract: Cell proliferation depends on the duplication of chromosomes followed by the segregation of duplicates (sister chromatids) to opposite poles of the cell prior to cell division (cytokinesis). How cells ensure that chromosome duplication, chromosome segregation, and cell division occur in the correct order and form an immortal reproductive cycle is one of the most fundamental questions in cell biology. Without such coordination, cells would not maintain a constant chromosome number and sexual reproduction as we know and love it would not be possible.

The prosurvival Bcl-2 homolog Bfl-1/A1 is a direct transcriptional target of NF-κB that blocks TNFα-induced apoptosis

Abstract: Bcl-2-family proteins are key regulators of the apoptotic response. Here, we demonstrate that the pro-survival Bcl-2 homolog Bfl-1/A1 is a direct transcriptional target of NF-kB. We show that bfl-1 gene expression is dependent on NF-kB activity and that it can substitute for NF-kB to suppress TNFa-induced apoptosis. bfl-1 promoter analysis identified an NF-kB site responsible for its Rel/NF-kB-dependent induction. The expression of bfl-1 in immune tissues supports the protective role of NF-kB in the immune system. The activation of Bfl-1 may be the means by which NF-kB functions in oncogenesis and promotes cell resistance to anti-cancer therapy.