Showing papers in "Molecular and Cellular Biology in 1998"

Identification and characterization of a family of mammalian methyl-CpG binding proteins.

Abstract: Methylation at the DNA sequence 5'-CpG is required for mouse development. MeCP2 and MBD1 (formerly PCM1) are two known proteins that bind specifically to methylated DNA via a related amino acid motif and that can repress transcription. We describe here three novel human and mouse proteins (MBD2, MBD3, and MBD4) that contain the methyl-CpG binding domain. MBD2 and MBD4 bind specifically to methylated DNA in vitro. Expression of MBD2 and MBD4 tagged with green fluorescent protein in mouse cells shows that both proteins colocalize with foci of heavily methylated satellite DNA. Localization is disrupted in cells that have greatly reduced levels of CpG methylation. MBD3 does not bind methylated DNA in vivo or in vitro. MBD1, MBD2, MBD3, and MBD4 are expressed in somatic tissues, but MBD1 and MBD2 expression is reduced or absent in embryonic stem cells which are known to be deficient in MeCP1 activity. The data demonstrate that MBD2 and MBD4 bind specifically to methyl-CpG in vitro and in vivo and are therefore likely to be mediators of the biological consequences of the methylation signal.

Functional Inactivation of the Retinoblastoma Protein Requires Sequential Modification by at Least Two Distinct Cyclin-cdk Complexes

Abstract: The retinoblastoma protein (pRb) acts to constrain the G1-S transition in mammalian cells. Phosphorylation of pRb in G1 inactivates its growth-inhibitory function, allowing for cell cycle progression. Although several cyclins and associated cyclin-dependent kinases (cdks) have been implicated in pRb phosphorylation, the precise mechanism by which pRb is phosphorylated in vivo remains unclear. By inhibiting selectively either cdk4/6 or cdk2, we show that endogenous D-type cyclins, acting with cdk4/6, are able to phosphorylate pRb only partially, a process that is likely to be completed by cyclin E-cdk2 complexes. Furthermore, cyclin E-cdk2 is unable to phosphorylate pRb in the absence of prior phosphorylation by cyclin D-cdk4/6 complexes. Complete phosphorylation of pRb, inactivation of E2F binding, and activation of E2F transcription occur only after sequential action of at least two distinct G1 cyclin kinase complexes.

XRCC1 Is Specifically Associated with Poly(ADP-Ribose) Polymerase and Negatively Regulates Its Activity following DNA Damage

Abstract: The genomic integrity of cells is controlled by a network of protein factors that assess the status of the genome and either cause progression of proliferation or induce a halt in the cell cycle. In eukaryotes, DNA strand breaks, introduced either directly by ionizing radiation or indirectly following enzymatic incision of a DNA lesion, trigger the synthesis of poly(ADP-ribose) by the enzyme poly(ADP-ribose) polymerase (PARP) (1, 13, 39). At the site of breakage, PARP catalyzes the transfer of the ADP-ribose moiety from its substrate, NAD+, to a limited number of protein acceptors involved in chromatin architecture and DNA metabolism, including the enzyme itself. These modified proteins, which carry long chains of negatively charged ADP-ribose polymers, lose their affinity for DNA and are thus inactivated. The short half-life of the polymer is attributed to the high activity of poly(ADP-ribose) glycohydrolase, which cleaves the ribose-ribose bond (28, 30). Therefore, poly(ADP-ribosylation) is an immediate but transient postranslational modification of nuclear proteins, induced by DNA-damaging agents. The physiological role of PARP has been much debated in the last decade, but recent molecular and genetic approaches, including expression of either a dominant-negative mutant (26, 36, 44) or antisense oligonucleotides (14), have clearly implicated PARP in the base excision repair (BER) pathway. A more definitive assessment of PARP function was recently provided by the generation of PARP-deficient mice by homologous recombination (35, 53). We found that PARP−/− mice are hypersensitive to monofunctional alkylating agents and γ-irradiation and display a marked genomic instability (sister chromatid exchanges and chromatid and chromosome breaks) following DNA damage (35). Interestingly, γ-irradiation of these mice causes acute toxicity of the epithelia of their small intestines (35), as has been observed with other DNA damage and signalling and repair enzyme deficiencies (2, 3), thus emphasizing the crucial function of DNA surveillance programs of rapidly dividing cells. Similar results indicating that PARP is important for the maintenance of genomic stability following environmental or experimental stress were recently obtained (54). In this work, we have used the two-hybrid system to identify genes encoding proteins that putatively interact with PARP and are involved in its biological function. The human PARP cDNA fused to the LexA-encoding DNA-binding domain (DBD) was used as bait to screen a HeLa cDNA library fused with the activation domain of Gal4. This screening resulted in the identification of the BER pathway protein XRCC1 (X-ray repair cross-complementing 1) as a factor that associates with PARP. This interaction was further confirmed by in vivo experiments with glutathione S-transferase (GST)-tagged fusion proteins expressed in Cos-7 and HeLa cells. XRCC1 and PARP were found to interact via their respective BRCT (BRCA1 C terminus) modules (4, 9) and via an additional site located in the N-terminal zinc-finger domain of PARP. This association dramatically decreased the catalytic activity of PARP without modifying its nick sensor function. Therefore, the association of PARP with XRCC1, a partner of DNA ligase III (7, 8) and DNA polymerase β (25), is suggestive of a role in the detection and protection of a DNA strand break and the subsequent targeting of a BER complex to the damaged site.

Virus-Dependent Phosphorylation of the IRF-3 Transcription Factor Regulates Nuclear Translocation, Transactivation Potential, and Proteasome-Mediated Degradation

Abstract: The interferon regulatory factors (IRF) consist of a growing family of related transcription proteins first identified as regulators of the alpha beta interferon (IFN-alpha/beta) gene promoters, as well as the interferon-stimulated response element (ISRE) of some IFN-stimulated genes. IRF-3 was originally identified as a member of the IRF family based on homology with other IRF family members and on binding to the ISRE of the ISG15 promoter. IRF-3 is expressed constitutively in a variety of tissues, and the relative levels of IRF-3 mRNA do not change in virus-infected or IFN-treated cells. In the present study, we demonstrate that following Sendai virus infection, IRF-3 is posttranslationally modified by protein phosphorylation at multiple serine and threonine residues, which are located in the carboxy terminus of IRF-3. A combination of IRF-3 deletion and point mutations localized the inducible phosphorylation sites to the region -ISNSHPLSLTSDQ- between amino acids 395 and 407; point mutation of residues Ser-396 and Ser-398 eliminated virus-induced phosphorylation of IRF-3 protein, although residues Ser-402, Thr-404, and Ser-405 were also targets. Phosphorylation results in the cytoplasm-to-nucleus translocation of IRF-3, DNA binding, and increased transcriptional activation. Substitution of the Ser-Thr sites with the phosphomimetic Asp generated a constitutively active form of IRF-3 that functioned as a very strong activator of promoters containing PRDI-PRDIII or ISRE regulatory elements. Phosphorylation also appears to represent a signal for virus-mediated degradation, since the virus-induced turnover of IRF-3 was prevented by mutation of the IRF-3 Ser-Thr cluster or by proteasome inhibitors. Interestingly, virus infection resulted in the association of IRF-3 with the CREB binding protein (CBP) coactivator, as detected by coimmunoprecipitation with anti-CBP antibody, an interaction mediated by the C-terminal domains of both proteins. Mutation of residues Ser-396 and Ser-398 in IRF-3 abrogated its binding to CBP. These results are discussed in terms of a model in which virus-inducible, C-terminal phosphorylation of IRF-3 alters protein conformation to permit nuclear translocation, association with transcriptional partners, and primary activation of IFN- and IFN-responsive genes.

Identification and characterization of pancreatic eukaryotic initiation factor 2 α-subunit kinase, PEK, involved in translational control

Abstract: In response to various environmental stresses, eukaryotic cells down-regulate protein synthesis by phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2alpha). In mammals, the phosphorylation was shown to be carried out by eIF-2alpha kinases PKR and HRI. We report the identification and characterization of a cDNA from rat pancreatic islet cells that encodes a new related kinase, which we term pancreatic eIF-2alpha kinase, or PEK. In addition to a catalytic domain with sequence and structural features conserved among eIF-2alpha kinases, PEK contains a distinctive amino-terminal region 550 residues in length. Using recombinant PEK produced in Escherichia coli or Sf-9 insect cells, we demonstrate that PEK is autophosphorylated on both serine and threonine residues and that the recombinant enzyme can specifically phosphorylate eIF-2alpha on serine-51. Northern blot analyses indicate that PEK mRNA is expressed in all tissues examined, with highest levels in pancreas cells. Consistent with our mRNA assays, PEK activity was predominantly detected in pancreas and pancreatic islet cells. The regulatory role of PEK in protein synthesis was demonstrated both in vitro and in vivo. The addition of recombinant PEK to reticulocyte lysates caused a dose-dependent inhibition of translation. In the Saccharomyces model system, PEK functionally substituted for the endogenous yeast eIF-2alpha kinase, GCN2, by a process requiring the serine-51 phosphorylation site in eIF-2alpha. We also identified PEK homologs from both Caenorhabditis elegans and the puffer fish Fugu rubripes, suggesting that this eIF-2alpha kinase plays an important role in translational control from nematodes to mammals.

Activation of the Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase Pathway by Conventional, Novel, and Atypical Protein Kinase C Isotypes

Abstract: To date, 11 members of the protein kinase C (PKC) superfamily have been identified (for reviews, see references 13, 28, 45, and 52). On the basis of their biochemical properties and sequence homologies, they have been divided into three groups: the conventional PKCs (cPKC-α, -β1, -β2, and -γ), which are activated in a diacylglycerol (DAG)- and calcium-dependent manner; the calcium-independent but DAG-dependent novel PKCs (nPKC-δ, -ɛ, -η, -θ, and -μ, also termed PKD); and a third group consisting of atypical PKCs (aPKC-ζ and -ι/λ). The members of this last group of isotypes are unresponsive to DAG and calcium and, in contrast to c- and nPKCs, do not respond to phorbol esters. The existence of this large family of PKC isotypes suggests that individual PKC isotypes likely have specific roles in signal transduction. We have been interested in determining if such specificity exists in the case of the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) cascade, by which PKC may mediate some of its effects on cell growth and differentiation. The MAPK cascade, which involves the kinases Raf, MAPK/ERK kinase (MEK), and ERK/MAPK, is ubiquitously expressed in mammalian cells and serves to couple various cell surface stimuli to the alteration of cell function. This cascade is implicated in both regulated cell proliferation (induced by growth factors) and deregulated proliferation (e.g., Ras transformation) as well as the control of differentiation (33, 54). Such actions are elicited at least in part through the translocation of activated MAPK to the nucleus, where it phosphorylates target molecules such as the transcription factors Elk-1 and SAP1, which consequently leads to alterations in gene expression (24). The mechanisms involved in the activation events for this MAPK cascade have been studied extensively and are well established for MEK and p42 MAPK (ERK2). In both cases, two phosphorylations within the activation loop of the kinase are required for activation, and these are catalyzed by the immediate upstream kinase (4, 47, 59). For instance, for p42 MAPK, it was shown that MEK phosphorylates a threonine (T) and a tyrosine (Y) residue within a characteristic TEY motif, causing activation. In contrast to these activation mechanisms, the regulation of Raf has proven substantially more complex. This protein kinase is regulated in part through interaction with membrane-associated GTP-Ras and in part by phosphorylation (33, 37, 42). Furthermore, it is possible that other modifications and/or associations, such as dimerization of Raf molecules or association with 14-3-3 proteins, regulate Raf function (17, 19, 29, 35). Among the mechanisms involved, there is evidence for the operation of both PKC-dependent and PKC-independent pathways of Raf activation in response to agonists (49). Much evidence for the involvement of PKC in Raf activation comes from the action of the tumor promoters of the phorbol ester class. Acute treatment with phorbol esters leads to a rapid activation of p42 MAPK in most cell types (25, 50). Since PKC is the major receptor for these tumor promoters, it has been implicated in the activation of the ERK/MAPK pathway and the consequent triggering of cellular responses such as cell differentiation and proliferation (18, 23, 40, 41, 44). More-direct evidence for the involvement of PKC in regulating this pathway has come from coexpression studies in insect cells, which have reported that PKC-α, -β1/2, and -γ alone can induce Raf autophosphorylation, peptide phosphorylation, or MEK phosphorylation (38, 51). While early studies employed diverse criteria for assessment of Raf activation, the identification of MEK-1 as a physiological substrate for Raf has provided a robust assay for agonist-induced Raf activation. Using this assay and employing dominant-negative and constitutively active PKC mutants, we here define the potential for these proteins in activation of the MAPK cascade, which is a prerequisite in the mediation of PKC effects, e.g., cell proliferation. We demonstrate here that PKC can be rate limiting for MAPK activation in mammalian cells. Furthermore, it is shown that all PKC isotypes tested (α, β1, δ, ɛ, η, and ζ) have the capacity to activate p42 MAPK and MEK. Additionally, we have been able to show that there are at least two mechanisms involved in activation of the ERK/MAPK pathway by PKCs: cPKC-α and nPKC-η use a Raf-dependent pathway to activate MEK and MAPK, while aPKC-ζ leads to MEK activation in a manner independent of Raf activation. Furthermore, PKC-α (and PKC-β1) is shown to induce a novel desensitization effect in c-Raf activation which prevents further activation by growth factors. These data indicate the operation of a distinct control by conventional PKCs of Raf function. In light of the effects of PKC isotypes on c-Raf mutants, the mechanism of PKC-dependent activation of this pathway is discussed.

Effects of p21(Cip1/Waf1) at both the G1/S and the G2/M cell cycle transitions: pRB is a critical determinant in blocking DNA replication and in preventing endoreduplication

Abstract: It has been proposed that the functions of the cyclin-dependent kinase inhibitors p21(Cip1/Waf1) and p27Kip1 are limited to cell cycle control at the G1/S-phase transition and in the maintenance of cellular quiescence To test the validity of this hypothesis, p21 was expressed in a diverse panel of cell lines, thus isolating the effects of p21 activity from the pleiotropic effects of upstream signaling pathways that normally induce p21 expression The data show that at physiological levels of accumulation, p21, in addition to its role in negatively regulating the G1/S transition, contributes to regulation of the G2/M transition Both G1- and G2-arrested cells were observed in all cell types, with different preponderances Preponderant G1 arrest in response to p21 expression correlated with the presence of functional pRb G2 arrest was more prominent in pRb-negative cells The arrest distribution did not correlate with the p53 status, and proliferating-cell nuclear antigen (PCNA) binding activity of p21 did not appear to be involved, since p27, which lacks a PCNA binding domain, produced similar arrest distributions [corrected], DNA endoreduplication occurred in pRb-negative but not in pRb-positive cells, suggesting that functional pRb is necessary to prevent DNA replication in p21 G2-arrested cells These results suggest that the primary target of the Cip/Kip family of inhibitors leading to efficient G1 arrest as well as to blockade of DNA replication from either G1 or G2 phase is the pRb regulatory system Finally, the tendency of Rb-negative cells to undergo endoreduplication cycles when p21 is expressed may have negative implications in the therapy of Rb-negative cancers with genotoxic agents that activate the p53/p21 pathway

The nuclear orphan receptor car-retinoid x receptor heterodimer activates the phenobarbital-responsive enhancer module of the cyp2b gene

Abstract: PBREM, the phenobarbital-responsive enhancer module of the cytochrome P-450 Cyp2b10 gene, contains two potential nuclear receptor binding sites, NR1 and NR2 Consistent with the finding that anti-retinoid X receptor (RXR) could supershift the NR1-nuclear protein complex, DNA affinity chromatography with NR1 oligonucleotides enriched the nuclear orphan receptor RXR from the hepatic nuclear extracts of phenobarbital-treated mice In addition to RXR, the nuclear orphan receptor CAR was present in the same enriched fraction In the phenobarbital-treated mice, the binding of both CAR and RXR was rapidly increased before the induction of CYP2B10 mRNA In vitro-translated CAR bound to NR1, but only in the presence of similarly prepared RXR PBREM was synergistically activated by transfection of CAR and RXR in HepG2 and HEK293 cells when the NR1 site was functional A CAR-RXR heterodimer has thus been characterized as a trans-acting factor for the phenobarbital-inducible Cyp2b10 gene

Stat3 Activation by Src Induces Specific Gene Regulation and Is Required for Cell Transformation

Abstract: Signal transducers and activators of transcription (STATs) are latent cytoplasmic transcription factors that were first identified as mediators of cellular responses to interferons (reviewed in references 12, 16 and 35). Signaling induced by the interaction of interferons and other cytokines with their cognate receptors is initiated by a cascade of events, including receptor aggregation and activation of Janus protein tyrosine kinases (JAKs) associated with the receptors. Subsequently, STAT proteins are recruited to the receptor-JAK complexes and activated by tyrosine phosphorylation, which promotes the formation of homodimers or heterodimers of STAT family members. Activated STATs, in turn, translocate to the nucleus and bind to specific DNA response elements that regulate gene expression. There are at least seven genes in the mammalian genome known to encode different STAT family members, which are activated in various combinations in response to stimulation by numerous cytokines (12, 16, 35). It has become evident that, in addition to cytokines, mitogenic growth factors, such as platelet-derived growth factor and epidermal growth factor, also induce STAT signaling, particularly Stat1, Stat3, and Stat5 (21, 35). An emerging concept is that normal signaling by STAT proteins is involved in control of diverse biological processes regulated by cytokines and growth factors, including cell differentiation, proliferation, development, and apoptosis (2, 4, 10, 13, 19, 20, 22, 23, 26, 29, 31, 37, 39, 40, 50). Increasingly, evidence that indicates an association between abnormal activation of STAT signaling and oncogenesis has also been accumulating. For example, we and other investigators have demonstrated constitutive activation of Stat1, Stat3, Stat5, and Stat6 in cells transformed by Src, Abl, and various other oncoproteins and tumor viruses (7, 8, 11, 14, 24, 25, 47, 51). In the context of human cancer, there is a high frequency of activation of Stat1, Stat3, and Stat5 in breast carcinoma cells (14, 32, 42) and in lymphoid malignancies, including lymphomas and leukemias (15, 43, 49). Although these findings suggest a role for STAT signaling in cell transformation as well as in human cancer, direct evidence for the obligatory requirement of STAT signaling in oncogenesis has not been reported previously. Because transformation of mammalian fibroblasts by viral Src (v-Src) specifically induces constitutive activation of one STAT family member, Stat3, this system is ideal for investigating the role of Stat3 signaling in oncogenesis (7, 47). The embryonic lethality of targeted disruption of the Stat3 gene (39), however, precludes generation of viable Stat3-deficient mouse models for these studies. An alternative approach for disrupting Stat3 function is to make use of Stat3 dominant negative proteins that interfere with Stat3 signaling. One such variant of Stat3, known as Stat3β, has been shown to block Stat3 function in response to interleukin 5 (IL-5) stimulation (5). Stat3β is a naturally occurring splice variant with a deletion in the C-terminal portion that harbors the transcriptional activation domain and the Ser-727 residue, phosphorylation of which is required for efficient transcriptional activation (44, 45). As a result of this C-terminal deletion, dimers formed with human Stat3β lack transcriptional activity (5). In some studies, however, mouse Stat3β has been shown to activate the promoters of certain genes in a cell type-dependent manner (33, 34), suggesting that the dominant negative effect of Stat3β may be cell type specific. To investigate Stat3-mediated gene regulation by v-Src and the role of Stat3 in oncogenesis, we disrupted Stat3 signaling by using human Stat3β. We report that in NIH 3T3 fibroblasts transiently expressing luciferase reporter constructs, v-Src induced Stat3-specific transcriptional activation. As a potent dominant negative modulator of Stat3-mediated signaling, Stat3β effectively blocked Stat3-specific gene expression induced by v-Src in these cells. Furthermore, cotransfection of expression vectors encoding Stat3β and v-Src suppressed cell transformation of NIH 3T3 fibroblasts, and stable cell lines overexpressing Stat3β were found to be resistant to transformation by v-Src. Our findings establish that activation of Stat3 by v-Src induces specific gene regulation and provide evidence for the requirement of Stat3 signaling in cell transformation by the Src oncoprotein.

Stat3 Activation Is Required for Cellular Transformation by v-src

Abstract: Stat3 activation has been associated with cytokine-induced proliferation, anti-apoptosis, and transformation. Constitutively activated Stat3 has been found in many human tumors as well as v-abl- and v-src-transformed cell lines. Because of these correlations, we examined directly the relationship of activated Stat3 to cellular transformation and found that wild-type Stat3 enhances the transforming potential of v-src while three dominant negative Stat3 mutants inhibit v-src transformation. Stat3 wild-type or mutant proteins did not affect v-ras transformation. We conclude that Stat3 has a necessary role in v-src transformation.

Control of PKR Protein Kinase by Hepatitis C Virus Nonstructural 5A Protein: Molecular Mechanisms of Kinase Regulation

Abstract: The PKR protein kinase is a critical component of the cellular antiviral and antiproliferative responses induced by interferons. Recent evidence indicates that the nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) can repress PKR function in vivo, possibly allowing HCV to escape the antiviral effects of interferon. NS5A presents a unique tool by which to study the molecular mechanisms of PKR regulation in that mutations within a region of NS5A, termed the interferon sensitivity-determining region (ISDR), are associated with sensitivity of HCV to the antiviral effects of interferon. In this study, we investigated the mechanisms of NS5A-mediated PKR regulation and the effect of ISDR mutations on this regulatory process. We observed that the NS5A ISDR, though necessary, was not sufficient for PKR interactions; we found that an additional 26 amino acids (aa) carboxyl to the ISDR were required for NS5A-PKR complex formation. Conversely, we localized NS5A binding to within PKR aa 244 to 296, recently recognized as a PKR dimerization domain. Consistent with this observation, we found that NS5A from interferon-resistant HCV genotype 1b disrupted kinase dimerization in vivo. NS5A-mediated disruption of PKR dimerization resulted in repression of PKR function and inhibition of PKR-mediated eIF-2alpha phosphorylation. Introduction of multiple ISDR mutations abrogated the ability of NS5A to bind to PKR in mammalian cells and to inhibit PKR in a yeast functional assay. These results indicate that mutations within the PKR-binding region of NS5A, including those within the ISDR, can disrupt the NS5A-PKR interaction, possibly rendering HCV sensitive to the antiviral effects of interferon. We propose a model of PKR regulation by NS5A which may have implications for therapeutic strategies against HCV.

Disruption of Higher-Order Folding by Core Histone Acetylation Dramatically Enhances Transcription of Nucleosomal Arrays by RNA Polymerase III

Abstract: We have examined the effects of core histone acetylation on the transcriptional activity and higher-order folding of defined 12-mer nucleosomal arrays. Purified HeLa core histone octamers containing an average of 2, 6, or 12 acetates per octamer (8, 23, or 46% maximal site occupancy, respectively) were assembled onto a DNA template consisting of 12 tandem repeats of a 208-bp Lytechinus 5S rRNA gene fragment. Reconstituted nucleosomal arrays were transcribed in a Xenopus oocyte nuclear extract and analyzed by analytical hydrodynamic and electrophoretic approaches to determine the extent of array compaction. Results indicated that in buffer containing 5 mM free Mg2+ and 50 mM KCl, high levels of acetylation (12 acetates/octamer) completely inhibited higher-order folding and concurrently led to a 15-fold enhancement of transcription by RNA polymerase III. The molecular mechanisms underlying the acetylation effects on chromatin condensation were investigated by analyzing the ability of differentially acetylated nucleosomal arrays to fold and oligomerize. In MgCl2-containing buffer the folding of 12-mer nucleosomal arrays containing an average of two or six acetates per histone octamer was indistinguishable, while a level of 12 acetates per octamer completely disrupted the ability of nucleosomal arrays to form higher-order folded structures at all ionic conditions tested. In contrast, there was a linear relationship between the extent of histone octamer acetylation and the extent of disruption of Mg2+-dependent oligomerization. These results have yielded new insight into the molecular basis of acetylation effects on both transcription and higher-order compaction of nucleosomal arrays.

Direct interaction of SRY-related protein SOX9 and steroidogenic factor 1 regulates transcription of the human anti-Müllerian hormone gene.

Abstract: For proper male sexual differentiation, anti-Mullerian hormone (AMH) must be tightly regulated during embryonic development to promote regression of the Mullerian duct. However, the molecular mechanisms specifying the onset of AMH in male mammals are not yet clearly defined. A DNA-binding element for the steroidogenic factor 1 (SF-1), a member of the orphan nuclear receptor family, located in the AMH proximal promoter has recently been characterized and demonstrated as being essential for AMH gene activation. However, the requirement for a specific promoter environment for SF-1 activation as well as the presence of conserved cis DNA-binding elements in the AMH promoter suggest that SF-1 is a member of a combinatorial protein-protein and protein-DNA complex. In this study, we demonstrate that the canonical SOX-binding site within the human AMH proximal promoter can bind the transcription factor SOX9, a Sertoli cell factor closely associated with Sertoli cell differentiation and AMH expression. Transfection studies with COS-7 cells revealed that SOX9 can cooperate with SF-1 in this activation process. In vitro and in vivo protein-binding studies indicate that SOX9 and SF-1 interact directly via the SOX9 DNA-binding domain and the SF-1 C-terminal region, respectively. We propose that the two transcription factors SOX9 and SF-1 could both be involved in the expression of the AMH gene, in part as a result of their respective binding to the AMH promoter and in part because of their ability to interact with each other. Our work thus identifies SOX9 as an interaction partner of SF-1 that could be involved in the Sertoli cell-specific expression of AMH during embryogenesis.

Nuclear Export Is Required for Degradation of Endogenous p53 by MDM2 and Human Papillomavirus E6

Abstract: The MDM2 oncoprotein targets the p53 tumor suppressor protein for degradation when the two proteins are expressed in cells. The regulation of p53 levels by MDM2 requires the ability of MDM2 to be exported from the nucleus by utilizing its nuclear export signal (NES). The drug leptomycin B (LMB) blocks the formation of nuclear export complexes consisting of CRM1, RanGTP, and NES-containing proteins. It is predicted that LMB should inhibit nuclear-cytoplasmic shuttling by MDM2 and subsequently stabilize p53. This communication demonstrates that LMB treatment of various cell lines led to an increase in the steady-state levels of the p53 protein as a result of an increase in its stability. The stabilized p53 protein localized to the nucleus and was an active transcription factor. These results indicate that the low steady-state levels of p53 in the absence of DNA damage result from p53's nuclear export for cytoplasmic degradation. LMB also led to p53 stabilization in cell lines that contain human papillomavirus (HPV) DNA and express HPV E6, a protein that targets p53 for degradation. MDM2 is not necessary for E6-dependent degradation of p53, as evidenced by the observation that E6 promoted p53 degradation in cells lacking endogenous MDM2. In addition, LMB reduced E6's ability to degrade p53 in the absence of MDM2, demonstrating that complete degradation of p53 by E6 requires nuclear export and therefore likely occurs in cytoplasmic proteasomes. These data suggest that the nuclear export of p53 to the cytoplasm for degradation is a general mechanism for regulating p53 levels.

Translation of vascular endothelial growth factor mRNA by internal ribosome entry: implications for translation under hypoxia.

Abstract: Vascular endothelial growth factor (VEGF) is a hypoxia-inducible angiogenic growth factor that promotes compensatory angiogenesis in circumstances of oxygen shortage. The requirement for translational regulation of VEGF is imposed by the cumbersome structure of the 5′ untranslated region (5′UTR), which is incompatible with efficient translation by ribosomal scanning, and by the physiologic requirement for maximal VEGF production under conditions of hypoxia, where overall protein synthesis is compromised. Using bicistronic reporter gene constructs, we show that the 1,014-bp 5′UTR of VEGF contains a functional internal ribosome entry site (IRES). Efficient cap-independent translation is maintained under hypoxia, thereby securing efficient production of VEGF even under unfavorable stress conditions. To identify sequences within the 5′UTR required for maximal IRES activity, deletion mutants were analyzed. Elimination of the majority (851 nucleotides) of internal 5′UTR sequences not only maintained full IRES activity but also generated a significantly more potent IRES. Activity of the 163-bp long “improved” IRES element was abrogated, however, following substitution of a few bases near the 5′ terminus as well as substitutions close to the translation start codon. Both the full-length 5′UTR and its truncated version function as translational enhancers in the context of a monocistronic mRNA.

Regulation of the p85/p110 Phosphatidylinositol 3′-Kinase: Stabilization and Inhibition of the p110α Catalytic Subunit by the p85 Regulatory Subunit

Abstract: We propose a novel model for the regulation of the p85/pl10alpha phosphatidylinositol 3'-kinase. In insect cells, the p110alpha catalytic subunit is active as a monomer but its activity is decreased by coexpression with the p85 regulatory subunit. Similarly, the lipid kinase activity of recombinant glutathione S-transferase (GST)-p110alpha is reduced by 65 to 85% upon in vitro reconstitution with p85. Incubation of p110alpha/p85 dimers with phosphotyrosyl peptides restored activity, but only to the level of monomeric p110alpha. These data show that the binding of phosphoproteins to the SH2 domains of p85 activates the p85/p110alpha dimers by inducing a transition from an inhibited to a disinhibited state. In contrast, monomeric p110 had little activity in HEK 293T cells, and its activity was increased 15- to 20-fold by coexpression with p85. However, this apparent requirement for p85 was eliminated by the addition of a bulky tag to the N terminus of p110alpha or by the growth of the HEK 293T cells at 30 degrees C. These nonspecific interventions mimicked the effects of p85 on p110alpha, suggesting that the regulatory subunit acts by stabilizing the overall conformation of the catalytic subunit rather than by inducing a specific activated conformation. This stabilization was directly demonstrated in metabolically labeled HEK 293T cells, in which p85 increased the half-life of p110. Furthermore, p85 protected p110 from thermal inactivation in vitro. Importantly, when we examined the effect of p85 on GST-p110alpha in mammalian cells at 30 degrees C, culture conditions that stabilize the catalytic subunit and that are similar to the conditions used for insect cells, we found that p85 inhibited p110alpha. Thus, we have experimentally distinguished two effects of p85 on p110alpha: conformational stabilization of the catalytic subunit and inhibition of its lipid kinase activity. Our data reconcile the apparent conflict between previous studies of insect versus mammalian cells and show that p110alpha is both stabilized and inhibited by dimerization with p85.

The Regulatory Particle of the Saccharomyces cerevisiae Proteasome

Abstract: The proteasome is a multisubunit protease responsible for degrading proteins conjugated to ubiquitin. The 670-kDa core particle of the proteasome contains the proteolytic active sites, which face an interior chamber within the particle and are thus protected from the cytoplasm. The entry of substrates into this chamber is thought to be governed by the regulatory particle of the proteasome, which covers the presumed channels leading into the interior of the core particle. We have resolved native yeast proteasomes into two electrophoretic variants and have shown that these represent core particles capped with one or two regulatory particles. To determine the subunit composition of the regulatory particle, yeast proteasomes were purified and analyzed by gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Resolution of the individual polypeptides revealed 17 distinct proteins, whose identities were determined by amino acid sequence analysis. Six of the subunits have sequence features of ATPases (Rpt1 to Rpt6). Affinity chromatography was used to purify regulatory particles from various strains, each of which expressed one of the ATPases tagged with hexahistidine. In all cases, multiple untagged ATPases copurified, indicating that the ATPases assembled together into a heteromeric complex. Of the remaining 11 subunits that we have identified (Rpn1 to Rpn3 and Rpn5 to Rpn12), 8 are encoded by previously described genes and 3 are encoded by genes not previously characterized for yeasts. One of the previously unidentified subunits exhibits limited sequence similarity with deubiquitinating enzymes. Overall, regulatory particles from yeasts and mammals are remarkably similar, suggesting that the specific mechanistic features of the proteasome have been closely conserved over the course of evolution.

Nerve growth factor activates extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways to stimulate CREB serine 133 phosphorylation.

Abstract: The mechanisms by which growth factor-induced signals are propagated to the nucleus, leading to the activation of the transcription factor CREB, have been characterized. Nerve growth factor (NGF) was found to activate multiple signaling pathways that mediate the phosphorylation of CREB at the critical regulatory site, serine 133 (Ser-133). NGF activates the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases (MAPKs), which in turn activate the pp90 ribosomal S6 kinase (RSK) family of Ser/Thr kinases, all three members of which were found to catalyze CREB Ser-133 phosphorylation in vitro and in vivo. In addition to the ERK/RSK pathway, we found that NGF activated the p38 MAPK and its downstream effector, MAPK-activated protein kinase 2 (MAPKAP kinase 2), resulting in phosphorylation of CREB at Ser-133. Inhibition of either the ERK/RSK or the p38/MAPKAP kinase 2 pathway only partially blocked NGF-induced CREB Ser-133 phosphorylation, suggesting that either pathway alone is sufficient for coupling the NGF signal to CREB activation. However, inhibition of both the ERK/RSK and the p38/MAPKAP kinase 2 pathways completely abolished NGF-induced CREB Ser-133 phosphorylation. These findings indicate that NGF activates two distinct MAPK pathways, both of which contribute to the phosphorylation of the transcription factor CREB and the activation of immediate-early genes.

A Dominant-Negative Inhibitor of CREB Reveals that It Is a General Mediator of Stimulus-Dependent Transcription of c-fos

Abstract: Several studies have characterized the upstream regulatory region of c-fos, and identified cis-acting elements termed the cyclic AMP (cAMP) response elements (CREs) that are critical for c-fos transcription in response to a variety of extracellular stimuli. Although several transcription factors can bind to CREs in vitro, the identity of the transcription factor(s) that activates the c-fos promoter via the CRE in vivo remains unclear. To help identify the trans-acting factors that regulate stimulus-dependent transcription of c-fos via the CREs, dominant-negative (D-N) inhibitor proteins that function by preventing DNA binding of B-ZIP proteins in a dimerization domain-dependent fashion were developed. A D-N inhibitor of CREB, termed A-CREB, was constructed by fusing a designed acidic amphipathic extension onto the N terminus of the CREB leucine zipper domain. The acidic extension of A-CREB interacts with the basic region of CREB forming a coiled-coil extension of the leucine zipper and thus prevents the basic region of wild-type CREB from binding to DNA. Other D-N inhibitors generated in a similar manner with the dimerization domains of Fos, Jun, C/EBP, ATF-2, or VBP did not block CREB DNA binding activity, nor did they inhibit transcriptional activation of a minimal promoter containing a single CRE in PC12 cells. A-CREB inhibited activation of CRE-mediated transcription evoked by three distinct stimuli: forskolin, which increases intracellular cAMP; membrane depolarization, which promotes Ca 21 influx; and nerve growth factor (NGF). A-CREB completely inhibited cAMP-mediated, but only partially inhibited Ca 21 - and NGF-mediated, transcription of a reporter gene containing 750 bp of the native c-fos promoter. Moreover, glutamate induction of c-fos expression in primary cortical neurons was dependent on CREB. In contrast, induction of c-fos transcription by UV light was not inhibited by A-CREB. Lastly, A-CREB attenuated NGF induction of morphological differentiation in PC12 cells. These results suggest that CREB or its closely related family members are general mediators of stimulus-dependent transcription of c-fos and are required for at least some of the long-term actions of NGF.

Characterization of the p53-Dependent Postmitotic Checkpoint following Spindle Disruption

Abstract: The proper execution of events in the eukaryotic cell cycle is regulated by a number of different checkpoints. For example, to ensure stable maintenance of the genome, cells arrest in G1 or G2 upon detection of DNA damage, providing time for DNA repair before the initiation of DNA synthesis or entry into mitosis (11). Other checkpoints function during mitosis to monitor successful assembly of the spindle and control the initiation of metaphase, thus protecting the cell from chromosome missegregation (37). Genes that are required for cellular arrest at different checkpoints have been identified, demonstrating that each checkpoint is a genetic pathway activated by specific signals. The inactivation of checkpoint genes leads to increased mutation rate, chromosome loss, or changes in ploidy, depending on the genes affected (34). Interestingly, some genes that are mutated in human cancers are involved in checkpoint functions, suggesting that without such controls in place, the resulting genetic damage predisposes cells to malignancy (38). The p53 tumor suppressor gene is mutated in over half of all sporadic human cancers. p53 has an essential role in the G1 checkpoint in response to DNA-damaging agents such as radiation (20, 21, 23). Functional analysis of the p53 protein has shown that it is a transcription factor with sequence-specific DNA binding activity (12, 22, 42). After DNA damage, p53 activates the transcription of several downstream target genes, including p21, an inhibitor of cyclin-dependent kinases (CDKs) (10). The induction of p21 causes subsequent arrest in the G1 phase of the cell cycle by binding of cyclin-CDK complexes (14, 15, 41). Additional p53 target genes are likely to cooperate with p21 in implementing G1 arrest, as p21-deficient mouse cells are only partially defective in their DNA damage arrest response, while p53-deficient cells are completely defective (2, 7). Recently, p53 has been proposed to have an additional, novel function as a checkpoint at mitosis. Wild-type fibroblasts arrest when mitotic spindle assembly is disrupted by the addition of drugs which bind microtubules. However, studies have shown that p53-deficient fibroblasts fail to arrest under such conditions but instead undergo a new round of DNA synthesis in the absence of cell division, becoming polyploid (5, 8, 33). This phenotype is very similar to that observed in yeast strains that have inactivated spindle assembly checkpoints. Saccharomyces cerevisiae strains with mutations in the MAD or BUB genes, which monitor spindle assembly, become polyploid when treated with spindle inhibitors (17, 27). Based on this similarity, one might conclude that, like the MAD and BUB gene products, p53 monitors spindle integrity and can thus be termed a mitotic checkpoint. Also, additional data have suggested that the spindle components themselves may be under p53 regulation, as p53 has been shown to localize to the centrosome (1) and p53−/− mouse embryonic fibroblasts (MEFs) contain abnormal numbers of centrosomes (13). However, later work has shown that in cells treated with spindle inhibitors, p53 is neither expressed during mitosis nor required for mitotic arrest (33). Instead, expression of p53 protein occurs only after cells exit mitotic arrest and progress to an interphase state while retaining 4N DNA content. These data argue that p53 does not function as a mitotic checkpoint but acts at some subsequent point in the cell cycle to induce arrest following a defect in M phase. In this study, we have characterized in greater detail the p53-dependent checkpoint following disruption of the mitotic spindle with microtubule-destabilizing drugs. Upon examining the response of wild-type and p53-deficient mouse embryo fibroblasts to spindle inhibitors, we observed that cells of either genotype underwent a transient arrest at mitosis and subsequently progressed into a G1-like state without ever completing cell division. Wild-type MEFs remained arrested in this state, while p53−/− MEFs initiated another round of DNA synthesis. Using time-lapse videomicroscopy, we were able to define the length of mitotic arrest and establish precisely the timing of S phase reentry in p53−/− MEFs after exit from mitotic arrest. We also demonstrated a requirement for the p53 target gene product, p21, in executing cell cycle arrest after spindle disruption. Upon further characterization of arrested cells, we determined that cells expressed molecular markers associated with the G1 phase of the cell cycle, despite having 4N DNA content. These data demonstrate that the p53-dependent checkpoint in cells with disrupted mitotic spindles has strong similarity to the p53-dependent checkpoint in G1 following DNA damage. Our data suggest that rather than having a novel role as a spindle checkpoint, p53 functions in the G1 phase of the cell cycle and via the same downstream pathway in murine cells which have sustained either DNA damage or microtubule disruption.

CCAAT/enhancer binding protein alpha is a regulatory switch sufficient for induction of granulocytic development from bipotential myeloid progenitors

Abstract: According to the current view of hematopoiesis, all blood cell types derive from a common pluripotent stem cell (65). In the adult, the stem cells are found in bone marrow, where they divide to produce more stem cells (self-renewal) and various precursor cells committed to a single lineage which terminally differentiate to morphologically and functionally distinct erythroid, myeloid, or lymphoid cells. Within the myeloid compartment, the same committed precursor can give rise to monocytic or granulocytic cells. This raises a question: what are the molecular mechanisms that dictate the fate of the common precursor to one or the other of these two diverse myeloid lineages? Genetic manipulations such as gene knockout and gene transfer experiments provide increasing evidence that transcription factors are involved in execution of the differentiation program of a cell. Determination of the developmental role of a number of transcription factors in blood development has been achieved by gene disruption experiments. These studies indicated that the GATA-1 and GATA-2, SCL, PU.1, Ikaros, c-myb, and AML1 genes, among others, are key regulators of hematopoiesis (reviewed in references 63 and 68). Inactivation of these genes had multilineage effects. Complementary to these knockout experiments are expression studies which identified certain transcription factors as master regulators of development, defined as genes which once activated would establish a specific cell type. For example, ectopic expression of MyoD in diverse cell types converts them to muscle cells (17). Similarly, expression of the B-cell transcription factor Oct-2 or the helix-loop-helix protein E47 in non-B cells induces B cell-like phenotypes (55, 59). In addition, thanks to advances in cell and molecular biological techniques, isolation and analysis of single primary cells, including functional stem cells, is feasible, and their developmental patterns can be studied as well (12). CCAAT/enhancer binding protein α (C/EBPα) was initially identified in liver and adipose tissue, where it was found to be important for terminal differentiation (8, 16, 22, 39, 44, 75). More recently it was shown to be also expressed in early myeloid cells (12, 61, 68). In addition, a number of granulocyte-specific genes, including granulocyte colony-stimulating factor (G-CSF) receptor (64), neutrophil elastase (52), and myeloperoxidase (23, 52) genes, have been shown to be regulated by C/EBPα. Gene targeting experiments revealed a specific defect in the hematopoietic system of C/EBPα knockout mice. The C/EBPα null phenotype was characterized by lack of mature granulocytes, with all the other blood cell types present, including monocytes and peritoneal macrophages (81). These results strongly point to a critical role of C/EBPα in granulocytic differentiation (reviewed in reference 68). In the present study, we have investigated the expression pattern of C/EBPα in the hematopoietic system during monocytic and granulocytic differentiation. We also examined the effect of induced overexpression of C/EBPα on the differentiation program of early bipotential myeloid cells. Our results demonstrate that the C/EBPα gene is activated at the stage of myeloid commitment and is specifically expressed in granulocytic cells. Increased levels of C/EBPα expressed from an inducible promoter construct directed differentiation along the granulocytic pathway, as determined by morphological criteria. Furthermore, ectopic expression of C/EBPα resulted in upregulation of mRNA of the granulocyte-restricted genes encoding the G-CSF receptor and C/EBPɛ, as well as secondary granule protein genes lactoferrin and human neutrophil collagenase. Our findings identify C/EBPα as the molecular switch during early hematopoietic developmental events that directs cells to the granulocytic pathway.

Aberrant Recruitment of the Nuclear Receptor Corepressor-Histone Deacetylase Complex by the Acute Myeloid Leukemia Fusion Partner ETO

Abstract: Nuclear receptor corepressor (CoR)-histone deacetylase (HDAC) complex recruitment is indispensable for the biological activities of the retinoic acid receptor fusion proteins of acute promyelocytic leukemias We report here that ETO (eight-twenty-one or MTG8), which is fused to the acute myelogenous leukemia 1 (AML1) transcription factor in t(8;21) AML, interacts via its zinc finger region with a conserved domain of the corepressors N-CoR and SMRT and recruits HDAC in vivo The fusion protein AML1-ETO retains the ability of ETO to form stable complexes with N-CoR/SMRT and HDAC Deletion of the ETO C terminus abolishes CoR binding and HDAC recruitment and severely impairs the ability of AML1-ETO to inhibit differentiation of hematopoietic precursors These data indicate that formation of a stable complex with CoR–HDAC is crucial to the activation of the leukemogenic potential of AML1 by ETO and suggest that aberrant recruitment of corepressor complexes is a general mechanism of leukemogenesis

ETO, a Target of t(8;21) in Acute Leukemia, Interacts with the N-CoR and mSin3 Corepressors

Abstract: t(8;21) is one of the most frequent translocations associated with acute myeloid leukemia. It produces a chimeric protein, acute myeloid leukemia-1 (AML-1)-eight-twenty-one (ETO), that contains the amino-terminal DNA binding domain of the AML-1 transcriptional regulator fused to nearly all of ETO. Here we demonstrate that ETO interacts with the nuclear receptor corepressor N-CoR, the mSin3 corepressors, and histone deacetylases. Endogenous ETO also cosediments on sucrose gradients with mSin3A, N-CoR, and histone deacetylases, suggesting that it is a component of one or more corepressor complexes. Deletion mutagenesis indicates that ETO interacts with mSin3A independently of its association with N-CoR. Single amino acid mutations that impair the ability of ETO to interact with the central portion of N-CoR affect the ability of the t(8;21) fusion protein to repress transcription. Finally, AML-1/ETO associates with histone deacetylase activity and a histone deacetylase inhibitor impairs the ability of the fusion protein to repress transcription. Thus, t(8;21) fuses a component of a corepressor complex to AML-1 to repress transcription.

Regulation of Insulin-Stimulated Glucose Transporter GLUT4 Translocation and Akt Kinase Activity by Ceramide

Abstract: The sphingomyelin derivative ceramide is a signaling molecule implicated in numerous physiological events. Recently published reports indicate that ceramide levels are elevated in insulin-responsive tissues of diabetic animals and that agents which trigger ceramide production inhibit insulin signaling. In the present series of studies, the short-chain ceramide analog C2-ceramide inhibited insulin-stimulated glucose transport by approximately 50% in 3T3-L1 adipocytes, with similar reductions in hormone-stimulated translocation of the insulin-responsive glucose transporter (GLUT4) and insulin-responsive aminopeptidase. C2-ceramide also inhibited phosphorylation and activation of Akt, a molecule proposed to mediate multiple insulin-stimulated metabolic events. C2-ceramide, at concentrations which antagonized activation of both glucose uptake and Akt, had no effect on the tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) or the amounts of p85 protein and phosphatidylinositol kinase activity that immunoprecipitated with anti-IRS-1 or antiphosphotyrosine antibodies. Moreover, C2-ceramide also inhibited stimulation of Akt by platelet-derived growth factor, an event that is IRS-1 independent. C2-ceramide did not inhibit insulin-stimulated phosphorylation of mitogen-activated protein kinase or pp70 S6-kinase, and it actually stimulated phosphorylation of the latter in the absence of insulin. Various pharmacological agents, including the immunosuppressant rapamycin, the protein synthesis inhibitor cycloheximide, and several protein kinase C inhibitors, were without effect on ceramide's inhibition of Akt. These studies demonstrate ceramide's capacity to inhibit activation of Akt and imply that this is a mechanism of antagonism of insulin-dependent physiological events, such as the peripheral activation of glucose transport and the suppression of apoptosis.

Identification and Characterization of a Constitutively Active STAT5 Mutant That Promotes Cell Proliferation

Abstract: Stimulation of cytokine receptors leads to activation of multiple signal transduction pathways, including the Ras-Raf-MEK-mitogen-activated protein kinase (MAPK) and the JAK-STAT pathways (14, 28, 34, 42, 44). The latter signaling pathway was originally found downstream of the interferon receptors and is now recognized as a common pathway downstream of most cytokine receptors. Upon stimulation with cytokines, receptor-associated JAKs are activated and phosphorylate STAT factors on tyrosine residues. The phosphorylated STAT molecules then form homo- or heterodimers through SH2-mediated interactions and translocate into nuclei to activate transcription of various target genes. Seven members of the STAT family (STAT1 through 4, -5A, -5B, and -6) are known; STAT5A and STAT5B are closely related. With the exception of STAT4 and STAT6, which were shown to be specifically activated by only one or two cytokines, interleukin 12 (IL-12) or both IL-4 and IL-13, respectively (13, 15), most of the other STATs are activated by multiple cytokines. In particular, both STAT5A and STAT5B are activated by numerous cytokines, including prolactin, IL-2, IL-3, IL-5, IL-7, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, M-CSF, erythropoietin (Epo), thrombopoietin, and growth hormone (GH). Using the receptor for the human GM-CSF as a model system, members of our group previously showed that activation of the Ras-Raf-MEK-MAPK pathway inhibits apoptosis while the region of the GM-CSF receptor, which is responsible for activation of JAK2 and STAT5 and induction of c-myc expression, plays a role in DNA synthesis (18, 41). Members of our group also demonstrated that a dominant negative STAT5 protein partially inhibited IL-3-induced cell proliferation in Ba/F3 cells, suggesting that STAT5A is involved in cell proliferation (29). A similar conclusion was reached by others who observed that IL-2 and Epo receptors defective in STAT5 activation (5, 7, 9) were also deficient in supporting a proliferative signal. In contrast, others have concluded that STAT5 is not involved in cell proliferation since mutant IL-2 (8) and Epo receptors (37) which cannot activate STAT5 are still capable of transmitting an attenuated proliferative signal. These differing interpretations of generally similar data indicate a point of controversy. Recently, mice nullizygous for STAT5A and STAT5B have been generated (24, 45). Since these mice exhibit apparently normal hematopoiesis, the contribution of STAT5 activation to proliferation or to any other biological function, such as differentiation in normal hematopoietic tissues, remains unclear. In this paper, we report the identification and characterization of a constitutively active form of STAT5 by PCR-driven mutagenesis followed by retrovirus-mediated expression screening. The mutant STAT5 protein is constitutively phosphorylated on its tyrosine residues, is localized in the nucleus, and is transcriptionally active in the absence of growth factor stimulation. Expression of the mutant STAT5 in IL-3-dependent cells is capable of inducing cytokine-independent growth.

Classification of gas5 as a multi-small-nucleolar-RNA (snoRNA) host gene and a member of the 5'-terminal oligopyrimidine gene family reveals common features of snoRNA host genes.

Abstract: In the nucleolus of eukaryotic cells, ribosomal DNA is transcribed by RNA polymerase I into long precursor (pre-rRNA) transcripts, which are modified by methylation and pseudo-uridylation, cleaved to yield 18S, 5.8S, and 28S rRNAs, and then assembled into the mature large and small ribosomal subunits prior to export to the cytoplasm (for reviews see references 22, 26, and 77). A large number of small nucleolar ribonucleoprotein (snoRNP) particles have emerged as key players in this biosynthetic process. Currently more than 70 snoRNA species have been identified (for reviews see references 51, 75, and 82). All snoRNAs, with the exception of MRP RNA, can be divided into two classes: those that possess boxes C (RUGAUGA) and D (CUGA), which are required for association with the abundant nucleolar autoantigen fibrillarin (see reference 51), and those that possess boxes H (ANANNA) and ACA, which mediate the binding of Gar1 protein (4, 8, 24, 37). Only a few snoRNAs have been found to be required for growth in yeast (U3 [6, 29], U14 [43], MRP [14, 73], snR10 [80, 81], and snR30 [5, 54]) or for specific pre-rRNA cleavage events in Xenopus oocytes (U3 [34, 72], U8 [63], and U22 [85]). Recently, box C/D snoRNAs and box H/ACA snoRNAs were found to target specific sites in pre-rRNA for 2′-O-methylation and pseudouridylation, respectively (for reviews see references 46, 48, 62, 75, and 82). These modification reactions are mediated by extensive regions (10 to 21 nt) of complementarity between the so-called “antisense” snoRNAs and sequences flanking the rRNA sites to be modified. Specifically, U24, U20, and U25 were shown to direct site-specific ribose methylation of pre-rRNA in HeLa cells (39), yeast (13), and Xenopus oocytes (87), respectively, and snR8, snR3, snR33, and snR5 (among others) were demonstrated to target pre-rRNA for pseudouridylation in yeast (23, 57). The presence of ∼200 modified nucleotides in vertebrate rRNA (47) suggests that more than half of the antisense snoRNAs remain to be identified. A unique feature of snoRNAs is that most are encoded within the introns of protein-coding genes (reviewed in reference 51). This economic use of introns is commonplace among intron-rich organisms, such as vertebrates, where antisense snoRNAs have been found to be exclusively intron encoded. By contrast, in Saccharomyces cerevisiae many snoRNAs are produced from independent transcription units. A vertebrate host gene intron encodes only a single snoRNA, whereas in yeast and plants, some snoRNA genes are located in polycistronic arrays without exons separating the snoRNA sequences (41). In all species investigated, the intron-encoded snoRNAs are transcribed from their host genes by RNA polymerase II as portions of the pre-mRNA. The functional snoRNAs are then produced by exonucleolytic trimming that follows either splicing (12, 38, 86) or endonucleolytic cleavage of intron sequences (9, 10). The mode by which snoRNA sequences became inserted into the introns of their host genes is not known. Interestingly, the host gene for a particular snoRNA can differ even among closely related vertebrates, suggesting that intron-encoded snoRNAs may be highly mobile genetic elements (see reference 51). Likewise, the reason particular genes have been chosen as hosts for intron-encoded snoRNAs has been unclear. Initially, it appeared that all snoRNA host genes generate protein products that function in ribosome biogenesis or in translation; ribosomal proteins (rp) L1, L5, L7a, S8, nucleolin, and eIF4AI are a few examples (see references 66, 68, 67, 59, 58, and 24, respectively). Such genetic organization could provide coregulation of protein components of the translational machinery and snoRNAs, which contribute to rRNA maturation (76). However, the discovery of other snoRNA host genes lacking obvious ribosome-related functions (for example, ATP synthase β [39]) suggested that host genes may have been chosen merely to meet the need for transcription rates high enough to produce a sufficient level of snoRNAs (∼104 copies/cell) to base pair with the cell’s nascent pre-rRNA molecules. UHG (U22 host gene) is an unusual snoRNA host gene because it does not appear to specify a protein product. It generates, in addition to U22, seven different box C/D antisense snoRNAs (U25 to U31) (84). Comparison of mouse and human UHG sequences revealed that its introns are more conserved than its exons, suggesting that the snoRNAs may be the only functional portions of the transcript. Both human and mouse UHG messages are riddled with stop codons in all three reading frames; the longest open reading frames (ORFs) would produce peptides of 51 and 40 amino acids for human and mouse UHG, respectively. Nonetheless, the UHG transcript resembles a typical rp mRNA in that it begins with a C residue, is spliced and polyadenylated, and is associated with ribosomes. However, unlike rp mRNAs, the spliced UHG RNA is almost undetectable in HeLa cells. Inhibition of translation in HeLa cells with the initiation inhibitor pactamycin or elongation inhibitors cycloheximide or puromycin results in a 15-fold increase in the level of spliced UHG transcript (84). This link between the levels of UHG RNA and active translation, in conjunction with its numerous stop codons, suggests that it may be a candidate for the nonsense-mediated decay pathway (49, 84). Here we report the identification of a second member of the UHG class of snoRNA host genes. Growth arrest-specific transcript 5 (gas5) was initially discovered in a screen for potential tumor suppressor genes expressed at high levels during growth arrest (74). The murine gas5 gene produces a ubiquitous, polyadenylated, alternatively spliced message which is almost undetectable in actively growing cells yet is highly expressed in cells undergoing serum starvation or density arrest (15, 16). We demonstrate that gas5 is a multi-snoRNA host gene which encodes 9 (in mouse) or 10 (in human) antisense snoRNAs. By mapping the 5′ end of the gas5 transcript and comparing it with other known snoRNA host genes, we observe that all known snoRNA host genes exhibit characteristics which define the 5′TOP (terminal oligopyrimidine) class of genes. We provide evidence that membership in the 5′TOP family explains why the abundance of the gas5 spliced product is growth dependent. Furthermore, the discovery that all snoRNA host genes contain 5′TOP sequences may illuminate why certain genes have been selected to serve as snoRNA host genes.

Multiple Grb2-Mediated Integrin-Stimulated Signaling Pathways to ERK2/Mitogen-Activated Protein Kinase: Summation of Both c-Src- and Focal Adhesion Kinase-Initiated Tyrosine Phosphorylation Events

Abstract: Fibronectin receptor integrin-mediated cell adhesion triggers intracellular signaling events such as the activation of the Ras/mitogen-activated protein (MAP) kinase cascade. In this study, we show that the nonreceptor protein-tyrosine kinases (PTKs) c-Src and focal adhesion kinase (FAK) can be independently activated after fibronectin (FN) stimulation and that their combined activity promotes signaling to extracellular signal-regulated kinase 2 (ERK2)/MAP kinase through multiple pathways upstream of Ras. FN stimulation of NIH 3T3 fibroblasts promotes c-Src and FAK association in the Triton-insoluble cell fraction, and the time course of FN-stimulated ERK2 activation paralleled that of Grb2 binding to FAK at Tyr-925 and Grb2 binding to Shc. Cytochalasin D treatment of fibroblasts inhibited FN-induced FAK in vitro kinase activity and signaling to ERK2, but it only partially inhibited c-Src activation. Treatment of fibroblasts with protein kinase C inhibitors or with the PTK inhibitor herbimycin A or PP1 resulted in reduced Src PTK activity, no Grb2 binding to FAK, and lowered levels of ERK2 activation. FN-stimulated FAK PTK activity was not significantly affected by herbimycin A treatment and, under these conditions, FAK autophosphorylation promoted Shc binding to FAK. In vitro, FAK directly phosphorylated Shc Tyr-317 to promote Grb2 binding, and in vivo Grb2 binding to Shc was observed in herbimycin A-treated fibroblasts after FN stimulation. Interestingly, c-Src in vitro phosphorylation of Shc promoted Grb2 binding to both wild-type and Phe-317 Shc. In vivo, Phe-317 Shc was tyrosine phosphorylated after FN stimulation of human 293T cells and its expression did not inhibit signaling to ERK2. Surprisingly, expression of Phe-925 FAK with Phe-317 Shc also did not block signaling to ERK2, whereas FN-stimulated signaling to ERK2 was inhibited by coexpression of an SH3 domain-inactivated mutant of Grb2. Our studies show that FN receptor integrin signaling upstream of Ras and ERK2 does not follow a linear pathway but that, instead, multiple Grb2-mediated interactions with Shc, FAK, and perhaps other yet-to-be-determined phosphorylated targets represent parallel signaling pathways that cooperate to promote maximal ERK2 activation.

The Mitochondrial Permeability Transition Is Required for Tumor Necrosis Factor Alpha-Mediated Apoptosis and Cytochrome c Release

Abstract: This study assesses the controversial role of the mitochondrial permeability transition (MPT) in apoptosis. In primary rat hepatocytes expressing an IκB superrepressor, tumor necrosis factor alpha (TNFα) induced apoptosis as shown by nuclear morphology, DNA ladder formation, and caspase 3 activation. Confocal microscopy showed that TNFα induced onset of the MPT and mitochondrial depolarization beginning 9 h after TNFα treatment. Initially, depolarization and the MPT occurred in only a subset of mitochondria; however, by 12 h after TNFα treatment, virtually all mitochondria were affected. Cyclosporin A (CsA), an inhibitor of the MPT, blocked TNFα-mediated apoptosis and cytochrome c release. Caspase 3 activation, cytochrome c release, and apoptotic nuclear morphological changes were induced after onset of the MPT and were prevented by CsA. Depolarization and onset of the MPT were blocked in hepatocytes expressing ΔFADD, a dominant negative mutant of Fas-associated protein with death domain (FADD), or crmA, a natural serpin inhibitor of caspases. In contrast, Asp-Glu-Val-Asp-cho, an inhibitor of caspase 3, did not block depolarization or onset of the MPT induced by TNFα, although it inhibited cell death completely. In conclusion, the MPT is an essential component in the signaling pathway for TNFα-induced apoptosis in hepatocytes which is required for both cytochrome c release and cell death and functions downstream of FADD and crmA but upstream of caspase 3.

Modulation of transcriptional regulation by LEF-1 in response to Wnt-1 signaling and association with beta-catenin.

Abstract: Wnt signaling is thought to be mediated via interactions between beta-catenin and members of the LEF-1/TCF family of transcription factors. Here we study the mechanism of transcriptional regulation by LEF-1 in response to a Wnt-1 signal under conditions of endogenous beta-catenin in NIH 3T3 cells, and we examine whether association with beta-catenin is obligatory for the function of LEF-1. We find that Wnt-1 signaling confers transcriptional activation potential upon LEF-1 by association with beta-catenin in the nucleus. By mutagenesis, we identified specific residues in LEF-1 important for interaction with beta-catenin, and we delineated two transcriptional activation domains in beta-catenin whose function is augmented in specific association with LEF-1. Finally, we show that a Wnt-1 signal and beta-catenin association are not required for the architectural function of LEF-1 in the regulation of the T-cell receptor alpha enhancer, which involves association of LEF-1 with a different cofactor, ALY. Thus, LEF-1 can assume diverse regulatory functions by association with different proteins.

The Syk Protein Tyrosine Kinase Is Essential for Fcγ Receptor Signaling in Macrophages and Neutrophils

Abstract: The cytoplasmic protein tyrosine kinase Syk has two amino-terminal SH2 domains that engage phosphorylated immunoreceptor tyrosine-based activation motifs in the signaling subunits of immunoreceptors. Syk, in conjunction with Src family kinases, has been implicated in immunoreceptor signaling in both lymphoid and myeloid cells. We have investigated the role of Syk in Fcgamma receptor (FcgammaR)-dependent and -independent responses in bone marrow-derived macrophages and neutrophils by using mouse radiation chimeras reconstituted with fetal liver cells from Syk-/- embryos. Chimeric mice developed an abdominal hemorrhage starting 2 to 3 months after transplantation that was ultimately lethal. Syk-deficient neutrophils derived from the bone marrow were incapable of generating reactive oxygen intermediates in response to FcgammaR engagement but responded normally to tetradecanoyl phorbol acetate stimulation. Syk-deficient macrophages were defective in phagocytosis induced by FcgammaR but showed normal phagocytosis in response to complement. The tyrosine phosphorylation of multiple cellular polypeptides, including the FcgammaR gamma chain, as well as Erk2 activation, was compromised in Syk-/- macrophages after FcgammaR stimulation. In contrast, the induction of nitric oxide synthase in macrophages stimulated with lipopolysaccharide and gamma interferon was not dependent on Syk. Surprisingly, Syk-deficient macrophages were impaired in the ability to survive or proliferate on plastic petri dishes. Taken together, these results suggest that Syk has specific physiological roles in signaling from FcgammaRs in neutrophils and macrophages and raise the possibility that in vivo, Syk is involved in signaling events other than those mediated by immunoreceptors.