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H F Lodish

Publications and source records attributed to H F Lodish.

At least 55 records · Page 3Linked to original sources
PubMed1999

Expression cloning of LDLB, a gene essential for normal Golgi function and assembly of the ldlCp complex.

The Chinese hamster ovary (CHO) cell mutants ldlC and ldlB, which exhibit almost identical phenotypes, define two genes required for multiple steps in the normal medial and trans Golgi-associated processing of glycoconjugates. The LDLC gene encodes ldlCp, an approximately 80-kDa protein, which in wild-type, but not ldlB, cells associates reversibly with the cytoplasmic surface of the Golgi apparatus. Here, we have used a retrovirus-based expression cloning system to clone a murine cDNA, LDLB, that corrects the pleiotropic mutant phenotypes of ldlB cells. The corresponding mRNA was not detected in ldlB mutants. LDLB encodes an approximately 110-kDa protein, ldlBp, which lacks homology to known proteins and contains no common structural motifs. Database searches identified short segments of homology to sequences from Drosophila melanogaster, Arabidopsis thaliana, and Caenorhabditis elegans, and the essentially full-length homologous human sequence (82% identity); however, as was the case for ldlCp, no homologue was identified in Saccharomyces cerevisiae. We have found that in wild-type cell cytosols, ldlCp is a component of an approximately 950-kDa "ldlCp complex," which is smaller, approximately 700 kDa, in ldlB cytosols. Normal assembly of this complex is ldlBp-dependent and may be required for Golgi association of ldlCp and for the normal activities of multiple luminal Golgi processes.

3T3 Cells↗
PubMed1999

Interaction of the Ski oncoprotein with Smad3 regulates TGF-beta signaling.

TGF-beta treatment of cells induces a variety of physiologic responses, including growth inhibition, differentiation, and induction of apoptosis. TGF-beta induces phosphorylation and nuclear translocation of Smad3. We describe here the association of Smad3 with the nuclear protooncogene protein Ski in response to the activation of TGF-beta signaling. Association with Ski represses transcriptional activation by Smad3, and overexpression of Ski renders cells resistant to the growth-inhibitory effects of TGF-beta. The transcriptional repression as well as the growth resistance to TGF-beta by overexpression of Ski can be overcome by overexpression of Smad3. These results demonstrate that Ski is a novel component of the TGF-beta signaling pathway and shed light on the mechanism of action of the Ski oncoprotein.

Animals↗
PubMed1999

Identification of the major intestinal fatty acid transport protein.

While intestinal transport systems for metabolites such as carbohydrates have been well characterized, the molecular mechanisms of fatty acid (FA) transport across the apical plasmalemma of enterocytes have remained largely unclear. Here, we show that FATP4, a member of a large family of FA transport proteins (FATPs), is expressed at high levels on the apical side of mature enterocytes in the small intestine. Further, overexpression of FATP4 in 293 cells facilitates uptake of long chain FAs with the same specificity as enterocytes, while reduction of FATP4 expression in primary enterocytes by antisense oligonucleotides inhibits FA uptake by 50%. This suggests that FATP4 is the principal fatty acid transporter in enterocytes and may constitute a novel target for antiobesity therapy.

Binding, Competitive↗
PubMed1999

Growth factor independence and BCR/ABL transformation: promise and pitfalls of murine model systems and assays.

The expression of the BCR-ABL fusion oncoprotein in primitive hematopoietic cells results in chronic myeloid leukemia. Over the past decade studies of several in vitro and in vivo cell systems revealed multiple signal transduction pathways activated by BCR-ABL. However, the precise function of BCR-ABL in the pathogenesis of CML is still unclear. The goal of this review is to synthesize data on intracellular signaling in the context of the diverse murine assay systems employed. We emphasize the importance of in vivo assays and assays using primary cells in understanding the biology of CML and the molecular mechanisms by which BCR-ABL exerts its effects.

Animals↗
PubMed1998

Zinc is essential for binding of p56(lck) to CD4 and CD8alpha.

Binding of the protein tyrosine kinase p56(lck) to T-cell co-receptors CD4 and CD8alpha is necessary for T-lymphocyte development and activation. Association of p56(lck) with CD4 requires two conserved cysteine residues in the cytosolic domain of CD4 and two in the amino terminus of p56(lck), consistent with the notion that these four residues coordinate a single metal atom (1-5). Here we demonstrate that Zn2+ is essential for complex formation. In an in vitro binding reaction, Zn2+ mediates p56(lck) association with a glutathione S-transferase (GST) fusion protein containing the cytosolic domains of CD4 or CD8alpha; no other metals tested support binding. Treatment of preformed GST-CD4.p56(lck) dimers with the Zn2+ chelators 1,10-O-phenanthroline or 8-hydroxyquinoline-5-sulfonic acid results in dissociation of GST-CD4 from p56(lck), consistent with the finding of Huse et al. (5) that Zn2+ is contained within similar complexes. Furthermore, we show that, within live cells, CD4.p56(lck) and CD8alpha.p56(lck) interactions occur in a zinc-dependent fashion. Specifically, pretreatment of the human Jurkat T-cell line with membrane permeable zinc chelators disrupts CD4.p56(lck) complexes, and treatment of COS cells co-expressing CD8alpha and p56(lck) with such chelators likewise leads to dissociation of CD8alpha.p56(lck) complexes. CD4. p56(lck) and CD8alpha.p56(lck) represent the first examples of intracellular proteins that require zinc as a bridge for heterodimerization.

Animals↗
PubMed1998

Synergistic cooperation of TFE3 and smad proteins in TGF-beta-induced transcription of the plasminogen activator inhibitor-1 gene.

Members of the TGF-beta superfamily influence a broad range of biological activities including stimulation of wound healing and inhibition of cell growth. TGF-beta signals through type I and II receptor serine/ threonine kinases and induces transcription of many genes including plasminogen activator inhibitor-1 (PAI-1). To identify proteins that participate in TGF-beta-induced gene expression, we developed a novel retrovirus-mediated expression cloning strategy; and using this approach, we established that transcription factor microE3 (TFE3) is involved in TGF-beta-induced activation of the PAI-1 promoter. We showed that TFE3 binds to an E-box sequence in PE2, a 56-bp promoter fragment of the PAI-1 promoter, and that mutation of this sequence abolishes both TFE3 binding as well as TGF-beta-dependent activation. TFE3 and Smad3 synergistically activate the PE2 promoter and phosphorylated Smad3 and Smad4 bind to a sequence adjacent to the TFE3-binding site in this promoter. Binding of both TFE3 and the Smad proteins to their cognate sequences is indispensable for TGF-beta-inducible activation of the PE2 promoter. Hence, TFE3 is an important transcription factor in at least one TGF-beta-activated signal transduction pathway.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗
PubMed1998

The prolactin receptor rescues EpoR-/- erythroid progenitors and replaces EpoR in a synergistic interaction with c-kit.

We recently showed that a retrovirally transduced prolactin receptor (PrlR) efficiently supports the differentiation of wild-type burst-forming unit erythroid (BFU-e) and colony-forming unit erythroid (CFU-e) progenitors in response to prolactin and in the absence of erythropoietin (Epo). To examine directly whether the Epo receptor (EpoR) expressed by wild-type erythroid progenitors was essential for their terminal differentiation, we infected EpoR-/- progenitors with retroviral constructs encoding either the PrlR or a chimeric receptor containing the extracellular domain of the PrlR and intracellular domain of EpoR. In response to prolactin, both receptors were equally efficient in supporting full differentiation of the EpoR-/- progenitors into erythroid colonies in vitro. Therefore, there is no requirement for an EpoR-unique signal in erythroid differentiation; EpoR signaling has no instructive role in red blood cell differentiation. A synergistic interaction between EpoR and c-kit is essential for the production of normal numbers of red blood cells, as demonstrated by the severe anemia of mice mutant for either c-kit or its ligand, stem cell factor. We show that the addition of stem cell factor potentiates the ability of the PrlR to support differentiation of both EpoR-/- and wild-type CFU-e progenitors. This synergism is quantitatively equivalent to that observed between c-kit and EpoR. Therefore, there is no requirement for an EpoR-unique signal in the synergistic interaction between c-kit and EpoR.

Animals↗
PubMed1998

A family of fatty acid transporters conserved from mycobacterium to man.

Long chain fatty acids (LCFAs) are an important source of energy for most organisms. They also function as blood hormones, regulating key metabolic functions such as hepatic glucose production. Although LCFAs can diffuse through the hydrophobic core of the plasma membrane into cells, this nonspecific transport cannot account for the high affinity and specific transport of LCFAs exhibited by cells such as cardiac muscle, hepatocytes, and adipocytes. Transport of LCFAs across the plasma membrane is facilitated by fatty acid transport protein (FATP), a plasma membrane protein that increases LCFA uptake when expressed in cultured mammalian cells [Schaffer, J. E. & Lodish, H. F. (1994) Cell 79, 427-436]. Here, we report the identification of four novel murine FATPs, one of which is expressed exclusively in liver and another only in liver and kidney. Both genes increase fatty acid uptake when expressed in mammalian cells. All five murine FATPs have homologues in humans in addition to a sixth FATP gene. FATPs are found in such diverse organisms as Fugu rubripes, Caenorhabditis elegans, Drosophila melanogaster, Saccharomyces cerevisiae, and Mycobacterium tuberculosis. The function of the FATP gene family is conserved throughout evolution as the C. elegans and mycobacterial FATPs facilitate LCFA uptake when overexpressed in COS cells or Escherichia coli, respectively. The identification of this evolutionary conserved fatty acid transporter family will allow us to gain a better understanding of the mechanisms whereby LCFAs traverse the lipid bilayer as well as yield insight into the control of energy homeostasis and its dysregulation in diseases such as diabetes and obesity.

Amino Acid Sequence↗
PubMed1998

Oligomeric structure of type I and type II transforming growth factor beta receptors: homodimers form in the ER and persist at the plasma membrane.

Transforming growth factor beta (TGF-beta) signaling involves interactions of at least two different receptors, types I (TbetaRI) and II (TbetaRII), which form ligand-mediated heteromeric complexes. Although we have shown in the past that TbetaRII in the absence of ligand is a homodimer on the cell surface, TbetaRI has not been similarly investigated, and the site of complex formation is not known for either receptor. Several studies have indicated that homomeric interactions are involved in TGF-beta signaling and regulation, emphasizing the importance of a detailed understanding of the homooligomerization of TbetaRI or TbetaRII. Here we have combined complementary approaches to study these homomeric interactions in both naturally expressing cell lines and cells cotransfected with various combinations of epitope-tagged type I or type II receptors. We used sedimentation velocity of metabolically labeled receptors on sucrose gradients to show that both TbetaRI and TbetaRII form homodimer-sized complexes in the endoplasmic reticulum, and we used coimmunoprecipitation studies to demonstrate the existence of type I homooligomers. Using a technique based on antibody-mediated immunofluorescence copatching of receptors carrying different epitope tags, we have demonstrated ligand-independent homodimers of TbetaRI on the surface of live cells. Soluble forms of both receptors are secreted as monomers, indicating that the ectodomains are not sufficient to mediate homodimerization, although TGF-beta1 is able to promote dimerization of the type II receptor ectodomain. These findings may have important implications for the regulation of TGF-beta signaling.

Activin Receptors, Type I↗
PubMed1998

The anemic Friend virus gp55 envelope protein induces erythroid differentiation in fetal liver colony-forming units-erythroid.

The gp55 envelope proteins of the spleen focus-forming virus initiate erythroleukemia in adult mice. Because the gp55 from the polycythemic strain (gp55-P), but not from the anemic strain (gp55-A), activates the erythropoietin receptor (EpoR) for proliferation of hematopoietic cell lines, the mechanism by which gp55-A initiates erythroleukemia has remained a mystery. We show here that gp55-A activates the EpoR in fetal liver cells. In contrast to previous studies using bone marrow cells from phenylhydrazine-treated, anemic mice, we find that both gp55-A and gp55-P induce erythroid differentiation from colony-forming unit-erythroid (CFU-E) progenitors in fetal liver cells. The effects on CFU-Es of both gp55-A and -P are mediated by the EpoR, because no colonies are seen upon expression of either gp55 in EpoR-/- fetal liver cells. However, only gp55-P induces erythroid bursts from burst-forming unit-erythroid progenitors and only gp55-P induces Epo independence in Epo-dependent cell lines. Using chimeric gp55 P/A proteins, we extend earlier work showing that the transmembrane sequence determines the capacity of gp55 proteins to differentially activate EpoR signaling. We discuss the possibilities for different signaling capacities of gp55-A and -P in fetal liver and bone marrow-derived erythroid progenitor cells.

Animals↗
PubMed1998

Tumor necrosis factor receptor (TNFR)-associated factor 2A (TRAF2A), a TRAF2 splice variant with an extended RING finger domain that inhibits TNFR2-mediated NF-kappaB activation.

We describe here the identification and characterization of tumor necrosis factor receptor (TNFR)-associated factor 2A (TRAF2A), a splice variant of the TRAF2 molecule utilized for signal transduction by members of the TNFR family. TRAF2A and TRAF2 cDNAs are identical in sequence with the exception of an extra 21 base pairs of sequence encoding a 7-amino acid insert within the TRAF2A RING finger domain. TRAF2A mRNA expression is regulated in a tissue-specific manner, with relative TRAF2A mRNA levels being highest in spleen and lowest in brain. TRAF2A protein is capable of binding to the cytoplasmic domain of TNFR2 (p75) and is detectable in T-lymphoma cells stably transfected with the TRAF2A cDNA. Unlike TRAF2, TRAF2A has a short half-life ( approximately 100 min) in these cells and is expressed at only low levels in transiently transfected COS-7 cells. However, TRAF2A levels in transiently transfected COS-7 cells approach those of TRAF2 upon coexpression with TRAF1 and/or TRAF2, indicating that TRAF2A stability is regulated by the binding of other TRAF family proteins. Also in contrast to TRAF2, TRAF2A is unable to stimulate NF-kappaB activity when overexpressed in 293 cells and acts as a dominant inhibitor of TNFR2-dependent NF-kappaB activation. TRAF2A thus represents a novel signal transduction protein, the expression of which can act to inhibit TRAF2-dependent NF-kappaB activation.

Alternative Splicing↗
PubMed1998

Tyrosine residues within the intracellular domain of the erythropoietin receptor mediate activation of AP-1 transcription factors.

Binding of erythropoietin (Epo) to the Epo receptor (EpoR) initiates a signaling cascade resulting in tyrosine phosphorylation of several proteins and induction of AP-1 transcription factor(s). While Epo is known to activate c-fos gene expression, the mechanism of AP-1 activation is unknown. Here we show that AP-1 activation by Epo requires tyrosine kinase activity and also de novo protein synthesis. Using a mutant EpoR containing no cytosolic tyrosine residues, and a set of eight mutants containing a single cytosolic tyrosine residue, we show that multiple EpoR tyrosines, thought to activate multiple intracellular signal transduction proteins, can mediate AP-1 activation. An EpoR containing only tyrosine 343 or tyrosine 464 supports a maximal level of AP-1 activation. We also show that AP-1 activation does not require maximal STAT5 activation and may occur via a STAT5-independent signaling pathway.

Cycloheximide↗
PubMed1998

Cloning of cell-specific secreted and surface proteins by subtractive antibody screening.

To identify and clone genes that encode cell- or tissue-specific secreted and surface proteins, a polyclonal antiserum was raised against a complex mixture of surface or secreted proteins from the target cell, followed by immunodepletion of antibodies that recognize proteins from a nontarget cell or tissue. The depleted antiserum is used to screen bacteriophage cDNA expression libraries. Because of our interest in how adipocytes communicate with other cells, we have used this method to clone cDNAs encoding secreted and plasma membrane proteins that are induced during adipocyte differentiation. We describe several of these, including a novel plasma membrane-associated protein, S3-12.

3T3 Cells↗
PubMed1997

Transforming growth factor beta-induced phosphorylation of Smad3 is required for growth inhibition and transcriptional induction in epithelial cells.

Drosophila Mad proteins are intracellular signal transducers of decapentaplegic (dpp), the Drosophila transforming growth factor beta (TGF-beta)/bone morphogenic protein (BMP) homolog. Studies in which the mammalian Smad homologs were transiently overexpressed in cultured cells have implicated Smad2 in TGF-beta signaling, but the physiological relevance of the Smad3 protein in signaling by TGF-beta receptors has not been established. Here we stably expressed Smad proteins at controlled levels in epithelial cells using a novel approach that combines highly efficient retroviral gene transfer and quantitative cell sorting. We show that upon TGF-beta treatment Smad3 becomes rapidly phosphorylated at the SSVS motif at its very C terminus. Either attachment of an epitope tag to the C terminus or replacement of these three serine residues with alanine abolishes TGF-beta-induced Smad3 phosphorylation; these proteins act in a dominant-negative fashion to block the antiproliferative effect of TGF-beta in mink lung epithelial cells. A Smad3 protein in which the three C-terminal serines have been replaced by aspartic acids is also a dominant inhibitor of TGF-beta signaling, but can activate plasminogen activator inhibitor 1 (PAI-1) transcription in a ligand-independent fashion when its nuclear localization is forced by transient overexpression. Phosphorylation of the three C-terminal serine residues of Smad3 by an activated TGF-beta receptor complex is an essential step in signal transduction by TGF-beta for both inhibition of cell proliferation and activation of the PAI-1 promoter.

Amino Acid Sequence↗
PubMed1997

The prolactin receptor and severely truncated erythropoietin receptors support differentiation of erythroid progenitors.

Activation of the erythropoietin receptor is essential for the survival, proliferation, and differentiation of erythroid progenitors. To understand the role of erythropoietin receptor (EpoR) activation in erythroid differentiation, we infected primary erythroid progenitors with high-titer retrovirus encoding the non-hematopoietic prolactin receptor. The infected progenitors responded to prolactin in the absence of Epo by generating fully differentiated erythroid colonies. Therefore, differentiation of erythroid progenitors does not require an intracellular signal generated uniquely by the EpoR; the EpoR does not have an instructive role in erythroid differentiation. We also infected primary erythroid progenitors with retrovirus encoding chimeric receptors containing the extracellular domain of PrlR and the intracellular domain of either the wild-type or truncated EpoRs. A chimeric receptor containing only the membrane-proximal 136 amino acids of the EpoR cytoplasmic domain efficiently supported prolactin-dependent differentiation of erythroid progenitors. Substitution of the single cytoplasmic domain tyrosine in this receptor with phenylalanine (Y343F) eliminated its ability to support differentiation. The minimal EpoR cytoplasmic domain required for erythroid differentiation is therefore the same as that previously reported to be sufficient to support cell proliferation (D'Andrea, A. D., Yoshimura, A., Youssoufian, H., Zon, L. I., Koo, J. W., and Lodish, H. F. (1991) Mol. Cell. Biol. 11, 1980-1987; Miura, O., D'Andrea, A. D., Kabat, D., and Ihle, J. N. (1991) Mol. Cell. Biol. 11, 4895-4902; He, T.-C., Jiang, N., Zhuang, H., Quelle, D. E., and Wojchowski, D. M. (1994) J. Biol. Chem. 269, 18291-18294).

Animals↗