Coordinate regulation of lipogenic gene expression by androgens: Evidence for a cascade mechanism involving sterol regulatory element binding proteins

To gain more insight into the molecular mechanisms by which androgens stimulate lipogenesis and induce a marked accumulation of neutral lipids in the human prostate cancer cell line LNCaP, we studied their impact on the expression of lipogenic enzymes. Northern blot analysis of the steady-state mRNA levels of seven different lipogenic enzymes revealed that androgens coordinately stimulate the expression of enzymes belonging to the two major lipogenic pathways: fatty acid synthesis and cholesterol synthesis. In view of the important role of the recently characterized sterol regulatory element binding proteins (SREBPs) in the coordinate induction of lipogenic genes, we examined whether the observed effects of androgens on lipogenic gene expression are mediated by these transcription factors. Our findings indicate that androgens stimulate the expression of SREBP transcripts and precursor proteins and enhance the nuclear content of the mature active form of the transcription factor. Moreover, by using the fatty acid synthase gene as an experimental paradigm we demonstrate that the presence of an SREBP-binding site is essential for its regulation by androgens. These data support the hypothesis that SREBPs are involved in the coordinate regulation of lipogenic gene expression by androgens and provide evidence for the existence of a cascade mechanism of androgen-regulated gene expression.

Differential binding to HLA-C of p50-activating and p58-inhibitory natural killer cell receptors

Natural killer (NK) cell cytotoxicity is regulated in large part by the expression of NK cell receptors able to bind class I major histocompatibility complex glycoproteins. The receptors associated with recognition of HLA-C allospecificities are the two-domain Ig-like molecules, p50 and p58 proteins, with highly homologous extracellular domains but differing in that they have either an activating or inhibitory function, respectively, depending on the transmembrane domain and cytoplasmic tails that they possess. We have compared the binding to HLA-Cw7 of an inhibitory p58 molecule, NKAT2, the highly homologous activating p50 molecule, clone 49, and a second activating p50 molecule, clone 39, which has homologies to both NKAT1 and NKAT2. NKAT2 binds to HLA-Cw7 with very rapid association and dissociation rates. However, the p50 receptors bind only very weakly, if at all, to HLA-C. The molecular basis of this difference is analyzed, and the functional significance of these observations is discussed.

AF5q31, a newly identified AF4-related gene, is fused to MLL in infant acute lymphoblastic leukemia with ins(5;11)(q31;q13q23)

Infant acute lymphoblastic leukemia (ALL) with MLL gene rearrangements is characterized by early pre-B phenotype (CD10−/CD19+) and poor treatment outcome. The t(4;11), creating MLL-AF4 chimeric transcripts, is the predominant 11q23 chromosome translocation in infant ALL and is associated with extremely poor prognosis as compared with other 11q23 translocations. We analyzed an infant early preB ALL with ins(5;11)(q31;q13q23) and identified the AF5q31 gene on chromosome 5q31 as a fusion partner of the MLL gene. The AF5q31 gene, which encoded a protein of 1,163 aa, was located in the vicinity of the cytokine cluster region of chromosome 5q31 and contained at least 16 exons. The AF5q31 gene was expressed in fetal heart, lung, and brain at relatively high levels and fetal liver at a low level, but the expression in these tissues decreased in adults. The AF5q31 protein was homologous to AF4-related proteins, including AF4, LAF4, and FMR2. The AF5q31 and AF4 proteins had three homologous regions, including the transactivation domain of AF4, and the breakpoint of AF5q31 was located within the region homologous to the transactivation domain of AF4. Furthermore, the clinical features of this patient with the MLL-AF5q31 fusion transcript, characterized by the early pre-B phenotype (CD10−/CD19+) and poor outcome, were similar to those of patients having MLL-AF4 chimeric transcripts. These findings suggest that AF5q31 and AF4 might define a new family particularly involved in the pathogenesis of 11q23-associated-ALL.

Chemical synthesis of the precursor molecule of the Aequorea green fluorescent protein, subsequent folding, and development of fluorescence

The present paper describes the total chemical synthesis of the precursor molecule of the Aequorea green fluorescent protein (GFP). The molecule is made up of 238 amino acid residues in a single polypeptide chain and is nonfluorescent. To carry out the synthesis, a procedure, first described in 1981 for the synthesis of complex peptides, was used. The procedure is based on performing segment condensation reactions in solution while providing maximum protection to the segment. The effectiveness of the procedure has been demonstrated by the synthesis of various biologically active peptides and small proteins, such as human angiogenin, a 123-residue protein analogue of ribonuclease A, human midkine, a 121-residue protein, and pleiotrophin, a 136-residue protein analogue of midkine. The GFP precursor molecule was synthesized from 26 fully protected segments in solution, and the final 238-residue peptide was treated with anhydrous hydrogen fluoride to obtain the precursor molecule of GFP containing two Cys(acetamidomethyl) residues. After removal of the acetamidomethyl groups, the product was dissolved in 0.1 M Tris⋅HCl buffer (pH 8.0) in the presence of DTT. After several hours at room temperature, the solution began to emit a green fluorescence (λmax = 509 nm) under near-UV light. Both fluorescence excitation and fluorescence emission spectra were measured and were found to have the same shape and maxima as those reported for native GFP. The present results demonstrate the utility of the segment condensation procedure in synthesizing large protein molecules such as GFP. The result also provides evidence that the formation of the chromophore in GFP is not dependent on any external cofactor.

TROSY in triple-resonance experiments: New perspectives for sequential NMR assignment of large proteins

The NMR assignment of 13C, 15N-labeled proteins with the use of triple resonance experiments is limited to molecular weights below ∼25,000 Daltons, mainly because of low sensitivity due to rapid transverse nuclear spin relaxation during the evolution and recording periods. For experiments that exclusively correlate the amide proton (1HN), the amide nitrogen (15N), and 13C atoms, this size limit has been previously extended by additional labeling with deuterium (2H). The present paper shows that the implementation of transverse relaxation-optimized spectroscopy ([15N,1H]-TROSY) into triple resonance experiments results in several-fold improved sensitivity for 2H/13C/15N-labeled proteins and approximately twofold sensitivity gain for 13C/15N-labeled proteins. Pulse schemes and spectra recorded with deuterated and protonated proteins are presented for the [15N, 1H]-TROSY-HNCA and [15N, 1H]-TROSY-HNCO experiments. A theoretical analysis of the HNCA experiment shows that the primary TROSY effect is on the transverse relaxation of 15N, which is only little affected by deuteration, and predicts sensitivity enhancements that are in close agreement with the experimental data.

Generation of secretable and nonsecretable interleukin 15 isoforms through alternate usage of signal peptides

Two isoforms of human interleukin 15 (IL-15) exist. One isoform has a shorter putative signal peptide (21 amino acids) and its transcript shows a tissue distribution pattern that is distinct from that of the alternative IL-15 isoform with a 48-aa signal peptide. The 21-aa signal isoform is preferentially expressed in tissues such as testis and thymus. Experiments using different combinations of signal peptides and mature proteins (IL-2, IL-15, and green fluorescent protein) showed that the short signal peptide regulates the fate of the mature protein by controlling the intracellular trafficking to nonendoplasmic reticulum sites, whereas the long signal peptide both regulates the rate of protein translation and functions as a secretory signal peptide. As a consequence, the IL-15 associated with the short signal peptide is not secreted, but rather is stored intracellularly, appearing in the nucleus and cytoplasmic components. Such production of an intracellular lymphokine is not typical of other soluble interleukin systems, suggesting a biological function for IL-15 as an intracellular molecule.

Genetic interactions with Rap1 and Ras1 reveal a second function for the Fat facets deubiquitinating enzyme in Drosophila eye development

The Drosophila fat facets gene encodes a deubiquitinating enzyme that regulates a cell communication pathway essential very early in eye development, prior to facet assembly, to limit the number of photoreceptor cells in each facet of the compound eye to eight. The Fat facets protein facilitates the production of a signal in cells outside the developing facets that inhibits neural development of particular facet precursor cells. Novel gain-of-function mutations in the Drosophila Rap1 and Ras1 genes are described herein that interact genetically with fat facets mutations. Analysis of these genetic interactions reveals that Fat facets has an additional function later in eye development involving Rap1 and Ras1 proteins. Moreover, the results suggest that undifferentiated cells outside the facet continue to influence facet assembly later in eye development.

Meis1 and pKnox1 bind DNA cooperatively with Pbx1 utilizing an interaction surface disrupted in oncoprotein E2a-Pbx1

E2a-Pbx1 is a chimeric transcription factor oncoprotein produced by the t(1;19) translocation in human pre-B cell leukemia. Class I Hox proteins bind DNA cooperatively with both Pbx proteins and oncoprotein E2a-Pbx1, suggesting that leukemogenesis by E2a-Pbx1 and Hox proteins may alter transcription of cellular genes regulated by Pbx–Hox motifs. Likewise, in murine myeloid leukemia, transcriptional coactivation of Meis1 with HoxA7/A9 suggests that Meis1–HoxA7/9 heterodimers may evoke aberrant gene transcription. Here, we demonstrate that both Meis1 and its relative, pKnox1, dimerize with Pbx1 on the same TGATTGAC motif selected by dimers of Pbx proteins and unidentified partner(s) in nuclear extracts, including those from t(1;19) pre-B cells. Outside their homeodomains, Meis1 and pKnox1 were highly conserved only in two motifs required for cooperativity with Pbx1. Like the unidentified endogenous partner(s), both Meis1 and pKnox1 failed to dimerize significantly with E2a-Pbx1. The Meis1/pKnox1-interaction domain in Pbx1 resided predominantly in a conserved N-terminal Pbx domain deleted in E2a-Pbx1. Thus, the leukemic potential of E2a-Pbx1 may require abrogation of its interaction with members of the Meis and pKnox families of transcription factors, permitting selective targeting of genes regulated by Pbx–Hox complexes. In addition, because most motifs bound by Pbx–Meis1/pKnox1 were not bound by Pbx1–Hox complexes, the leukemic potential of Meis1 in myeloid leukemias may involve shifting Pbx proteins from promoters containing Pbx–Hox motifs to those containing Pbx–Meis motifs.

Efficient gene tagging in Arabidopsis thaliana using a gene trap approach

Large quantities of DNA sequence information about plant genes are rapidly accumulating in public databases, but to progress from DNA sequence to biological function a mutant allele for each of the genes ideally should be available. Here we describe a gene trap construct that allowed us to disrupt transcribed genes with a high efficiency in Arabidopsis thaliana. In the T-DNA vector used, the expression of a bacterial reporter gene coding for neomycin phosphotransferase II (nptII) depends on the in vivo generation of a translation fusion upon the T-DNA integration into the Arabidopsis genome. Analysis of 20 selected transgenic lines showed that 12 lines are T-DNA insertion mutants. The disrupted genes analyzed encoded ribosomal proteins (three lines), aspartate tRNA synthase, DNA ligase, basic-domain leucine zipper DNA binding protein, ATP-binding cassette transporter, and five proteins of unknown function. Four tagged genes were new for Arabidopsis. The results presented here suggest that gene trapping, using nptII as a reporter gene, can be as high as 80% and opens novel perspectives for systematic gene tagging in A. thaliana.

Cleavage Furrows Formed between Centrosomes Lacking an Intervening Spindle and Chromosomes Contain Microtubule Bundles, INCENP, and CHO1 but Not CENP-E

PtK1 cells containing two independent mitotic spindles can cleave between neighboring centrosomes, in the absence of an intervening spindle, as well as at the spindle equators. We used same-cell video, immunofluorescence, and electron microscopy to compare the structure and composition of normal equatorial furrows with that of ectopic furrows formed between spindles. As in controls, ectopic furrows contained midbodies composed of microtubule bundles and an electron-opaque matrix. Despite the absence of an intervening spindle and chromosomes, the midbodies associated with ectopic furrows also contained the microtubule-bundling protein CHO1 and the chromosomal passenger protein INCENP. However, CENP-E, another passenger protein, was not found in ectopic furrows but was always present in controls. We also examined cells in which the ectopic furrow initiated but relaxed. Although relaxing furrows contained overlapping microtubules from opposing centrosomes, they lacked microtubule bundles as well as INCENP and CHO1. Together these data suggest that the mechanism defining the site of furrow formation during mitosis in vertebrates does not depend on the presence of underlying microtubule bundles and chromosomes or on the stable association of INCENP or CHO1. The data also suggest that the completion of cytokinesis requires the presence of microtubule bundles and specific proteins (e.g., INCENP, CHO1, etc.) that do not include CENP-E.

Protein Interactions with the Glucose Transporter Binding Protein GLUT1CBP That Provide a Link between GLUT1 and the Cytoskeleton

Subcellular targeting and the activity of facilitative glucose transporters are likely to be regulated by interactions with cellular proteins. This report describes the identification and characterization of a protein, GLUT1 C-terminal binding protein (GLUT1CBP), that binds via a PDZ domain to the C terminus of GLUT1. The interaction requires the C-terminal four amino acids of GLUT1 and is isoform specific because GLUT1CBP does not interact with the C terminus of GLUT3 or GLUT4. Most rat tissues examined contain both GLUT1CBP and GLUT1 mRNA, whereas only small intestine lacked detectable GLUT1CBP protein. GLUT1CBP is also expressed in primary cultures of neurons and astrocytes, as well as in Chinese hamster ovary, 3T3-L1, Madin–Darby canine kidney, Caco-2, and pheochromocytoma-12 cell lines. GLUT1CBP is able to bind to native GLUT1 extracted from cell membranes, self-associate, or interact with the cytoskeletal proteins myosin VI, α-actinin-1, and the kinesin superfamily protein KIF-1B. The presence of a PDZ domain places GLUT1CBP among a growing family of structural and regulatory proteins, many of which are localized to areas of membrane specialization. This and its ability to interact with GLUT1 and cytoskeletal proteins implicate GLUT1CBP in cellular mechanisms for targeting GLUT1 to specific subcellular sites either by tethering the transporter to cytoskeletal motor proteins or by anchoring the transporter to the actin cytoskeleton.

Microdomain-dependent Regulation of Lck and Fyn Protein-Tyrosine Kinases in T Lymphocyte Plasma Membranes

Src family protein-tyrosine kinases are implicated in signaling via glycosylphosphatidylinositol (GPI)-anchored receptors. Both kinds of molecules reside in opposite leaflets of the same sphingolipid-enriched microdomains in the lymphocyte plasma membrane without making direct contact. Under detergent-free conditions, we isolated a GPI-enriched plasma membrane fraction, also containing transmembrane proteins, selectively associated with sphingolipid microdomains. Nonionic detergents released the transmembrane proteins, yielding core sphingolipid microdomains, limited amounts of which could also be obtained by detergent-free subcellular fractionation. Protein-tyrosine kinase activity in membranes containing both GPI-anchored and transmembrane proteins was much lower than in core sphingolipid microdomains but was strongly reactivated by nonionic detergents. The inhibitory mechanism acting on Lck and Fyn kinases in these membranes was independent of the protein-tyrosine phosphatase CD45 and was characterized as a mixed, noncompetitive one. We propose that in lymphocyte plasma membranes, Lck and Fyn kinases exhibit optimal activity when juxtaposed to the GPI- and sphingolipid-enriched core microdomains but encounter inhibitory conditions in surrounding membrane areas that are rich in glycerophospholipids and contain additional transmembrane proteins.

The Cell Surface Flocculin Flo11 Is Required for Pseudohyphae Formation and Invasion by Saccharomyces cerevisiae

Diploid yeast develop pseudohyphae in response to nitrogen starvation, while haploid yeast produce invasive filaments which penetrate the agar in rich medium. We have identified a gene, FLO11, that encodes a cell wall protein which is critically required for both invasion and pseudohyphae formation in response to nitrogen starvation. FLO11 encodes a cell surface flocculin with a structure similar to the class of yeast serine/threonine-rich GPI-anchored cell wall proteins. Cells of the Saccharomyces cerevisiae strain Σ1278b with deletions of FLO11 do not form pseudohyphae as diploids nor invade agar as haploids. In rich media, FLO11 is regulated by mating type; it is expressed in haploid cells but not in diploids. Upon transfer to nitrogen starvation media, however, FLO11 transcripts accumulate in diploid cells, but not in haploids. Overexpression of FLO11 in diploid cells, which are otherwise not invasive, enables them to invade agar. Thus, the mating type repression of FLO11 in diploids grown in rich media suffices to explain the inability of these cells to invade. The promoter of FLO11 contains a consensus binding sequence for Ste12p and Tec1p, proteins known to cooperatively activate transcription of Ty1 elements and the TEC1 gene during development of pseudohyphae. Yeast with a deletion of STE12 does not express FLO11 transcripts, indicating that STE12 is required for FLO11 expression. These ste12-deletion strains also do not invade agar. However, the ability to invade can be restored by overexpressing FLO11. Activation of FLO11 may thus be the primary means by which Ste12p and Tec1p cause invasive growth.

The Golgi and Endoplasmic Reticulum Remain Independent during Mitosis in HeLa Cells

Partitioning of the mammalian Golgi apparatus during cell division involves disassembly at M-phase. Despite the importance of the disassembly/reassembly pathway in Golgi biogenesis, it remains unclear whether mitotic Golgi breakdown in vivo proceeds by direct vesiculation or involves fusion with the endoplasmic reticulum (ER). To test whether mitotic Golgi is fused with the ER, we compared the distribution of ER and Golgi proteins in interphase and mitotic HeLa cells by immunofluorescence microscopy, velocity gradient fractionation, and density gradient fractionation. While mitotic ER appeared to be a fine reticulum excluded from the region containing the spindle-pole body, mitotic Golgi appeared to be dispersed small vesicles that penetrated the area containing spindle microtubules. After cell disruption, M-phase Golgi was recovered in two size classes. The major breakdown product, accounting for at least 75% of the Golgi, was a population of 60-nm vesicles that were completely separated from the ER using velocity gradient separation. The minor breakdown product was a larger, more heterogenously sized, membrane population. Double-label fluorescence analysis of these membranes indicated that this portion of mitotic Golgi also lacked detectable ER marker proteins. Therefore we conclude that the ER and Golgi remain distinct at M-phase in HeLa cells. To test whether the 60-nm vesicles might form from the ER at M-phase as the result of a two-step vesiculation pathway involving ER–Golgi fusion followed by Golgi vesicle budding, mitotic cells were generated with fused ER and Golgi by brefeldin A treatment. Upon brefeldin A removal, Golgi vesicles did not emerge from the ER. In contrast, the Golgi readily reformed from similarly treated interphase cells. We conclude that Golgi-derived vesicles remain distinct from the ER in mitotic HeLa cells, and that mitotic cells lack the capacity of interphase cells for Golgi reemergence from the ER. These experiments suggest that mitotic Golgi breakdown proceeds by direct vesiculation independent of the ER.

Mechanisms Governing the Activation and Trafficking of Yeast G Protein-coupled Receptors

We have addressed the mechanisms governing the activation and trafficking of G protein-coupled receptors (GPCRs) by analyzing constitutively active mating pheromone receptors (Ste2p and Ste3p) of the yeast Saccharomyces cerevisiae. Substitution of the highly conserved proline residue in transmembrane segment VI of these receptors causes constitutive signaling. This proline residue may facilitate folding of GPCRs into native, inactive conformations, and/or mediate agonist-induced structural changes leading to G protein activation. Constitutive signaling by mutant receptors is suppressed upon coexpression with wild-type, but not G protein coupling-defective, receptors. Wild-type receptors may therefore sequester a limiting pool of G proteins; this apparent “precoupling” of receptors and G proteins could facilitate signal production at sites where cell surface projections form during mating partner discrimination. Finally, rather than being expressed mainly at the cell surface, constitutively active pheromone receptors accumulate in post-endoplasmic reticulum compartments. This is in contrast to other defective membrane proteins, which apparently are targeted by default to the vacuole. We suggest that the quality-control mechanism that retains receptors in post-endoplasmic reticulum compartments may normally allow wild-type receptors to fold into their native, fully inactive conformations before reaching the cell surface. This may ensure that receptors do not trigger a response in the absence of agonist.