Phosphorylation of the rRNA transcription factor upstream binding factor promotes its association with TATA binding protein

rRNA synthesis by RNA polymerase I requires both the promoter selectivity factor 1, which is composed of TATA binding protein (TBP) and three TBP-associated factors, and the activator upstream binding factor (UBF). Whereas there is strong evidence implicating a role for phosphorylation of UBF in the control of growth-induced increases in rRNA transcription, the mechanism of this effect is not known. Results of immunoprecipitation studies with TBP antibodies showed increased recovery of phosphorylated UBF from growth-stimulated smooth muscle cells. Moreover, using an immobilized protein-binding assay, we found that phosphorylation of UBF in vivo in response to stimulation with different growth factors or in vitro with smooth muscle cell nuclear extract increased its binding to TBP. Finally, we demonstrated that UBF–TBP binding depended on the C-terminal ‘acidic tail’ of UBF that was hyperphosphorylated at multiple serine sites after growth factor stimulation. Results of these studies suggest that phosphorylation of UBF and subsequent binding to TBP represent a key regulatory step in control of growth-induced increases in rRNA synthesis.

Karyopherin β2 mediates nuclear import of a mRNA binding protein

We have cloned and sequenced cDNA for human karyopherin β2, also known as transportin. In a solution binding assay, recombinant β2 bound directly to recombinant nuclear mRNA-binding protein A1. Binding was inhibited by a peptide representing A1’s previously characterized M9 nuclear localization sequence (NLS), but not by a peptide representing a classical NLS. As previously shown for karyopherin β1, karyopherin β2 bound to several nucleoporins containing characteristic peptide repeat motifs. In a solution binding assay, both β1 and β2 competed with each other for binding to immobilized repeat nucleoporin Nup98. In digitonin-permeabilized cells, β2 was able to dock A1 at the nuclear rim and to import it into the nucleoplasm. At low concentrations of β2, there was no stimulation of import by the exogenous addition of the GTPase Ran. However, at higher concentrations of β2 there was marked stimulation of import by Ran. Import was inhibited by the nonhydrolyzable GTP analog guanylyl imidodiphosphate by a Ran mutant that is unable to hydrolyze GTP and also by wheat germ agglutinin. Consistent with the solution binding results, karyopherin β2 inhibited karyopherin α/β1-mediated import of a classical NLS containing substrate and, vice versa, β1 inhibited β2-mediated import of A1 substrate, suggesting that the two import pathways merge at the level of docking of β1 and β2 to repeat nucleoporins.

Insulin-like growth factor binding protein 2 is a growth inhibitory protein conserved in zebrafish

Fish serum contains several specific binding proteins for insulin-like growth factors (IGFBPs). The structure and physiological function of these fish IGFBPs are unknown. Here we report the complete primary sequence of a zebrafish IGFBP deduced from cDNA clones isolated by library screening and rapid amplification of cDNA ends. The full-length 1,757-bp cDNA encodes a protein of 276 aa, which contains a putative 22-residue signal peptide and a 254-residue mature protein. The mature zebrafish IGFBP has a predicted molecular size of 28,440 Da and shows high sequence identity with human IGFBP-2 (52%). The sequence identities with other human IGFBPs are <37%. Chinese hamster ovary cells stably transfected with the zebrafish IGFBP-2 cDNA secreted a 31-kDa protein, which bound to IGF-I and IGF-II with high affinity, but did not bind to Des(1–3)IGF-I or insulin. Northern blot analyses revealed that the zebrafish IGFBP-2 transcript is a 1.8-kb band expressed in many embryonic and adult tissues. In adult zebrafish, IGFBP-2 mRNA levels were greatly reduced by growth hormone treatment but increased by prolonged fasting. When overexpressed or added to cultured zebrafish and mammalian cells, the zebrafish IGFBP-2 significantly inhibited IGF-I-stimulated cell proliferation and DNA synthesis. These results indicate that zebrafish IGFBP-2 is a negative growth regulator acting downstream in the growth hormone-IGF-I axis.

Synthesis of medium-chain-length polyhydroxyalkanoates in Arabidopsis thaliana using intermediates of peroxisomal fatty acid β-oxidation

Polyhydroxyalkanoate (PHA) is a family of polymers composed primarily of R-3-hydroxyalkanoic acids. These polymers have properties of biodegradable thermoplastics and elastomers. Medium-chain-length PHAs (MCL-PHAs) are synthesized in bacteria by using intermediates of the β-oxidation of alkanoic acids. To assess the feasibility of producing MCL-PHAs in plants, Arabidopsis thaliana was transformed with the PhaC1 synthase from Pseudomonas aeruginosa modified for peroxisome targeting by addition of the carboxyl 34 amino acids from the Brassica napus isocitrate lyase. Immunocytochemistry demonstrated that the modified PHA synthase was appropriately targeted to leaf-type peroxisomes in light-grown plants and glyoxysomes in dark-grown plants. Plants expressing the PHA synthase accumulated electron-lucent inclusions in the glyoxysomes and leaf-type peroxisomes, as well as in the vacuole. These inclusions were similar to bacterial PHA inclusions. Analysis of plant extracts by GC and mass spectrometry demonstrated the presence of MCL-PHA in transgenic plants to approximately 4 mg per g of dry weight. The plant PHA contained saturated and unsaturated 3-hydroxyalkanoic acids ranging from six to 16 carbons with 41% of the monomers being 3-hydroxyoctanoic acid and 3-hydroxyoctenoic acid. These results indicate that the β-oxidation of plant fatty acids can generate a broad range of R-3-hydroxyacyl-CoA intermediates that can be used to synthesize MCL-PHAs.

HIV-1 Tat transactivator recruits p300 and CREB-binding protein histone acetyltransferases to the viral promoter

In cells infected with HIV type 1 (HIV-1), the integrated viral promoter is present in a chromatin-bound conformation and is transcriptionally silent in the absence of stimulation. The HIV-1 Tat protein binds to a stem-loop structure at the 5′ end of viral mRNA and relieves this inhibition by inducing a remodeling of the nucleosome arrangement downstream of the transcription-initiation site. Here we show that Tat performs this activity by recruiting to the viral long terminal repeat (LTR) the transcriptional coactivator p300 and the closely related CREB-binding protein (CBP), having histone acetyltransferase (HAT) activity. Tat associates with HAT activity in human nuclear extracts and binds to p300 and CBP both in vitro and in vivo. Integrity of the basic domain of Tat is essential for this interaction. By a quantitative chromatin immunoprecipitation assay we show that the delivery of recombinant Tat induces the association of p300 and CBP with the chromosomally integrated LTR promoter. Expression of human p300 in both human and rodent cells increases the levels of Tat transactivation of the integrated LTR. These results reinforce the evidence that p300 and CBP have a pivotal function at both cellular and viral promoters and demonstrate that they also can be recruited by an RNA-targeted activator. Additionally, these findings have important implications for the understanding of the mechanisms of HIV-1 latency and reactivation.

Phosphorylation of the immunosuppressant FK506-binding protein FKBP52 by casein kinase II: Regulation of HSP90-binding activity of FKBP52

FKBP52 (HSP56, p59, HBI) is the 59-kDa immunosuppressant FK506-binding protein and has peptidyl prolyl isomerase as well as a chaperone-like activity in vitro. FKBP52 associates with the heat shock protein HSP90 and is included in the steroid hormone receptor complexes in vivo. FKBP52 possesses a well conserved phosphorylation site for casein kinase II (CK2) that was previously shown to be associated with HSP90. Here we examined whether FKBP52 is phosphorylated by CK2 both in vivo and in vitro. Recombinant rabbit FKBP52 was phosphorylated by purified CK2. We expressed and purified deletion mutants of FKBP52 to determine the site(s) phosphorylated by CK2. Thr-143 in the hinge I region was identified as the major phosphorylation site for CK2. A synthetic peptide corresponding to this region was phosphorylated by CK2, and the peptide competitively inhibited the phosphorylation of other substrates by CK2. The [32P]phosphate labeling of FKBP52-expressing cells revealed that the same site is also phosphorylated in vivo. FK506 binding to FKBP52 did not affect the phosphorylation by CK2 and, conversely, the FK506-binding activity of FKBP52 was not affected by the phosphorylation. Most importantly, CK2-phosphorylated FKBP52 did not bind to HSP90. These results indicate that CK2 phosphorylates FKBP52 both in vitro and in vivo and thus may regulate the protein composition of chaperone-containing complexes such as those of steroid receptors and certain protein kinases.

The Stem-Loop Binding Protein (SLBP1) Is Present in Coiled Bodies of the Xenopus Germinal Vesicle

The stem-loop binding protein (SLBP1) binds the 3′ stem-loop of histone pre-mRNA and is required for efficient processing of histone transcripts in the nucleus. We examined the localization of SLBP1 in the germinal vesicle of Xenopus laevis oocytes. In spread preparations of germinal vesicle contents, an anti-SLBP1 antibody stained coiled bodies and specific chromosomal loci, including terminal granules, axial granules, and some loops. After injection of myc-tagged SLBP1 transcripts into the oocyte cytoplasm, newly translated myc-SLBP1 protein was detectable in coiled bodies within 4 h and in terminal and axial granules by 8 h. To identify the region(s) of SLBP1 necessary for subnuclear localization, we subcloned various parts of the SLBP1 cDNA and injected transcripts of these into the cytoplasm of oocytes. We determined that 113 amino acids at the carboxy terminus of SLBP1 are sufficient for coiled body localization and that disruption of a previously defined RNA-binding domain did not alter this localization. Coiled bodies also contain the U7 small nuclear ribonucleoprotein particle (snRNP), which participates in cleavage of the 3′ end of histone pre-mRNA. The colocalization of SLBP1 and the U7 snRNP in the coiled body suggests coordinated control of their functions, perhaps through a larger histone-processing particle. Some coiled bodies are attached to the lampbrush chromosomes at the histone gene loci, consistent with the view that coiled bodies in the oocyte recruit histone-processing factors to the sites of histone pre-mRNA transcription. The non-histone chromosomal sites at which SLBP1 is found include the genes coding for 5 S rRNA, U1 snRNA, and U2 snRNA, suggesting a wider role for SLBP1 in the biosynthesis of small non-spliced RNAs.

Syndapin I, a Synaptic Dynamin-binding Protein that Associates with the Neural Wiskott-Aldrich Syndrome Protein

The GTPase dynamin has been clearly implicated in clathrin-mediated endocytosis of synaptic vesicle membranes at the presynaptic nerve terminal. Here we describe a novel 52-kDa protein in rat brain that binds the proline-rich C terminus of dynamin. Syndapin I (synaptic, dynamin-associated protein I) is highly enriched in brain where it exists in a high molecular weight complex. Syndapin I can be involved in multiple protein–protein interactions via a src homology 3 (SH3) domain at the C terminus and two predicted coiled-coil stretches. Coprecipitation studies and blot overlay analyses revealed that syndapin I binds the brain-specific proteins dynamin I, synaptojanin, and synapsin I via an SH3 domain-specific interaction. Coimmunoprecipitation of dynamin I with antibodies recognizing syndapin I and colocalization of syndapin I with dynamin I at vesicular structures in primary neurons indicate that syndapin I associates with dynamin I in vivo and may play a role in synaptic vesicle endocytosis. Furthermore, syndapin I associates with the neural Wiskott-Aldrich syndrome protein, an actin-depolymerizing protein that regulates cytoskeletal rearrangement. These characteristics of syndapin I suggest a molecular link between cytoskeletal dynamics and synaptic vesicle recycling in the nerve terminal.

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.

Effects of Age on the Posttranscriptional Regulation of CCAAT/Enhancer Binding Protein α and CCAAT/Enhancer Binding Protein β Isoform Synthesis in Control and LPS-Treated Livers

The CCAAT/enhancer binding protein α (C/EBPα) and CCAAT/enhancer binding protein β (C/EBPβ) mRNAs are templates for the differential translation of several isoforms. Immunoblotting detects C/EBPαs with molecular masses of 42, 38, 30, and 20 kDa and C/EBPβs of 35, 20, and ∼8.5 kDa. The DNA-binding activities and pool levels of p42C/EBPα and p30C/EBPα in control nuclear extracts decrease significantly whereas the binding activity and protein levels of the 20-kDa isoforms increase dramatically with LPS treatment. Our studies suggest that the LPS response involves alternative translational initiation at specific in-frame AUGs, producing specific C/EBPα and C/EBPβ isoform patterns. We propose that alternative translational initiation occurs by a leaky ribosomal scanning mechanism. We find that nuclear extracts from normal aged mouse livers have decreased p42C/EBPα levels and binding activity, whereas those of p20C/EBPα and p20C/EBPβ are increased. However, translation of 42-kDa C/EBPα is not down-regulated on polysomes, suggesting that aging may affect its nuclear translocation. Furthermore, recovery of the C/EBPα- and C/EBPβ-binding activities and pool levels from an LPS challenge is delayed significantly in aged mouse livers. Thus, aged livers have altered steady-state levels of C/EBPα and C/EBPβ isoforms. This result suggests that normal aging liver exhibits characteristics of chronic stress and a severe inability to recover from an inflammatory challenge.

The Small GTP-binding Protein R-Ras Can Influence Integrin Activation by Antagonizing a Ras/Raf-initiated Integrin Suppression Pathway

The rapid modulation of ligand-binding affinity (“activation”) is a central property of the integrin family of cell adhesion receptors. The small GTP-binding protein Ras and its downstream effector kinase Raf-1 suppress integrin activation. In this study we explored the relationship between Ras and the closely related small GTP-binding protein R-Ras in modulating the integrin affinity state. We found that R-Ras does not seem to be a direct activator of integrins in Chinese hamster ovary cells. However, we observed that GTP-bound R-Ras strongly antagonizes the Ras/Raf-initiated integrin suppression pathway. Furthermore, this reversal of the Ras/Raf suppressor pathway does not seem to be via a competition between Ras and R-Ras for common downstream effectors or via an inhibition of Ras/Raf-induced MAP kinase activation. Thus, R-Ras and Ras may act in concert to regulate integrin affinity via the activation of distinct downstream effectors.

The Phosphatidylinositol 3-Phosphate Binding Protein Vac1p Interacts with a Rab GTPase and a Sec1p Homologue to Facilitate Vesicle-mediated Vacuolar Protein Sorting

Activated GTP-bound Rab proteins are thought to interact with effectors to elicit vesicle targeting and fusion events. Vesicle-associated v-SNARE and target membrane t-SNARE proteins are also involved in vesicular transport. Little is known about the functional relationship between Rabs and SNARE protein complexes. We have constructed an activated allele of VPS21, a yeast Rab protein involved in vacuolar protein sorting, and demonstrated an allele-specific interaction between Vps21p and Vac1p. Vac1p was found to bind the Sec1p homologue Vps45p. Although no association between Vps21p and Vps45p was seen, a genetic interaction between VPS21 and VPS45 was observed. Vac1p contains a zinc-binding FYVE finger that may bind phosphatidylinositol 3-phosphate [PtdIns(3)P]. In other FYVE domain proteins, this motif and PtdIns(3)P are necessary for membrane association. Vac1 proteins with mutant FYVE fingers still associated with membranes but showed vacuolar protein sorting defects and reduced interactions with Vps45p and activated Vps21p. Vac1p membrane association was not dependent on PtdIns(3)P, Pep12p, Vps21p, Vps45p, or the PtdIns 3-kinase, Vps34p. Vac1p FYVE finger mutant missorting phenotypes were suppressed by a defective allele of VPS34. These data indicate that PtdIns(3)P may perform a regulatory role, possibly involved in mediating Vac1p protein–protein interactions. We propose that activated-Vps21p interacts with its effector, Vac1p, which interacts with Vps45p to regulate the Golgi to endosome SNARE complex.

A General RNA-Binding Protein Complex That Includes the Cytoskeleton-associated Protein MAP 1A

Association of mRNA with the cytoskeleton represents a fundamental aspect of RNA physiology likely involved in mRNA transport, anchoring, translation, and turnover. We report the initial characterization of a protein complex that binds RNA in a sequence-independent but size-dependent manner in vitro. The complex includes a ∼160-kDa protein that is bound directly to mRNA and that appears to be either identical or highly related to a ∼1600-kDa protein that binds directly to mRNA in vivo. In addition, the microtubule-associated protein, MAP 1A, a cytoskeletal associated protein is a component of this complex. We suggest that the general attachment of mRNA to the cytoskeleton may be mediated, in part, through the formation of this ribonucleoprotein complex.

Activation of Utrophin Promoter by Heregulin via the ets-related Transcription Factor Complex GA-binding Protein α/β

Utrophin/dystrophin-related protein is the autosomal homologue of the chromosome X-encoded dystrophin protein. In adult skeletal muscle, utrophin is highly enriched at the neuromuscular junction. However, the molecular mechanisms underlying regulation of utrophin gene expression are yet to be defined. Here we demonstrate that the growth factor heregulin increases de novo utrophin transcription in muscle cell cultures. Using mutant reporter constructs of the utrophin promoter, we define the N-box region of the promoter as critical for heregulin-mediated activation. Using this region of the utrophin promoter for DNA affinity purification, immunoblots, in vitro kinase assays, electrophoretic mobility shift assays, and in vitro expression in cultured muscle cells, we demonstrate that ets-related GA-binding protein α/β transcription factors are activators of the utrophin promoter. Taken together, these results suggest that the GA-binding protein α/β complex of transcription factors binds and activates the utrophin promoter in response to heregulin-activated extracellular signal–regulated kinase in muscle cell cultures. These findings suggest methods for achieving utrophin up-regulation in Duchenne’s muscular dystrophy as well as mechanisms by which neurite-derived growth factors such as heregulin may influence the regulation of utrophin gene expression and subsequent enrichment at the neuromuscular junction of skeletal muscle.

Characterization of Mayven, a Novel Actin-binding Protein Predominantly Expressed in Brain

The cytoskeleton plays an important role in neuronal morphogenesis. We have identified and characterized a novel actin-binding protein, termed Mayven, predominantly expressed in brain. Mayven contains a BTB (broad complex, tramtrack, bric-a-brac)/POZ (poxvirus, zinc finger) domain-like structure in the predicted N terminus and “kelch repeats” in the predicted C-terminal domain. Mayven shares 63% identity (77% similarity) with the Drosophila ring canal (“kelch”) protein. Somatic cell-hybrid analysis indicated that the human Mayven gene is located on chromosome 4q21.2, whereas the murine homolog gene is located on chromosome 8. The BTB/POZ domain of Mayven can self-dimerize in vitro, which might be important for its interaction with other BTB/POZ-containing proteins. Confocal microscopic studies of endogenous Mayven protein revealed a highly dynamic localization pattern of the protein. In U373-MG astrocytoma/glioblastoma cells, Mayven colocalized with actin filaments in stress fibers and in patchy cortical actin-rich regions of the cell margins. In primary rat hippocampal neurons, Mayven is highly expressed in the cell body and in neurite processes. Binding assays and far Western blotting analysis demonstrated association of Mayven with actin. This association is mediated through the “kelch repeats” within the C terminus of Mayven. Depolarization of primary hippocampal neurons with KCl enhanced the association of Mayven with actin. This increased association resulted in dynamic changes in Mayven distribution from uniform to punctate localization along neuronal processes. These results suggest that Mayven functions as an actin-binding protein that may be translocated along axonal processes and might be involved in the dynamic organization of the actin cytoskeleton in brain cells.