Pontin52, an interaction partner of β-catenin, binds to the TATA box binding protein

β-catenin, the vertebrate homolog of the Drosophila Armadillo protein, has been shown to have dual cellular functions, as a component of both the cadherin-catenin cell adhesion complex and the Wnt signaling pathway. At Wnt signaling, β-catenin becomes stabilized in the cytoplasm and subsequently available for interaction with transcription factors of the lymphocyte enhancer factor-1/T-cell factor family, resulting in a nuclear localization of β-catenin. Although β-catenin does not bind DNA directly, its carboxyl- and amino-terminal regions exhibit a transactivating activity still not well understood molecularly. Here we report the identification of an interaction partner of β-catenin, a nuclear protein designated Pontin52. Pontin52 binds β-catenin in the region of Armadillo repeats 2–5 and, more importantly, also binds the TATA box binding protein. We provide evidence for an in vivo multiprotein complex composed of Pontin52, β-catenin, and lymphocyte enhancer factor-1/T-cell factor. Our results suggest involvement of Pontin52 in the nuclear function of β-catenin.

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.

Dual role of the nuclear factor of activated T cells insert region in DNA recognition and cooperative contacts to activator protein 1

The transcription factors nuclear factor of activated T cells (NFAT) and activator protein 1 (AP-1) coordinately regulate cytokine gene expression in activated T-cells by binding to closely juxtaposed sites in cytokine promoters. The structural basis for cooperative binding of NFAT and AP-1 to these sites, and indeed for the cooperative binding of transcription factors to composite regulatory elements in general, is not well understood. Mutagenesis studies have identified a segment of AP-1, which lies at the junction of its DNA-binding and dimerization domains (basic region and leucine zipper, respectively), as being essential for protein–protein interactions with NFAT in the ternary NFAT/AP-1/DNA complex. In a model of the ternary complex, the segment of NFAT nearest AP-1 is the Rel insert region (RIR), a feature that is notable for its hypervariability in size and in sequence amongst members of the Rel transcription factor family. Here we have used mutational analysis to study the role of the NFAT RIR in binding to DNA and AP-1. Parallel yeast one-hybrid screening assays in combination with alanine-scanning mutagenesis led to the identification of four amino acid residues in the RIR of NFAT2 (also known as NFATC1 or NFATc) that are essential for cooperativity with AP-1 (Ile-544, Glu-545, Thr-551, and Ile-553), and three residues that are involved in interactions with DNA (Lys-538, Arg-540, and Asn-541). These results were confirmed and extended through in vitro binding assays. We thus conclude that the NFAT RIR plays an essential dual role in DNA recognition and cooperative binding to AP-1 family transcription factors.

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.

The amphiphysin-like protein 1 (ALP1) interacts functionally with the cABL tyrosine kinase and may play a role in cytoskeletal regulation

cABL is a protooncogene, activated in a subset of human leukemias, whose protein product is a nonreceptor tyrosine kinase of unknown function. cABL has a complex structure that includes several domains and motifs found in proteins implicated in signal transduction pathways. An approach to elucidate cABL function is to identify proteins that interact directly with cABL and that may serve as regulators or effectors of its activity. To this end, a protein-interaction screen of a phage expression library was undertaken to identify proteins that interact with specific domains of cABL. An SH3-domain-containing protein has been identified that interacts with sequences in the cABL carboxyl terminus. The cDNA encoding ALP1 (amphiphysin-like protein 1) was isolated from a 16-day mouse embryo. ALP1 has high homology to BIN1, a recently cloned myc-interacting protein, and also shows significant homology to amphiphysin, a neuronal protein cloned from human and chicken. The amino terminus has homology to two yeast proteins, Rvs167 and Rvs161, which are involved in cell entry into stationary phase and cytoskeletal organization. ALP1 binds cABL in vitro and in vivo. Expression of ALP1 results in morphological transformation of NIH 3T3 fibroblasts in a cABL-dependent manner. The properties of ALP1 suggest that it may be involved in possible cytoskeletal functions of the cABL kinase. Additionally, these results provide further evidence for the importance of the cABL carboxyl terminus and its binding proteins in the regulation of cABL function.

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.

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.

Thyroid hormone receptor-associated proteins and general positive cofactors mediate thyroid hormone receptor function in the absence of the TATA box-binding protein-associated factors of TFIID

Coactivators previously implicated in ligand-dependent activation functions by thyroid hormone receptor (TR) include p300 and CREB-binding protein (CBP), the steroid receptor coactivator-1 (SRC-1)-related family of proteins, and the multicomponent TR-associated protein (TRAP) complex. Here we show that two positive cofactors (PC2 and PC4) derived from the upstream stimulatory activity (USA) cofactor fraction act synergistically to mediate thyroid hormone (T3)-dependent activation either by TR or by a TR-TRAP complex in an in vitro system reconstituted with purified factors and DNA templates. Significantly, the TRAP-mediated enhancement of activation by TR does not require the TATA box-binding protein-associated factors of TFIID. Furthermore, neither the pleiotropic coactivators CBP and p300 nor members of the SRC-1 family were detected in either the TR-TRAP complex or the other components of the in vitro assay system. These results show that activation by TR at the level of naked DNA templates is enhanced by cooperative functions of the TRAP coactivators and the general coactivators PC2 and PC4, and they further indicate a potential functional redundancy between TRAPs and TATA box-binding protein-associated factors in TFIID. In conjunction with earlier studies on other nuclear receptor-interacting cofactors, the present study also suggests a multistep pathway, involving distinct sets of cofactors, for activation of hormone responsive genes.

Bruton’s tyrosine kinase activity is negatively regulated by Sab, the Btk-SH3 domain-binding protein

Bruton’s tyrosine kinase (Btk) is a cytoplasmic tyrosine kinase that is crucial for human and murine B cell development, and its deficiency causes human X-linked agammaglobulinemia and murine X-linked immunodeficiency. In this report, we describe the function of the Btk-binding protein Sab (SH3-domain binding protein that preferentially associates with Btk), which we reported previously as a newly identified Src homology 3 domain-binding protein. Sab was shown to inhibit the auto- and transphosphorylation activity of Btk, which prompted us to propose that Sab functions as a transregulator of Btk. Forced overexpression of Sab in B cells led to the reduction of B cell antigen receptor-induced tyrosine phosphorylation of Btk and significantly reduced both early and late B cell antigen receptor-mediated events, including calcium mobilization, inositol 1,4,5-trisphosphate production, and apoptotic cell death, where the involvement of Btk activity has been demonstrated previously. Together, these results indicate the negative regulatory role of Sab in the B cell cytoplasmic tyrosine kinase pathway.

Role of the calcium-binding protein parvalbumin in short-term synaptic plasticity

GABAergic (GABA = γ-aminobutyric acid) neurons from different brain regions contain high levels of parvalbumin, both in their soma and in their neurites. Parvalbumin is a slow Ca2+ buffer that may affect the amplitude and time course of intracellular Ca2+ transients in terminals after an action potential, and hence may regulate short-term synaptic plasticity. To test this possibility, we have applied paired-pulse stimulations (with 30- to 300-ms intervals) at GABAergic synapses between interneurons and Purkinje cells, both in wild-type (PV+/+) mice and in parvalbumin knockout (PV−/−) mice. We observed paired-pulse depression in PV+/+ mice, but paired-pulse facilitation in PV−/− mice. In paired recordings of connected interneuron-Purkinje cells, dialysis of the presynaptic interneuron with the slow Ca2+ buffer EGTA (1 mM) rescues paired-pulse depression in PV−/− mice. These data show that parvalbumin potently modulates short-term synaptic plasticity.

β-Adrenergic receptor-induced activation of nerve growth factor gene transcription in rat cerebral cortex involves CCAAT/enhancer-binding protein δ

Stimulation of β-adrenergic receptors (BAR) by clenbuterol (CLE) increases nerve growth factor (NGF) biosynthesis in the rat cerebral cortex but not in other regions of the brain. We have explored the transcription mechanisms that may account for the cortex-specific activation of the NGF gene. Although the NGF promoter contains an AP-1 element, AP-1-binding activity in the cerebral cortex was not induced by CLE, suggesting that other transcription factors govern the brain area-specific induction of NGF. Because BAR activation increases cAMP levels, we examined the role of CCAAT/enhancer-binding proteins (C/EBP), some of which are known to be cAMP-inducible. In C6–2B glioma cells, whose NGF expression is induced by BAR agonists, (i) CLE increased C/EBPδ-binding activity, (ii) NGF mRNA levels were increased by overexpressing C/EBPδ, and (iii) C/EBPδ increased the activity of an NGF promoter–reporter construct. Moreover, DNase footprinting and deletion analyses identified a C/EBPδ site in the proximal region of the NGF promoter. C/EBPδ appears to be responsible for the BAR-mediated activation of the NGF gene in vivo, since CLE elicited a time-dependent increase in C/EBPδ-binding activity in the cerebral cortex only. Our data suggest that, while AP-1 may regulate basal levels of NGF expression, C/EBPδ is a critical component determining the area-specific expression of NGF in response to BAR stimulation.

Novel role of phosphorylation in Fe–S cluster stability revealed by phosphomimetic mutations at Ser-138 of iron regulatory protein 1

Animals regulate iron metabolism largely through the action of the iron regulatory proteins (IRPs). IRPs modulate mRNA utilization by binding to iron-responsive elements (IRE) in the 5′ or 3′ untranslated region of mRNAs encoding proteins involved in iron homeostasis or energy production. IRP1 is also the cytosolic isoform of aconitase. The activities of IRP1 are mutually exclusive and are modulated through the assembly/disassembly of its [4Fe–4S] cluster, reversibly converting it between an IRE-binding protein and cytosolic aconitase. IRP1 is also phosphoregulated by protein kinase C, but the mechanism by which phosphorylation posttranslationally increases IRE binding activity has not been fully defined. To investigate this, Ser-138 (S138), a PKC phosphorylation site, was mutated to phosphomimetic glutamate (S138E), aspartate (S138D), or nonphosphorylatable alanine (S138A). The S138E IRP1 mutant and, to a lesser extent, the S138D IRP1 mutant were impaired in aconitase function in yeast when grown aerobically but not when grown anaerobically. Purified wild-type and mutant IRP1s could be reconstituted to active aconitases anaerobically. However, when exposed to oxygen, the [4Fe–4S] cluster of the S138D and S138E mutants decayed 5-fold and 20-fold faster, respectively, than was observed for wild-type IRP1. Our findings suggest that stability of the Fe–S cluster of IRP1 can be regulated by phosphorylation and reveal a mechanism whereby the balance between the IRE binding and [4Fe–4S] forms of IRP1 can be modulated independently of cellular iron status. Furthermore, our results show that IRP1 can function as an oxygen-modulated posttranscriptional regulator of gene expression.

Repression of transcription mediated by dual elements in the CCAAT/enhancer binding protein α gene

During adipocyte differentiation, the expression of C/EBPα is activated, which in turn serves to transcriptionally activate numerous adipocyte genes. A previous search for cis elements that regulate transcription of the C/EBPα gene led to the identification of a potential repressive element within the proximal 5′ flanking region of the gene. Nuclear extracts from 3T3-L1 preadipocytes, but not adipocytes, were found to contain a factor, CUP (C/EBPα undifferentiated protein), that binds to this site (the CUP-1 site). In the present investigation, we show that C/EBPα promoter-luciferase constructs containing both the proximal 5′ flanking and the entire 5′ untranslated regions of the gene exhibit an expression pattern during adipocyte differentiation comparable to that of the endogenous C/EBPα gene. Mutation of the CUP-1 site in these constructs had little effect on reporter gene expression; however, when this mutation was combined with deletion of the 5′ untranslated region, reporter gene expression by preadipocytes was dramatically up-regulated. Consistent with this finding, a second CUP binding site (the CUP-2 site) was identified in the 5′ untranslated region. Although mutation of either CUP element in constructs containing both the 5′ flanking and 5′ untranslated region had little effect on reporter gene transcription, mutation of both CUP elements markedly activated transcription. Thus, it appears that dual CUP regulatory elements repress transcription of the C/EBPα gene prior to induction of the adipocyte differentiation program.

The interphotoreceptor retinoid binding protein gene in therian mammals: Implications for higher level relationships and evidence for loss of function in the marsupial mole

The subclass Theria of Mammalia includes marsupials (infraclass Metatheria) and placentals (infraclass Eutheria). Within each group, interordinal relationships remain unclear. One limitation of many studies is incomplete ordinal representation. Here, we analyze DNA sequences for part of exon 1 of the interphotoreceptor retinoid binding protein gene, including 10 that are newly reported, for representatives of all therian orders. Among placentals, the most robust clades are Cetartiodactyla, Paenungulata, and an expanded African clade that includes paenungulates, tubulidentates, and macroscelideans. Anagalida, Archonta, Altungulata, Hyracoidea + Perissodactyla, Ungulata, and the “flying primate” hypothesis are rejected by statistical tests. Among marsupials, the most robust clade includes all orders except Didelphimorphia. The phylogenetic placement of the monito del monte and the marsupial mole remains unclear. However, the marsupial mole sequence contains three frameshift indels and numerous stop codons in all three reading frames. Given that the interphotoreceptor retinoid binding protein gene is a single-copy gene that functions in the visual cycle and that the marsupial mole is blind with degenerate eyes, this finding suggests that phenotypic degeneration of the eyes is accompanied by parallel changes at the molecular level as a result of relaxed selective constraints.