The disease-resistance gene Pto and the fenthion-sensitivity gene fen encode closely related functional protein kinases.

Resistance to bacterial speck in tomato is governed by a gene-for-gene interaction in which a single resistance locus (Pto) in the plant responds to the expression of a specific avirulence gene (avrPto) in the pathogen. Disease susceptibility results if either Pto or avrPto are lacking from the corresponding organisms. Leaves of tomato cultivars that contain the Pto locus also exhibit a hypersensitive-like response upon exposure to an organophosphorous insecticide, fenthion. Recently, the Pto gene was isolated by a map-based cloning approach and was shown to be a member of a clustered multigene family with similarity to various protein-serine/threonine kinases. Another member of this family, termed Fen, was found to confer sensitivity to fenthion. The Pto protein shares 80% identity (87% similarity) with Fen. Here, Pto and Fen are shown to be functional protein kinases that probably participate in the same signal transduction pathway.

Differential modulation of Th1- and Th2-related cytokine mRNA expression by a synthetic peptide homologous to a conserved domain within retroviral envelope protein.

The influence of a synthetic retroviral peptide, CKS-17, on T helper type 1 (Th1)- or Th2-related cytokines was investigated in human blood mononuclear cells. Cells were stimulated with staphylococcal enterotoxin A, anti-CD3 plus anti-CD28 monoclonal antibodies, or lipopolysaccharide to induce cytokine mRNA. mRNA was detected by a reverse transcription-polymerase chain reaction or Northern blot analysis. CKS-17 down-regulated stimulant-induced mRNA accumulation for interferon gamma (IFN-gamma), interleukin (IL)-2, and p40 heavy and p35 light chains of IL-12, a cytokine that mediates development of Th1 response. CKS-17 up-regulated stimulant-induced mRNA accumulation of IL-10 and did not suppress Th2-related cytokine (IL-4, IL-5, IL-6, or IL-13) mRNA expression. A reverse sequence of CKS-17 peptide, used as a control, showed no such action. Anti-human IL-10 monoclonal antibody blocked ability of CKS-17 to inhibit mRNA accumulation for IFN-gamma but not the CKS-17 suppressive activity of IL-12 p40 heavy chain mRNA. Thus, CKS-17-mediated suppression of IFN-gamma mRNA expression is dependent upon augmentation of IL-10 production by CKS-17. This conserved component of several retroviral envelope proteins, CKS-17, may act as an immunomodulatory epitope responsible for cytokine dysregulation that leads to suppression of cellular immunity.

A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase.

E6-AP is a 100-kDa cellular protein that interacts with the E6 protein of the cancer-associated human papillomavirus types 16 and 18. The E6/E6-AP complex binds to and targets the p53 tumor-suppressor protein for ubiquitin-mediated proteolysis. E6-AP is an E3 ubiquitin-protein ligase which accepts ubiquitin from an E2 ubiquitin-conjugating enzyme in the form of a thioester and then directly transfers the ubiquitin to targeted substrates. The amino acid sequence of E6-AP shows similarity to a number of protein sequences over an approximately 350-aa region corresponding to the carboxyl termini of both E6-AP and the E6-AP-related proteins. Of particular note is a conserved cysteine residue within the last 32-34 aa, which in E6-AP is likely to be the site of ubiquitin thioester formation. Two of the E6-AP-related proteins, a rat 100-kDa protein and a yeast 95-kDa protein (RSP5), both of previously unknown function, are shown here to form thioesters with ubiquitin. Mutation of the conserved cysteine residue of these proteins destroys their ability to accept ubiquitin. These data strongly suggest that the rat 100-kDa protein and RSP5, as well as the other E6-AP-related proteins, belong to a class of functionally related E3 ubiquitin-protein ligases, defined by a domain homologous to the E6-AP carboxyl terminus (hect domain).

Definition of amide protection factors for early kinetic intermediates in protein folding

Hydrogen–deuterium exchange experiments have been used previously to investigate the structures of well defined states of a given protein. These include the native state, the unfolded state, and any intermediates that can be stably populated at equilibrium. More recently, the hydrogen–deuterium exchange technique has been applied in kinetic labeling experiments to probe the structures of transiently formed intermediates on the kinetic folding pathway of a given protein. From these equilibrium and nonequilibrium studies, protection factors are usually obtained. These protection factors are defined as the ratio of the rate of exchange of a given backbone amide when it is in a fully solvent-exposed state (usually obtained from model peptides) to the rate of exchange of that amide in some state of the protein or in some intermediate on the folding pathway of the protein. This definition is straightforward for the case of equilibrium studies; however, it is less clear-cut for the case of transient kinetic intermediates. To clarify the concept for the case of burst-phase intermediates, we have introduced and mathematically defined two different types of protection factors: one is Pstruc, which is more related to the structure of the intermediate, and the other is Papp, which is more related to the stability of the intermediate. Kinetic hydrogen–deuterium exchange data from disulfide-intact ribonuclease A and from cytochrome c are discussed to explain the use and implications of these two definitions.

Region-dependent dynamics of cAMP response element-binding protein phosphorylation in the basal ganglia

The cAMP response element-binding protein (CREB) is an activity-dependent transcription factor that is involved in neural plasticity. The kinetics of CREB phosphorylation have been suggested to be important for gene activation, with sustained phosphorylation being associated with downstream gene expression. If so, the duration of CREB phosphorylation might serve as an indicator for time-sensitive plastic changes in neurons. To screen for regions potentially involved in dopamine-mediated plasticity in the basal ganglia, we used organotypic slice cultures to study the patterns of dopamine- and calcium-mediated CREB phosphorylation in the major subdivisions of the striatum. Different durations of CREB phosphorylation were evoked in the dorsal and ventral striatum by activation of dopamine D1-class receptors. The same D1 stimulus elicited (i) transient phosphorylation (≤15 min) in the matrix of the dorsal striatum; (ii) sustained phosphorylation (≤2 hr) in limbic-related structures including striosomes, the nucleus accumbens, the fundus striati, and the bed nucleus of the stria terminalis; and (iii) prolonged phosphorylation (up to 4 hr or more) in cellular islands in the olfactory tubercle. Elevation of Ca2+ influx by stimulation of L-type Ca2+ channels, NMDA, or KCl induced strong CREB phosphorylation in the dorsal striatum but not in the olfactory tubercle. These findings differentiate the response of CREB to dopamine and calcium signals in different striatal regions and suggest that dopamine-mediated CREB phosphorylation is persistent in limbic-related regions of the neonatal basal ganglia. The downstream effects activated by persistent CREB phosphorylation may include time-sensitive neuroplasticity modulated by dopamine.

Molecular cloning and characterization of an invertebrate cellular retinoic acid binding protein

We have cloned a cDNA and gene from the tobacco hornworm, Manduca sexta, which is related to the vertebrate cellular retinoic acid binding proteins (CRABPs). CRABPs are members of the superfamily of lipid binding proteins (LBPs) and are thought to mediate the effects of retinoic acid (RA) on morphogenesis, differentiation, and homeostasis. This discovery of a Manduca sexta CRABP (msCRABP) demonstrates the presence of a CRABP in invertebrates. Compared with bovine/murine CRABP I, the deduced amino acid sequence of msCRABP is 71% homologous overall and 88% homologous for the ligand binding pocket. The genomic organization of msCRABP is conserved with other CRABP family members and the larger LBP superfamily. Importantly, the promoter region contains a motif that resembles an RA response element characteristic of the promoter region of most CRABPs analyzed. Three-dimensional molecular modeling based on postulated structural homology with bovine/murine CRABP I shows msCRABP has a ligand binding pocket that can accommodate RA. The existence of an invertebrate CRABP has significant evolutionary implications, suggesting CRABPs appeared during the evolution of the LBP superfamily well before vertebrate/invertebrate divergence, instead of much later in evolution in selected vertebrates.

SIRE-1, a copia/Ty1-like retroelement from soybean, encodes a retroviral envelope-like protein

The soybean genome hosts a family of several hundred, relatively homogeneous copies of a large, copia/Ty1-like retroelement designated SIRE-1. A copy of this element has been recovered from a Glycine max genomic library. DNA sequence analysis of two SIRE-1 subclones revealed that SIRE-1 contains a long, uninterrupted, ORF between the 3′ end of the pol ORF and the 3′ long terminal repeat (LTR), a region that harbors the env gene in retroviral genomes. Conceptual translation of this second ORF produces a 70-kDa protein. Computer analyses of the amino acid sequence predicted patterns of transmembrane domains, α-helices, and coiled coils strikingly similar to those found in mammalian retroviral envelope proteins. In addition, a 65-residue, proline-rich domain is characterized by a strong amino acid compositional bias virtually identical to that of the 60-amino acid, proline-rich neutralization domain of the feline leukemia virus surface protein. The assignment of SIRE-1 to the copia/Ty1 family was confirmed by comparison of the conceptual translation of its reverse transcriptase-like domain with those of other retroelements. This finding suggests the presence of a proretrovirus in a plant genome and is the strongest evidence to date for the existence of a retrovirus-like genome closely related to copia/Ty1 retrotransposons.

Role for G protein-coupled receptor kinase in agonist-specific regulation of μ-opioid receptor responsiveness

The G protein-coupled μ-opioid receptor (μOR) mediates the physiological effects of endogenous opioid peptides as well as the structurally distinct opioid alkaloids morphine and etorphine. An intriguing feature of μOR signaling is the differential receptor trafficking and desensitization properties following activation by distinct agonists, which have been proposed as possible mechanisms related to opioid tolerance. Here we report that the ability of distinct opioid agonists to differentially regulate μOR internalization and desensitization is related to their ability to promote G protein-coupled receptor kinase (GRK)-dependent phosphorylation of the μOR. Although both etorphine and morphine effectively activate the μOR, only etorphine elicits robust μOR phosphorylation followed by plasma membrane translocation of β-arrestin and dynamin-dependent receptor internalization. In contrast, corresponding to its inability to cause μOR internalization, morphine is unable to either elicit μOR phosphorylation or stimulate β-arrestin translocation. However, upon the overexpression of GRK2, morphine gains the capacity to induce μOR phosphorylation, accompanied by the rescue of β-arrestin translocation and receptor sequestration. Moreover, overexpression of GRK2 also leads to an attenuation of morphine-mediated inhibition of adenylyl cyclase. These findings point to the existence of marked differences in the ability of different opioid agonists to promote μOR phosphorylation by GRK. These differences may provide the molecular basis underlying the different analgesic properties of opioid agonists and contribute to the distinct ability of various opioids to induce drug tolerance.

Delayed embryonic lethality in mice lacking protein phosphatase 2A catalytic subunit Cα

Protein phosphatase 2A (PP2A) is a multimeric enzyme, containing a catalytic subunit complexed with two regulatory subunits. The catalytic subunit PP2A C is encoded by two distinct and unlinked genes, termed Cα and Cβ. The specific function of these two catalytic subunits is unknown. To address the possible redundancy between PP2A and related phosphatases as well as between Cα and Cβ, the Cα subunit gene was deleted by homologous recombination. Homozygous null mutant mice are embryonically lethal, demonstrating that the Cα subunit gene is an essential gene. As PP2A exerts a range of cellular functions including cell cycle regulation and cell fate determination, we were surprised to find that these embryos develop normally until postimplantation, around embryonic day 5.5/6.0. While no Cα protein is expressed, we find comparable expression levels of PP2A C at a time when the embryo is degenerating. Despite a 97% amino acid identity, Cβ cannot completely compensate for the absence of Cα. Degenerated embryos can be recovered even at embryonic day 13.5, indicating that although embryonic tissue is still capable of proliferating, normal differentiation is significantly impaired. While the primary germ layers ectoderm and endoderm are formed, mesoderm is not formed in degenerating embryos.

Mahogany (mg) stimulates feeding and increases basal metabolic rate independent of its suppression of agouti

The mahogany (mg) locus originally was identified as a recessive suppressor of agouti, a locus encoding a skin peptide that modifies coat color by antagonizing the melanocyte-stimulating hormone receptor or MC1-R. Certain dominant alleles of agouti cause an obesity syndrome when ectopic expression of the peptide aberrantly antagonizes the MC4-R, a related melanocyte-stimulating hormone receptor expressed in hypothalamic circuitry and involved in the regulation of feeding behavior and metabolism. Recent work has demonstrated that mg, when homozygous, blocks not only the ability of agouti to induce a yellow coat color when expressed in the skin of the lethal yellow mouse (AY), but also the obesity resulting from ectopic expression of agouti in the brain. Detailed analysis of mg/mg AY/a animals, presented here, demonstrates that mg/mg blocks the obesity, hyperinsulinemia, and increased linear growth induced by ectopic expression of the agouti peptide. Remarkably, however, mg/mg did not reduce hyperphagia in the AY/a mouse. Furthermore, mg/mg induced hyperphagia and an increase in basal metabolic rate in the C57BL/6J mouse in the absence of AY. Consequently, although mahogany is broadly required for agouti peptide action, it also appears to be involved in the control of metabolic rate and feeding behavior independent of its suppression of agouti.

Targeting a SWI/SNF-related chromatin remodeling complex to the β-globin promoter in erythroid cells

Chromatin remodeling complexes such as the SWI/SNF complex make DNA accessible to transcription factors by disrupting nucleosomes. However, it is not known how such complexes are targeted to the promoter. For example, a SWI/SNF1-like chromatin remodeling complex erythroid Krüppel-like factor (EKLF) coactivator-remodeling complex 1 (E-RC1) disrupts the nucleosomes over the human β-globin promoter in an EKLF-dependent manner. However, it is not known whether E-RC1 is targeted specifically to the β-globin promoter or whether E-RC1 is randomly targeted, but its activity is evident only at the β-globin promoter. Because E-RC1 cannot remodel chromatin over the β-globin promoter without EKLF in vitro, it has been proposed that SWI/SNF1-like complexes such as E-RC1 are targeted specifically to the promoter by selectively interacting with promoter-associated transcription factors such as EKLF. In this report, we test this hypothesis in the cellular context by using the ProteIN POsition Identification with Nuclease Tail (PIN*POINT) assay. We find that the Brahma-related gene (BRG) 1 and BRG1-associated factor (BAF) 170 subunits of E-RC1 are both recruited near the transcription initiation site of the β-globin promoter. On transiently transfected templates, both the locus control region and the EKLF-binding site are important for their recruitment to the β-globin promoter in mouse erythroleukemia cells. When the β-globin promoter was linked to the cytomegalovirus enhancer, the E-RC1 complex was not recruited, suggesting that recruitment of the E-RC1 complex is not a general property of enhancers.

AtGRP7, a nuclear RNA-binding protein as a component of a circadian-regulated negative feedback loop in Arabidopsis thaliana

The endogenous clock that drives circadian rhythms is thought to communicate temporal information within the cell via cycling downstream transcripts. A transcript encoding a glycine-rich RNA-binding protein, Atgrp7, in Arabidopsis thaliana undergoes circadian oscillations with peak levels in the evening. The AtGRP7 protein also cycles with a time delay so that Atgrp7 transcript levels decline when the AtGRP7 protein accumulates to high levels. After AtGRP7 protein concentration has fallen to trough levels, Atgrp7 transcript starts to reaccumulate. Overexpression of AtGRP7 in transgenic Arabidopsis plants severely depresses cycling of the endogenous Atgrp7 transcript. These data establish both transcript and protein as components of a negative feedback circuit capable of generating a stable oscillation. AtGRP7 overexpression also depresses the oscillation of the circadian-regulated transcript encoding the related RNA-binding protein AtGRP8 but does not affect the oscillation of transcripts such as cab or catalase mRNAs. We propose that the AtGRP7 autoregulatory loop represents a “slave” oscillator in Arabidopsis that receives temporal information from a central “master” oscillator, conserves the rhythmicity by negative feedback, and transduces it to the output pathway by regulating a subset of clock-controlled transcripts.

The SH3p4/Sh3p8/SH3p13 protein family: Binding partners for synaptojanin and dynamin via a Grb2-like Src homology 3 domain

The GTPase dynamin I and the inositol 5-phosphatase synaptojanin are nerve terminal proteins implicated in synaptic vesicle recycling. Both proteins contain COOH-terminal proline-rich domains that can interact with a variety of Src homology 3 (SH3) domains. A major physiological binding partner for dynamin I and synaptojanin in the nervous system is amphiphysin I, an SH3 domain-containing protein also concentrated in nerve terminals. We have used the proline-rich tail of synaptojanin to screen a rat brain library by the two-hybrid method to identify additional interacting partners of synaptojanin. Three related proteins containing SH3 domains that are closely related to the SH3 domains of Grb2 were isolated: SH3p4, SH3p8, and SH3p13. Further biochemical studies demonstrated that the SH3p4/8/13 proteins bind to both synaptojanin and dynamin I. The SH3p4/8/13 transcripts are differentially expressed in tissues: SH3p4 mRNA was detected only in brain, SH3p13 mRNA was present in brain and testis, and the SH3p8 transcript was detected at similar levels in multiple tissues. Members of the SH3p4/8/13 protein family were found to be concentrated in nerve terminals, and pools of synaptojanin and dynamin I were coprecipitated from brain extracts with antibodies recognizing SH3p4/8/13. These findings underscore the important role of SH3-mediated interactions in synaptic vesicle recycling.

Identification of a new mammalian centrin gene, more closely related to Saccharomyces cerevisiae CDC31 gene

Among the numerous centrin isoforms identified by two-dimensional gel electrophoresis in human cells, an acidic and slow-migrating isoform is particularly enriched in a centrosome fraction. We report here that this isoform specifically reacts with antibodies raised against Saccharomyces cerevisiae Cdc31p and is present, as other centrin isoforms, in the distal lumen of centrioles. It is encoded by a new centrin gene, which we propose to name HsCEN3 (Homo sapiens centrin gene 3). This gene is more closely related to the yeast CDC31 gene, and shares less identity with algae centrin than HsCEN1 and HsCEN2. A murine CDC31-related gene was also found that shows 98% identity and 100% similarity with HsCEN3, demonstrating a higher interspecies conservation than the murine centrin gene MmCEN1 (Mus musculus centrin gene 1) with either HsCEN1, or HsCEN2. Finally, immunological data suggest that a CDC31-related gene could exist in amphibians and echinoderms as well. All together, our data suggest the existence of two divergent protein subfamilies in the current centrin family, which might be involved in distinct centrosome-associated functions. The possible implication of this new mammalian centrin gene in centrosome duplication is discussed.

MLL is fused to CBP, a histone acetyltransferase, in therapy-related acute myeloid leukemia with a t(11;16)(q23;p13.3)

The recurring translocation t(11;16)(q23;p13.3) has been documented only in cases of acute leukemia or myelodysplasia secondary to therapy with drugs targeting DNA topoisomerase II. We show that the MLL gene is fused to the gene that codes for CBP (CREB-binding protein), the protein that binds specifically to the DNA-binding protein CREB (cAMP response element-binding protein) in this translocation. MLL is fused in-frame to a different exon of CBP in two patients producing chimeric proteins containing the AT-hooks, methyltransferase homology domain, and transcriptional repression domain of MLL fused to the CREB binding domain or to the bromodomain of CBP. Both fusion products retain the histone acetyltransferase domain of CBP and may lead to leukemia by promoting histone acetylation of genomic regions targeted by the MLL AT-hooks, leading to transcriptional deregulation via aberrant chromatin organization. CBP is the first partner gene of MLL containing well defined structural and functional motifs that provide unique insights into the potential mechanisms by which these translocations contribute to leukemogenesis.