Desmuslin, an intermediate filament protein that interacts with α-dystrobrevin and desmin

Dystrobrevin is a component of the dystrophin-associated protein complex and has been shown to interact directly with dystrophin, α1-syntrophin, and the sarcoglycan complex. The precise role of α-dystrobrevin in skeletal muscle has not yet been determined. To study α-dystrobrevin's function in skeletal muscle, we used the yeast two-hybrid approach to look for interacting proteins. Three overlapping clones were identified that encoded an intermediate filament protein we subsequently named desmuslin (DMN). Sequence analysis revealed that DMN has a short N-terminal domain, a conserved rod domain, and a long C-terminal domain, all common features of type 6 intermediate filament proteins. A positive interaction between DMN and α-dystrobrevin was confirmed with an in vitro coimmunoprecipitation assay. By Northern blot analysis, we find that DMN is expressed mainly in heart and skeletal muscle, although there is some expression in brain. Western blotting detected a 160-kDa protein in heart and skeletal muscle. Immunofluorescent microscopy localizes DMN in a stripe-like pattern in longitudinal sections and in a mosaic pattern in cross sections of skeletal muscle. Electron microscopic analysis shows DMN colocalized with desmin at the Z-lines. Subsequent coimmunoprecipitation experiments confirmed an interaction with desmin. Our findings suggest that DMN may serve as a direct linkage between the extracellular matrix and the Z-discs (through plectin) and may play an important role in maintaining muscle cell integrity.

Toward a plant genomics initiative: Thoughts on the value of cross-species and cross-genera comparisons in the grasses

Comparative genomics offers unparalleled opportunities to integrate historically distinct disciplines, to link disparate biological kingdoms, and to bridge basic and applied science. Cross-species, cross-genera, and cross-kingdom comparisons are proving key to understanding how genes are structured, how gene structure relates to gene function, and how changes in DNA have given rise to the biological diversity on the planet. The application of genomics to the study of crop species offers special opportunities for innovative approaches for combining sequence information with the vast reservoirs of historical information associated with crops and their evolution. The grasses provide a particularly well developed system for the development of tools to facilitate comparative genetic interpretation among members of a diverse and evolutionarily successful family. Rice provides advantages for genomic sequencing because of its small genome and its diploid nature, whereas each of the other grasses provides complementary genetic information that will help extract meaning from the sequence data. Because of the importance of the cereals to the human food chain, developments in this area can lead directly to opportunities for improving the health and productivity of our food systems and for promoting the sustainable use of natural resources.

Characterization of a Red Beet Protein Homologous to the Essential 36-Kilodalton Subunit of the Yeast V-Type ATPase1

V-type proton-translocating ATPases (V-ATPases) (EC 3.6.1.3) are electrogenic proton pumps involved in acidification of endomembrane compartments in all eukaryotic cells. V-ATPases from various species consist of 8 to 12 polypeptide subunits arranged into an integral membrane proton pore sector (V0) and a peripherally associated catalytic sector (V1). Several V-ATPase subunits are functionally and structurally conserved among all species examined. In yeast, a 36-kD peripheral subunit encoded by the yeast (Saccharomyces cerevisiae) VMA6 gene (Vma6p) is required for stable assembly of the V0 sector as well as for V1 attachment. Vma6p has been characterized as a nonintegrally associated V0 subunit. A high degree of sequence similarity among Vma6p homologs from animal and fungal species suggests that this subunit has a conserved role in V-ATPase function. We have characterized a novel Vma6p homolog from red beet (Beta vulgaris) tonoplast membranes. A 44-kD polypeptide cofractionated with V-ATPase upon gel-filtration chromatography of detergent-solubilized tonoplast membranes and was specifically cross-reactive with anti-Vma6p polyclonal antibodies. The 44-kD polypeptide was dissociated from isolated tonoplast preparations by mild chaotropic agents and thus appeared to be nonintegrally associated with the membrane. The putative 44-kD homolog appears to be structurally similar to yeast Vma6p and occupies a similar position within the holoenzyme complex.

Actin-Bundling Protein Isolated from Pollen Tubes of Lily : Biochemical and Immunocytochemical Characterization

A 135-kD actin-bundling protein was purified from pollen tubes of lily (Lilium longiflorum) using its affinity to F-actin. From a crude extract of the pollen tubes, this protein was coprecipitated with exogenously added F-actin and then dissociated from F-actin by treating it with high-ionic-strength solution. The protein was further purified sequentially by chromatography on a hydroxylapatite column, a gel-filtration column, and a diethylaminoethyl-cellulose ion-exchange column. In the present study, this protein is tentatively referred to as P-135-ABP (Plant 135-kD Actin-Bundling Protein). By the elution position from a gel-filtration column, we estimated the native molecular mass of purified P-135-ABP to be 260 kD, indicating that it existed in a dimeric form under physiological conditions. This protein bound to and bundled F-actin prepared from chicken breast muscle in a Ca2+-independent manner. The binding of 135-P-ABP to actin was saturated at an approximate stoichiometry of 26 actin monomers to 1 dimer of P-135-ABP. By transmission electron microscopy of thin sections, we observed cross-bridges between F-actins with a longitudinal periodicity of 31 nm. Immunofluorescence microscopy using rhodamine-phalloidin and antibodies against the 135-kD polypeptide showed that P-135-ABP was colocalized with bundles of actin filaments in lily pollen tubes, leading us to conclude that it is the factor responsible for bundling the filaments.

Three Drought-Responsive Members of the Nonspecific Lipid-Transfer Protein Gene Family in Lycopersicon pennellii Show Different Developmental Patterns of Expression1

Genomic clones of two nonspecific lipid-transfer protein genes from a drought-tolerant wild species of tomato (Lycopersicon pennellii Corr.) were isolated using as a probe a drought- and abscisic acid (ABA)-induced cDNA clone (pLE16) from cultivated tomato (Lycopersicon esculentum Mill.). Both genes (LpLtp1 and LpLtp2) were sequenced and their corresponding mRNAs were characterized; they are both interrupted by a single intron at identical positions and predict basic proteins of 114 amino acid residues. Genomic Southern data indicated that these genes are members of a small gene family in Lycopersicon spp. The 3′-untranslated regions from LpLtp1 and LpLtp2, as well as a polymerase chain reaction-amplified 3′-untranslated region from pLE16 (cross-hybridizing to a third gene in L. pennellii, namely LpLtp3), were used as gene-specific probes to describe expression in L. pennellii through northern-blot analyses. All LpLtp genes were exclusively expressed in the aerial tissues of the plant and all were drought and ABA inducible. Each gene had a different pattern of expression in fruit, and LpLtp1 and LpLtp2, unlike LpLtp3, were both primarily developmentally regulated in leaf tissue. Putative ABA-responsive elements were found in the proximal promoter regions of LpLtp1 and LpLtp2.

Surface Localization of Zein Storage Proteins in Starch Granules from Maize Endosperm1 : Proteolytic Removal by Thermolysin and in Vitro Cross-Linking of Granule-Associated Polypeptides

Starch granules from maize (Zea mays) contain a characteristic group of polypeptides that are tightly associated with the starch matrix (C. Mu-Forster, R. Huang, J.R. Powers, R.W. Harriman, M. Knight, G.W. Singletary, P.L. Keeling, B.P. Wasserman [1996] Plant Physiol 111: 821–829). Zeins comprise about 50% of the granule-associated proteins, and in this study their spatial distribution within the starch granule was determined. Proteolysis of starch granules at subgelatinization temperatures using the thermophilic protease thermolysin led to selective removal of the zeins, whereas granule-associated proteins of 32 kD or above, including the waxy protein, starch synthase I, and starch-branching enzyme IIb, remained refractory to proteolysis. Granule-associated proteins from maize are therefore composed of two distinct classes, the surface-localized zeins of 10 to 27 kD and the granule-intrinsic proteins of 32 kD or higher. The origin of surface-localized δ-zein was probed by comparing δ-zein levels of starch granules obtained from homogenized whole endosperm with granules isolated from amyloplasts. Starch granules from amyloplasts contained markedly lower levels of δ-zein relative to granules prepared from whole endosperm, thus indicating that δ-zein adheres to granule surfaces after disruption of the amyloplast envelope. Cross-linking experiments show that the zeins are deposited on the granule surface as aggregates. In contrast, the granule-intrinsic proteins are prone to covalent modification, but do not form intermolecular cross-links. We conclude that individual granule intrinsic proteins exist as monomers and are not deposited in the form of multimeric clusters within the starch matrix.

Calcium-Dependent Protein Phosphorylation May Mediate the Gibberellic Acid Response in Barley Aleurone1

Peptide substrates of well-defined protein kinases were microinjected into aleurone protoplasts of barley (Hordeum vulgare L. cv Himalaya) to inhibit, and therefore identify, protein kinase-regulated events in the transduction of the gibberellin (GA) and abscisic acid signals. Syntide-2, a substrate designed for Ca2+- and calmodulin (CaM)-dependent kinases, selectively inhibited the GA response, leaving constitutive and abscisic acid-regulated events unaffected. Microinjection of syntide did not affect the GA-induced increase in cytosolic [Ca2+], suggesting that it inhibited GA action downstream of the Ca2+ signal. When photoaffinity-labeled syntide-2 was electroporated into protoplasts and cross-linked to interacting proteins in situ, it selectively labeled proteins of approximately 30 and 55 kD. A 54-kD, soluble syntide-2 phosphorylating protein kinase was detected in aleurone cells. This kinase was activated by Ca2+ and was CaM independent, but was inhibited by the CaM antagonist N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (250 μm), suggesting that it was a CaM-domain protein kinase-like activity. These results suggest that syntide-2 inhibits the GA response of the aleurone via an interaction with this kinase, implicating the 54-kD kinase as a Ca2+-dependent regulator of the GA response in these cells.

Guard Cells Possess a Calcium-Dependent Protein Kinase That Phosphorylates the KAT1 Potassium Channel1

Increasing evidence suggests that changes in cytosolic Ca2+ levels and phosphorylation play important roles in the regulation of stomatal aperture and as ion transporters of guard cells. However, protein kinases responsible for Ca2+ signaling in guard cells remain to be identified. Using biochemical approaches, we have identified a Ca2+-dependent protein kinase with a calmodulin-like domain (CDPK) in guard cell protoplasts of Vicia faba. Both autophosphorylation and catalytic activity of CDPK are Ca2+ dependent. CDPK exhibits a Ca2+-induced electrophoretic mobility shift and its Ca2+-dependent catalytic activity can be inhibited by the calmodulin antagonists trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide. Antibodies to soybean CDPKα cross-react with CDPK. Micromolar Ca2+ concentrations stimulate phosphorylation of several proteins from guard cells; cyclosporin A, a specific inhibitor of the Ca2+-dependent protein phosphatase calcineurin enhances the Ca2+-dependent phosphorylation of several soluble proteins. CDPK from guard cells phosphorylates the K+ channel KAT1 protein in a Ca2+-dependent manner. These results suggest that CDPK may be an important component of Ca2+ signaling in guard cells.

Hd6, a rice quantitative trait locus involved in photoperiod sensitivity, encodes the α subunit of protein kinase CK2

Hd6 is a quantitative trait locus involved in rice photoperiod sensitivity. It was detected in backcross progeny derived from a cross between the japonica variety Nipponbare and the indica variety Kasalath. To isolate a gene at Hd6, we used a large segregating population for the high-resolution and fine-scale mapping of Hd6 and constructed genomic clone contigs around the Hd6 region. Linkage analysis with P1-derived artificial chromosome clone-derived DNA markers delimited Hd6 to a 26.4-kb genomic region. We identified a gene encoding the α subunit of protein kinase CK2 (CK2α) in this region. The Nipponbare allele of CK2α contains a premature stop codon, and the resulting truncated product is undoubtedly nonfunctional. Genetic complementation analysis revealed that the Kasalath allele of CK2α increases days-to-heading. Map-based cloning with advanced backcross progeny enabled us to identify a gene underlying a quantitative trait locus even though it exhibited a relatively small effect on the phenotype.

The human formin-binding protein 17 (FBP17) interacts with sorting nexin, SNX2, and is an MLL-fusion partner in acute myelogeneous leukemia

We have cloned a fusion partner of the MLL gene at 11q23 and identified it as the gene encoding the human formin-binding protein 17, FBP17. It maps to chromosome 9q34 centromeric to ABL. The gene fusion results from a complex chromosome rearrangement that was resolved by fluorescence in situ hybridization with various probes on chromosomes 9 and 11 as an ins(11;9)(q23;q34)inv(11)(q13q23). The rearrangement resulted in a 5′-MLL/FBP17-3′ fusion mRNA. We retrovirally transduced murine-myeloid progenitor cells with MLL/FBP17 to test its transforming ability. In contrast to MLL/ENL, MLL/ELL and other MLL-fusion genes, MLL/FBP17 did not give a positive readout in a serial replating assay. Therefore, we assume that additional cooperating genetic abnormalities might be needed to establish a full malignant phenotype. FBP17 consists of a C-terminal Src homology 3 domain and an N-terminal region that is homologous to the cell division cycle protein, cdc15, a regulator of the actin cytoskeleton in Schizosaccharomyces pombe. Both domains are separated by a consensus Rho-binding motif that has been identified in different Rho-interaction partners such as Rhotekin and Rhophilin. We evaluated whether FBP17 and members of the Rho family interact in vivo with a yeast two-hybrid assay. None of the various Rho proteins tested, however, interacted with FBP17. We screened a human kidney library and identified a sorting nexin, SNX2, as a protein interaction partner of FBP17. These data provide a link between the epidermal growth factor receptor pathway and an MLL fusion protein.

An essential role for the interferon-inducible, double-stranded RNA-activated protein kinase PKR in the tumor necrosis factor-induced apoptosis in U937 cells.

Tumor necrosis factor alpha (TNF-alpha) is well-characterized for its necrotic action against tumor cells; however, it has been increasingly associated with an apoptosis-inducing potential on target cells. While the signaling events and the actual cytolytic mechanism(s) for both TNF-alpha-induced necrosis and apoptosis remain to be fully elucidated, we report here on (i) the ability of TNF-alpha to induce apoptosis in the promonocytic U937 cells, (ii) the discovery of a cross-talk between the TNF-alpha and the interferon signaling pathways, and (iii) the pivotal role of interferon-inducible, double-stranded RNA-activated protein kinase (PKR) in the induction of apoptosis by TNF-alpha. Our data from microscopy studies, trypan blue exclusion staining, and apoptotic DNA ladder electrophoresis revealed that a subclone derived from U937 and carrying a PKR antisense expression vector was resistant to TNF-alpha-induced apoptosis. Further, TNF-alpha initiated a generalized RNA degradation process in which the participation of PKR was required. Finally, the PKR gene is a candidate "death gene" since overexpression of this gene could bring about apoptosis in U937 cells.

Cross-talk between the insulin and angiotensin signaling systems.

Angiotensin II (AII), acting via its G-protein linked receptor, is an important regulator of cardiac, vascular, and renal function. Following injection of AII into rats, we find that there is also a rapid tyrosine phosphorylation of the major insulin receptor substrates 1 and 2 (IRS-1 and IRS-2) in the heart. This phenomenon appears to involve JAK2 tyrosine kinase, which associates with the AT1 receptor and IRS-1/IRS-2 after AII stimulation. AII-induced phosphorylation leads to binding of phosphatidylinositol 3-kinase (PI 3-kinase) to IRS-1 and IRS-2; however, in contrast to other ligands, AII injection results in an acute inhibition of both basal and insulin-stimulated PI 3-kinase activity. The latter occurs without any reduction in insulin receptor or IRS phosphorylation or in the interaction of the p85 and p110 subunits of PI 3-kinase with each other or with IRS-1/IRS-2. These effects of AII are inhibited by AT1 receptor antagonists. Thus, there is direct cross-talk between insulin and AII signaling pathways at the level of both tyrosine phosphorylation and PI 3-kinase activation. These interactions may play an important role in the association of insulin resistance, hypertension, and cardiovascular disease.

New approach for inhibiting Rev function and HIV-1 production using the influenza virus NS1 protein.

The Rev protein of HIV-1, which facilitates the nuclear export of HIV-1 pre-mRNAs, has been a target for antiviral therapy. Here we describe a new strategy for inhibiting Rev function and HIV-1 replication. In contrast to previous approaches, we use a wild-type rather than a mutant Rev protein and covalently link this Rev sequence to the NS1 protein of influenza A virus, a protein that inhibits the nuclear export of mRNAs. The NS1 protein contains an RNA-binding domain mutation (RM), so that the only functional RNA-binding domain in the chimeric protein (NS1RM-Rev) is in the Rev protein sequence. In the presence of the NS1RM-Rev chimeric protein, HIV-1 pre-mRNAs were retained in, rather than exported from, the nucleus. In addition, this chimeric protein effectively inhibited Rev function in trans in transfection experiments and effectively inhibited the production of HIV-1 in tissue culture cells transfected with an infectious molecular clone of HIV-1 DNA. The inhibitory activities of the NS1RM-Rev chimera were at least equivalent to those of the Rev M10 mutant protein, which has been considered to be the prototype trans inhibitor of Rev function and is currently in phase I clinical trials for the treatment of AIDS patients. We discuss (i) the potential for increasing the inhibitory activity of NS1-Rev chimeras against HIV-1 and (ii) the need for additional studies to evaluate these chimeras for the treatment of AIDS.

Conformational trapping in a membrane environment: a regulatory mechanism for protein activity?

Functional regulation of proteins is central to living organisms. Here it is shown that a nonfunctional conformational state of a polypeptide can be kinetically trapped in a lipid bilayer environment. This state is a metastable structure that is stable for weeks just above the phase transition temperature of the lipid. When the samples are incubated for several days at 68 degrees C, 50% of the trapped conformation converts to the minimum-energy functional state. This result suggests the possibility that another mechanism for functional regulation of protein activity may be available for membrane proteins: that cells may insert proteins into membranes in inactive states pending the biological demand for protein function.

The association between glycosylphosphatidylinositol-anchored proteins and heterotrimeric G protein alpha subunits in lymphocytes.

Glycosylphosphatidylinositol (GPI)-anchored proteins are nonmembrane spanning cell surface proteins that have been demonstrated to be signal transduction molecules. Because these proteins do not extend into the cytoplasm, the mechanism by which cross-linking of these molecules leads to intracellular signal transduction events is obscure. Previous analysis has indicated that these proteins are associated with src family member tyrosine kinases; however, the role this interaction plays in the generation of intracellular signals is not clear. Here we show that GPI-anchored proteins are associated with alpha subunits of heterotrimeric GTP binding proteins (G proteins) in both human and murine lymphocytes. When the GPI-anchored proteins CD59, CD48, and Thy-1 were immunoprecipitated from various cell lines or freshly isolated lymphocytes, all were found to be associated with a 41-kDa phosphoprotein that we have identified, by using specific antisera, as a mixture of tyrosine phosphorylated G protein alpha subunits: a small amount of Gialpha1, and substantial amounts of Gialpha2 and Gialpha3. GTP binding assays performed with immunoprecipitations of CD59 indicated that there was GTP-binding activity associated with this molecule. Thus, we have shown by both immunochemical and functional criteria that GPI-anchored proteins are physically associated with G proteins. These experiments suggest a potential role of G proteins in the transduction of signals generated by GPI-anchored molecules expressed on lymphocytes of both mouse and human.