Cell-Adhesive Responses to Tenascin-C Splice Variants Involve Formation of Fascin Microspikes

Tenascin-C is an adhesion-modulating matrix glycoprotein that has multiple effects on cell behavior. Tenascin-C transcripts are expressed in motile cells and at sites of tissue modeling during development, and alternative splicing generates variants that encode different numbers of fibronectin type III repeats. We have examined the in vivo expression and cell adhesive properties of two full-length recombinant tenascin-C proteins: TN-190, which contains the eight constant fibronectin type III repeats, and TN-ADC, which contains the additional AD2, AD1, and C repeats. In situ hybridization with probes specific for the AD2, AD1, and C repeats shows that these splice variants are expressed at sites of active tissue modeling and fibronectin expression in the developing avian feather bud and sternum. Transcripts incorporating the AD2, AD1, and C repeats are present in embryonic day 10 wing bud but not in embryonic day 10 lung. By using a panel of nine cell lines in attachment assays, we have found that C2C12, G8, and S27 myoblastic cells undergo concentration-dependent adhesion to both variants, organize actin microspikes that contain the actin-bundling protein fascin, and do not assemble focal contacts. On a molar basis, TN-ADC is more active than TN-190 in promoting cell attachment and irregular cell spreading. The addition of either TN-190 or TN-ADC in solution to C2C12, COS-7, or MG-63 cells adherent on fibronectin decreases cell attachment and results in decreased organization of actin microfilament bundles, with formation of cortical membrane ruffles and retention of residual points of substratum contact that contain filamentous actin and fascin. These data establish a biochemical similarity in the processes of cell adhesion to tenascin-C and thrombospondin-1, also an “antiadhesive” matrix component, and also demonstrate that both the adhesive and adhesion-modulating properties of tenascin-C involve similar biochemical events in the cortical cytoskeleton. In addition to these generic properties, TN-ADC is less active in adhesion modulation than TN-190. The coordinated expression of different tenascin-C transcripts during development may, therefore, provide appropriate microenvironments for regulated changes in cell shape, adhesion, and movement.

Heterogeneity of Mitochondrial Protein Biogenesis during Primary Leaf Development in Barley1

The natural developmental gradient of light-grown primary leaves of barley (Hordeum vulgare L.) was used to analyze the biogenesis of mitochondrial proteins in relation to the age and physiological changes within the leaf. The data indicate that the protein composition of mitochondria changes markedly during leaf development. Three distinct patterns of protein development were noted: group A proteins, consisting of the E1 β-subunit of the pyruvate dehydrogenase complex, ORF156, ORF577, alternative oxidase, RPS12, cytochrome oxidase subunits II and III, malic enzyme, and the α- and β-subunits of F1-ATPase; group B proteins, consisting of the E1 α-subunit of the pyruvate dehydrogenase complex, isocitrate dehydrogenase, HSP70A, cpn60C, and cpn60B; and group C proteins, consisting of the four subunits of the glycine decarboxylase complex (P, H, T, and L proteins), fumarase, and formate dehydrogenase. All of the proteins increased in concentration from the basal meristem to the end of the elongation zone (20.0 mm from the leaf base), whereupon group A proteins decreased, group B proteins increased to a maximum at 50 mm from the leaf base, and group C proteins increased to a maximum at the leaf tip. This study provides evidence of a marked heterogeneity of mitochondrial protein composition, reflecting a changing function as leaf cells develop photosynthetic and photorespiratory capacity.

The C-terminal SET domains of ALL-1 and TRITHORAX interact with the INI1 and SNR1 proteins, components of the SWI/SNF complex

The ALL-1 gene was discovered by virtue of its involvement in human acute leukemia. Its Drosophila homolog trithorax (trx) is a member of the trx-Polycomb gene family, which maintains correct spatial expression of the Antennapedia and bithorax complexes during embryogenesis. The C-terminal SET domain of ALL-1 and TRITHORAX (TRX) is a 150-aa motif, highly conserved during evolution. We performed yeast two hybrid screening of Drosophila cDNA library and detected interaction between a TRX polypeptide spanning SET and the SNR1 protein. SNR1 is a product of snr1, which is classified as a trx group gene. We found parallel interaction in yeast between the SET domain of ALL-1 and the human homolog of SNR1, INI1 (hSNF5). These results were confirmed by in vitro binding studies and by demonstrating coimmunoprecipitation of the proteins from cultured cells and/or transgenic flies. Epitope-tagged SNR1 was detected at discrete sites on larval salivary gland polytene chromosomes, and these sites colocalized with around one-half of TRX binding sites. Because SNR1 and INI1 are constituents of the SWI/SNF complex, which acts to remodel chromatin and consequently to activate transcription, the interactions we observed suggest a mechanism by which the SWI/SNF complex is recruited to ALL-1/trx targets through physical interactions between the C-terminal domains of ALL-1 and TRX and INI1/SNR1.

Association of Nonribosomal Nucleolar Proteins in Ribonucleoprotein Complexes during Interphase and Mitosis

rRNA precursors are bound throughout their length by specific proteins, as the pre-rRNAs emerge from the transcription machinery. The association of pre-rRNA with proteins as ribonucleoprotein (RNP) complexes persists during maturation of 18S, 5.8S, and 28S rRNA, and through assembly of ribosomal subunits in the nucleolus. Preribosomal RNP complexes contain, in addition to ribosomal proteins, an unknown number of nonribosomal nucleolar proteins, as well as small nucleolar RNA-ribonucleoproteins (sno-RNPs). This report describes the use of a specific, rapid, and mild immunopurification approach to isolate and analyze human RNP complexes that contain nonribosomal nucleolar proteins, as well as ribosomal proteins and rRNA. Complexes immunopurified with antibodies to nucleolin—a major nucleolar RNA-binding protein—contain several distinct specific polypeptides that include, in addition to nucleolin, the previously identified nucleolar proteins B23 and fibrillarin, proteins with electrophoretic mobilities characteristic of ribosomal proteins including ribosomal protein S6, and a number of additional unidentified proteins. The physical association of these proteins with one another is mediated largely by RNA, in that the complexes dissociate upon digestion with RNase. Complexes isolated from M-phase cells are similar in protein composition to those isolated from interphase cell nuclear extracts. Therefore, the predominant proteins that associate with nucleolin in interphase remain in RNP complexes during mitosis, despite the cessation of rRNA synthesis and processing in M-phase. In addition, precursor rRNA, as well as processed 18S and 28S rRNA and candidate rRNA processing intermediates, is found associated with the immunopurified complexes. The characteristics of the rRNP complexes described here, therefore, indicate that they represent bona fide precursors of mature cytoplasmic ribosomal subunits.

M Phase Phosphoprotein 10 Is a Human U3 Small Nucleolar Ribonucleoprotein Component

We have previously developed a novel technique for isolation of cDNAs encoding M phase phosphoproteins (MPPs). In the work described herein, we further characterize MPP10, one of 10 novel proteins that we identified, with regard to its potential nucleolar function. We show that by cell fractionation, almost all MPP10 was found in isolated nucleoli. By immunofluorescence, MPP10 colocalized with nucleolar fibrillarin and other known nucleolar proteins in interphase cells but was not detected in the coiled bodies stained for either fibrillarin or p80 coilin, a protein found only in the coiled body. When nucleoli were separated into fibrillar and granular domains by treatment with actinomycin D, almost all the MPP10 was found in the fibrillar caps, which contain proteins involved in rRNA processing. In early to middle M phase of the cell cycle, MPP10 colocalized with fibrillarin to chromosome surfaces. At telophase, MPP10 was found in cellular structures that resembled nucleolus-derived bodies and prenucleolar bodies. Some of these bodies lacked fibrillarin, a previously described component of nucleolus-derived bodies and prenucleolar bodies, however, and the bulk of MPP10 arrived at the nucleolus later than fibrillarin. To further examine the properties of MPP10, we immunoprecipitated it from cell sonicates. The resulting precipitates contained U3 small nucleolar RNA (snoRNA) but no significant amounts of other box C/D snoRNAs. This association of MPP10 with U3 snoRNA was stable to 400 mM salt and suggested that MPP10 is a component of the human U3 small nucleolar ribonucleoprotein.

The conserved SOCS box motif in suppressors of cytokine signaling binds to elongins B and C and may couple bound proteins to proteasomal degradation

The suppressors of cytokine signaling (SOCS) family of proteins act as intracellular inhibitors of several cytokine signal transduction pathways. Their expression is induced by cytokine activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway and they act as a negative feedback loop by subsequently inhibiting the JAK/STAT pathway either by direct interaction with activated JAKs or with the receptors. These interactions are mediated at least in part by the SH2 domain of SOCS proteins but these proteins also contain a highly conserved C-terminal homology domain termed the SOCS box. Here we show that the SOCS box mediates interactions with elongins B and C, which in turn may couple SOCS proteins and their substrates to the proteasomal protein degradation pathway. Analogous to the family of F-box-containing proteins, it appears that the SOCS proteins may act as adaptor molecules that target activated cell signaling proteins to the protein degradation pathway.

DNA-bound transcription factor complexes analysed by mass-spectrometry: binding of novel proteins to the human c-fos SRE and related sequences

Transcription factors control eukaryotic polymerase II function by influencing the recruitment of multiprotein complexes to promoters and their subsequent integrated function. The complexity of the functional ‘transcriptosome’ has necessitated biochemical fractionation and subsequent protein sequencing on a grand scale to identify individual components. As a consequence, much is now known of the basal transcription complex. In contrast, less is known about the complexes formed at distal promoter elements. The c-fos SRE, for example, is known to bind Serum Response Factor (SRF) and ternary complex factors such as Elk-1. Their interaction with other factors at the SRE is implied but, to date, none have been identified. Here we describe the use of mass-spectrometric sequencing to identify six proteins, SRF, Elk-1 and four novel proteins, captured on SRE duplexes linked to magnetic beads. This approach is generally applicable to the characterisation of nucleic acid-bound protein complexes and the post-translational modification of their components.

Allosteric modulation of Ca2+ channels by G proteins, voltage-dependent facilitation, protein kinase C, and Cavβ subunits

N-type and P/Q-type Ca2+ channels are inhibited by neurotransmitters acting through G protein-coupled receptors in a membrane-delimited pathway involving Gβγ subunits. Inhibition is caused by a shift from an easily activated “willing” (W) state to a more-difficult-to-activate “reluctant” (R) state. This inhibition can be reversed by strong depolarization, resulting in prepulse facilitation, or by protein kinase C (PKC) phosphorylation. Comparison of regulation of N-type Ca2+ channels containing Cav2.2a α1 subunits and P/Q-type Ca2+ channels containing Cav2.1 α1 subunits revealed substantial differences. In the absence of G protein modulation, Cav2.1 channels containing Cavβ subunits were tonically in the W state, whereas Cav2.1 channels without β subunits and Cav2.2a channels with β subunits were tonically in the R state. Both Cav2.1 and Cav2.2a channels could be shifted back toward the W state by strong depolarization or PKC phosphorylation. Our results show that the R state and its modulation by prepulse facilitation, PKC phosphorylation, and Cavβ subunits are intrinsic properties of the Ca2+ channel itself in the absence of G protein modulation. A common allosteric model of G protein modulation of Ca2+-channel activity incorporating an intrinsic equilibrium between the W and R states of the α1 subunits and modulation of that equilibrium by G proteins, Cavβ subunits, membrane depolarization, and phosphorylation by PKC accommodates our findings. Such regulation will modulate transmission at synapses that use N-type and P/Q-type Ca2+ channels to initiate neurotransmitter release.

Regulation of the myeloperoxidase enhancer binding proteins Pu1, C-EBP alpha, -beta, and -delta during granulocyte-lineage specification.

We have compared the molecular architecture and function of the myeloperoxidase upstream enhancer in multipotential versus granulocyte-committed hematopoietic progenitor cells. We show that the enhancer is accessible in multipotential cell chromatin but functionally incompetent before granulocyte commitment. Multipotential cells contain both Pu1 and C-EBP alpha as enhancer-binding activities. Pu1 is unphosphorylated in both multipotential and granulocyte-committed cells but is phosphorylated in B lymphocytes, raising the possibility that differential phosphorylation may play a role in specifying its lymphoid versus myeloid functions. C-EBP alpha exists as multiple phosphorylated forms in the nucleus of both multipotential and granulocyte-committed cells. C-EBP beta is unphosphorylated and cytoplasmically localized in multipotential cells but exists as a phosphorylated nuclear enhancer-binding activity in granulocyte-committed cells. Granulocyte colony-stimulating factor-induced granulocytic differentiation of multipotential progenitor cells results in activation of C-EBP delta expression and functional recruitment of C-EBP delta and C-EBP beta to the nucleus. Our results implicate Pu1 and the C-EBP family as critical regulators of myeloperoxidase gene expression and are consistent with a model in which a temporal exchange of C-EBP isoforms at the myeloperoxidase enhancer mediates the transition from a primed state in multipotential cells to a transcriptionally active configuration in promyelocytes.

Phosphorylated Ser/Arg-rich proteins: limiting factors in the assembly of 200S large nuclear ribonucleoprotein particles.

We have previously shown that specific nuclear pre-mRNA transcripts and their splicing products, as well as the general population of nuclear poly(A)+ RNA, are packaged in large nuclear ribonucleoprotein (InRNP) particles that sediment at the 200S region in sucrose gradients. The InRNP particles contain all uridine-rich small nuclear ribonucleoprotein complexes required for pre-mRNA splicing, as well as protein splicing factors. In this paper we show that all of the phosphorylated, mAb 104 detectable, Ser/Arg-rich essential splicing factors (SR proteins) in the nucleoplasm are integral components of the InRNP particles, whereas only part of the essential splicing factor U2AF65 (U2 snRNP auxiliary factor) and the polypyrimidine tract binding protein (PTB) are associated with these particles. This finding suggests a limiting role for SR proteins in the assembly of the InRNP particles. We further show that the structural integrity of InRNP particles is sensitive to variations in the phosphorylation levels of the SR proteins.

Haemonchus contortus GA1 antigens: related, phospholipase C-sensitive, apical gut membrane proteins encoded as a polyprotein and released from the nematode during infection.

It was previously shown that the Haemonchus contortus apical gut surface proteins p46, p52, and p100 induced protective immunity to challenge infections in goats. Here, it is shown that the three proteins are all encoded by a single gene (GA1) and initially expressed in adult parasites as a polyprotein (p100GA1). p46GA1 and p52GA1 are related proteins with 47% sequence identity, including a cysteine-containing region, which appears to confer secondary structure to these proteins, and a region with sequence similarity to bacterial Tolb proteins. GA1 protein expression is regulated during the life cycle at the level of transcript abundance. Only p52GA1 has characteristics of a glycosylinositolphospholipid membrane-anchored protein. However, both p46GA1 and p52GA1 were released from the gut membrane by phosphatidylinositol specific-phospholipase C, suggesting that p46GA1 membrane association depends on interactions with a glycosylinositolphospholipid gut membrane protein. Finally, GA1 proteins occur in abomasal mucus of infected lambs, demonstrating possible presentation to the host immune system during H. contortus infection. The results identify multiple characteristics of the GA1 proteins that should be considered for design of recombinant antigens for vaccine trials and that implicate a series of cellular processes leading to modification and expression of GA1 proteins at the nematode apical gut surface.

The C-terminal domain of the largest subunit of RNA polymerase II interacts with a novel set of serine/arginine-rich proteins.

Although transcription and pre-mRNA processing are colocalized in eukaryotic nuclei, molecules linking these processes have not previously been described. We have identified four novel rat proteins by their ability to interact with the repetitive C-terminal domain (CTD) of RNA polymerase II in a yeast two-hybrid assay. A yeast homolog of one of the rat proteins has also been shown to interact with the CTD. These CTD-binding proteins are all similar to the SR (serine/arginine-rich) family of proteins that have been shown to be involved in constitutive and regulated splicing. In addition to alternating Ser-Arg domains, these proteins each contain discrete N-terminal or C-terminal CTD-binding domains. We have identified SR-related proteins in a complex that can be immunoprecipitated from nuclear extracts with antibodies directed against RNA polymerase II. In addition, in vitro splicing is inhibited either by an antibody directed against the CTD or by wild-type but not mutant CTD peptides. Thus, these results suggest that the CTD and a set of CTD-binding proteins may act to physically and functionally link transcription and pre-mRNA processing.

Three-dimensional structure of human protein kinase C interacting protein 1, a member of the HIT family of proteins.

The three-dimensional structure of protein kinase C interacting protein 1 (PKCI-1) has been solved to high resolution by x-ray crystallography using single isomorphous replacement with anomalous scattering. The gene encoding human PKCI-1 was cloned from a cDNA library by using a partial sequence obtained from interactions identified in the yeast two-hybrid system between PKCI-1 and the regulatory domain of protein kinase C-beta. The PKCI-1 protein was expressed in Pichia pastoris as a dimer of two 13.7-kDa polypeptides. PKCI-1 is a member of the HIT family of proteins, shown by sequence identity to be conserved in a broad range of organisms including mycoplasma, plants, and humans. Despite the ubiquity of this protein sequence in nature, no distinct function has been shown for the protein product in vitro or in vivo. The PKCI-1 protomer has an alpha+beta meander fold containing a five-stranded antiparallel sheet and two helices. Two protomers come together to form a 10-stranded antiparallel sheet with extensive contacts between a helix and carboxy terminal amino acids of a protomer with the corresponding amino acids in the other protomer. PKCI-1 has been shown to interact specifically with zinc. The three-dimensional structure has been solved in the presence and absence of zinc and in two crystal forms. The structure of human PKCI-1 provides a model of this family of proteins which suggests a stable fold conserved throughout nature.

Radiometal labeling of recombinant proteins by a genetically engineered minimal chelation site: technetium-99m coordination by single-chain Fv antibody fusion proteins through a C-terminal cysteinyl peptide.

We describe a method to facilitate radioimaging with technetium-99m (99mTc) by genetic incorporation of a 99mTc chelation site in recombinant single-chain Fv (sFv) antibody proteins. This method relies on fusion of the sFv C terminus with a Gly4Cys peptide that specifically coordinates 99mTc. By using analogues of the 26-10 anti-digoxin sFv as our primary model, we find that addition of the chelate peptide, to form 26-10-1 sFv', does not alter the antigen-binding affinity of sFv. We have demonstrated nearly quantitative chelation of 0.5-50 mCi of 99mTc per mg of 26-10-1 sFv' (1 Ci = 37 GBq). These 99mTc-labeled sFv' complexes are highly stable to challenge with saline buffers, plasma, or diethylenetriaminepentaacetic acid. We find that the 99mTc-labeled 741F8-1 sFv', specific for the c-erbB-2 tumor-associated antigen, is effective in imaging human ovarian carcinoma in a scid mouse tumor xenograft model. This fusion chelate methodology should be applicable to diagnostic imaging with 99mTc and radioimmunotherapy with 186Re or 188Re, and its use could extend beyond the sFv' to other engineered antibodies, recombinant proteins, and synthetic peptides.