Self-regulated polymerization of the actin-related protein Arp1.
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The actin-related protein Arp1 (or centractin, actin RPV) is the major subunit of dynactin, a key component of the cytoplasmic dynein motor machinery [1] [2] [3]. Of the ubiquitously expressed members of the Arp superfamily, Arp1 is most similar to conventional actin [4] [5] [6] and, on the basis of conserved sequence features, is predicted to bind ATP and possibly polymerize. In vivo, all cytosolic Arp1 sediments at 20S [7] suggesting that it assembles into oligomers, most likely dynactin - a multiprotein complex known to contain eight or nine Arp1 monomers in a 37 nm filament [8]. The uniform length of Arp1 polymers suggests a novel assembly mechanism that may be governed by a 'ruler' activity. In dynactin, the Arp1 filament is bounded by actin-capping protein at one end and a heterotetrameric protein complex containing the p62 subunit (D.M. Eckley, S.R. Gill, J.B.B., J.E. Heuser, T.A.S., unpublished observations) at the other [8]. In the present study, we analyzed the behavior of highly purified, native Arp1. Arp1 was found to polymerize rapidly into short filaments that were similar, but not identical, in length to those in dynactin. With time, these filaments appeared to anneal to form longer assemblies but never attained the length of conventional actin filaments. (+info)
A steroid receptor coactivator, SRA, functions as an RNA and is present in an SRC-1 complex.
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Nuclear receptors play critical roles in the regulation of eukaryotic gene expression. We report the isolation and functional characterization of a novel transcriptional coactivator, termed steroid receptor RNA activator (SRA). SRA is selective for steroid hormone receptors and mediates transactivation via their amino-terminal activation function. We provide functional and mechanistic evidence that SRA acts as an RNA transcript; transfected SRA, unlike other steroid receptor coregulators, functions in the presence of cycloheximide, and SRA mutants containing multiple translational stop signals retain their ability to activate steroid receptor-dependent gene expression. Biochemical fractionation shows that SRA exists in distinct ribonucleoprotein complexes, one of which contains the nuclear receptor coactivator steroid receptor coactivator 1. We suggest that SRA may act to confer functional specificity upon multiprotein complexes recruited by liganded receptors during transcriptional activation. (+info)
The N-terminal transactivation domain of ATF2 is a target for the co-operative activation of the c-jun promoter by p300 and 12S E1A.
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The adenovirus E1A proteins activate the c-jun promoter through two Jun/ATF-binding sites, jun1 and jun2. P300, a transcriptional coactivator of several AP1 and ATF transcription factors has been postulated to play a role in this activation. Here, we present evidence that p300 can control c-jun transcription by acting as a cofactor for ATF2: (1) Over-expression of p300 was found to stimulate c-jun transcription both in the presence and absence of E1A. (2) Like E1A, p300 activates the c-jun promoter through the junl and jun2 elements and preferentially activates the N-terminal domain of ATF2. (3) Co-immunoprecipitation assays of crude cell extracts indicate that endogenous p300/CBP(-like) proteins and ATF2 proteins are present in a multiprotein complex that can bind specifically to the jun2 element. We further demonstrate that the Stress-Activated-Protein-Kinase (SAPK) target sites of ATF2, Thr69 and Thr71 are not required for the formation of the p300/CBP-ATF2 multiprotein complex. These data indicate that E1A does not inhibit all transcription activation functions of p300, and, in fact, cooperates with p300 in the activation of the ATF2 N-terminus. (+info)
Arabidopsis FUSCA5 encodes a novel phosphoprotein that is a component of the COP9 complex.
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The COP9 complex is a regulator essential for repression of light-mediated development in Arabidopsis. Using partial amino acid sequence data generated from purified COP9 complexes, we cloned the Arabidopsis cDNA encoding the 27-kD subunit of the COP9 complex and showed that it is encoded by the previously identified FUSCA5 (FUS5) locus. fus5 mutants exhibit constitutive photomorphogenic phenotypes similar to those of cop9 and fus6. Point mutations in FUS5 that led to a loss of FUS5 protein were detected in four fus5 allelic strains. FUS5 contains the PCI/PINT and mitogen-activated protein kinase kinase activation loop motifs and is highly conserved with the mammalian COP9 complex subunit 7 and the Aspergillus nidulans AcoB proteins. FUS5 is present in both complex and monomeric forms. In the COP9 complex, FUS5 may interact directly with FUS6 and COP9. Mutations in FUS6 and COP9 result in a shift in the electrophoretic mobility of FUS5. This shift can be mimicked by in vitro phosphorylation of FUS5 by plant extracts. These findings further support the hypothesis that the COP9 complex is a central and common regulator that may interact with multiple signaling pathways. (+info)
Identification of TATA-binding protein-free TAFII-containing complex subunits suggests a role in nucleosome acetylation and signal transduction.
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Recently we identified a novel human (h) multiprotein complex, called TATA-binding protein (TBP)-free TAFII-containing complex (TFTC), which is able to nucleate RNA polymerase II transcription and can mediate transcriptional activation. Here we demonstrate that TFTC, similar to other TBP-free TAFII complexes (yeast SAGA, hSTAGA, and hPCAF) contains the acetyltransferase hGCN5 and is able to acetylate histones in both a free and a nucleosomal context. The recently described TRRAP cofactor for oncogenic transcription factor pathways was also characterized as a TFTC subunit. Furthermore, we identified four other previously uncharacterized subunits of TFTC: hADA3, hTAFII150, hSPT3, and hPAF65beta. Thus, the polypeptide composition of TFTC suggests that TFTC is recruited to chromatin templates by activators to acetylate histones and thus may potentiate initiation and activation of transcription. (+info)
Maternal histone deacetylase is accumulated in the nuclei of Xenopus oocytes as protein complexes with potential enzyme activity.
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Reversible acetylation of core histones plays an important regulatory role in transcription and replication of chromatin. The acetylation status of chromatin is determined by the equilibrium between activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs). The Xenopus protein HDACm shows sequence homology to other putative histone deacetylases, but its mRNA is expressed only during early development. Both HDACm protein and acetylated non-chromosomal histones are accumulated in developing oocytes, indicating that the key components for histone deposition into new chromatin during blastula formation are in place by the end of oogenesis. Here we show that the 57 kDa HDACm protein undergoes steady accumulation in the nucleus, where it is organized in a multiprotein complex of approx. 300 kDa. A second, major component of the nuclear complex is the retinoblastoma-associated protein p48 (RbAp48/46), which may be used as an adaptor to contact acetylated histones in newly assembled chromatin. The nuclear complex has HDAC activity that is sensitive to trichostatin A, zinc ions and phosphatase treatment. The 57 kDa protein serves as a marker for total HDAC activity throughout oogenesis and early embryogenesis. The active HDACm complex and its acetylated histone substrates appear to be kept apart until after chromatin assembly has taken place. However, recombinant HDACm, injected into the cytoplasm of oocytes, not only is translocated to the nucleus, but also is free to interact with the endogenous chromatin. (+info)
Myb-related fission yeast cdc5p is a component of a 40S snRNP-containing complex and is essential for pre-mRNA splicing.
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Myb-related cdc5p is required for G(2)/M progression in the yeast Schizosaccharomyces pombe. We report here that all detectable cdc5p is stably associated with a multiprotein 40S complex. Immunoaffinity purification has allowed the identification of 10 cwf (complexed with cdc5p) proteins. Two (cwf6p and cwf10p) are members of the U5 snRNP; one (cwf9p) is a core snRNP protein. cwf8p is the apparent ortholog of the Saccharomyces cerevisiae splicing factor Prp19p. cwf1(+) is allelic to the prp5(+) gene defined by the S. pombe splicing mutant, prp5-1, and there is a strong negative genetic interaction between cdc5-120 and prp5-1. Five cwfs have not been recognized previously as important for either pre-mRNA splicing or cell cycle control. Further characterization of cwf1p, cwf2p, cwf3p, and cwf4p demonstrates that they are encoded by essential genes, cosediment with cdc5p at 40S, and coimmunoprecipitate with cdc5p. We further show that cdc5p associates with the U2, U5, and U6 snRNAs and that cells lacking cdc5(+) function are defective in pre-mRNA splicing. These data raise the possibility that the cdc5p complex is an intermediate in the assembly or disassembly of an active S. pombe spliceosome. (+info)
Kinase suppressor of Ras forms a multiprotein signaling complex and modulates MEK localization.
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Genetic screens for modifiers of activated Ras phenotypes have identified a novel protein, kinase suppressor of Ras (KSR), which shares significant sequence homology with Raf family protein kinases. Studies using Drosophila melanogaster and Caenorhabditis elegans predict that KSR positively regulates Ras signaling; however, the function of mammalian KSR is not well understood. We show here that two predicted kinase-dead mutants of KSR retain the ability to complement ksr-1 loss-of-function alleles in C. elegans, suggesting that KSR may have physiological, kinase-independent functions. Furthermore, we observe that murine KSR forms a multimolecular signaling complex in human embryonic kidney 293T cells composed of HSP90, HSP70, HSP68, p50(CDC37), MEK1, MEK2, 14-3-3, and several other, unidentified proteins. Treatment of cells with geldanamycin, an inhibitor of HSP90, decreases the half-life of KSR, suggesting that HSPs may serve to stabilize KSR. Both nematode and mammalian KSRs are capable of binding to MEKs, and three-point mutants of KSR, corresponding to C. elegans loss-of-function alleles, are specifically compromised in MEK binding. KSR did not alter MEK activity or activation. However, KSR-MEK binding shifts the apparent molecular mass of MEK from 44 to >700 kDa, and this results in the appearance of MEK in membrane-associated fractions. Together, these results suggest that KSR may act as a scaffolding protein for the Ras-mitogen-activated protein kinase pathway. (+info)