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PubMed1993

Rb may act as a transcriptional co-activator in undifferentiated F9 cells.

The reversible interaction of the retinoblastoma susceptibility gene product (Rb) with the cellular transcription factor E2F has recently been demonstrated. Activation of the adenovirus E2a promoter by the products of the viral E1a gene correlates with the ability of both early E1a proteins to sequester Rb, thereby releasing E2F from inactive complexes with this protein. The E2a promoter is also efficiently stimulated by a product (17.5 kDa) of the viral E4 gene. The specific interaction of this E4 protein with E2F results in the formation of complexes that bind cooperatively to the two neighboring E2F binding sites in the E2a promoter. We have previously shown that in undifferentiated F9 cells (F9EC) the E2a promoter is refractory to E2F-mediated activation by E1a, but not by E4. Using both band-shift and transfection experiments, we now demonstrate (i) that in F9EC cells the E4 product, in combination with E2F, recruits Rb into a stable multiprotein complex and (ii) that in these undifferentiated cells, as opposed to their differentiated counterpart, Rb is actively involved in the transcriptional stimulation of the E2a promoter by E4. Our results suggest that, depending on the cell state, Rb may behave either as a transcriptional activator (F9EC cells) or as a transcriptional inhibitor (differentiated F9 cells).

Adenovirus E2 Proteins↗
PubMed2005

Identification of heme binding protein complexes in murine erythroleukemic cells: study by a novel two-dimensional native separation -- liquid chromatography and electrophoresis.

In the current postgenomic era there is a growing interest in analysis of protein complexes in their native state. Here we present a novel two-dimensional separation technique for assessment of native protein complexes. The method combines native chromatography with native electrophoresis. The approach was used to study heme-binding protein complexes in murine erythroleukemia cells. The cells were metabolically labeled with [(59)Fe]-heme and cellular lysates were separated by anion-exchange chromatography. Fractions containing the (59)Fe isotope were collected, concentrated and further separated by native gel electrophoresis. A total of 13 radioactive protein bands were detected and analyzed by liquid chromatography-tandem mass spectrometry. Thirty-three individual proteins were identified and attributed to four novel multiprotein complexes representing four different 'snapshots' of cellular events involved in hemoglobin biosynthesis.

Animals↗
PubMed2000

A mechanism for translationally coupled mRNA turnover: interaction between the poly(A) tail and a c-fos RNA coding determinant via a protein complex.

mRNA turnover mediated by the major protein-coding-region determinant of instability (mCRD) of the c-fos proto-oncogene transcript illustrates a functional interplay between mRNA turnover and translation. We show that the function of mCRD depends on its distance from the poly(A) tail. Five mCRD-associated proteins were identified: Unr, a purine-rich RNA binding protein; PABP, a poly(A) binding protein; PAIP-1, a poly(A) binding protein interacting protein; hnRNP D, an AU-rich element binding protein; and NSAP1, an hnRNP R-like protein. These proteins form a multiprotein complex. Overexpression of these proteins stabilized mCRD-containing mRNA by impeding deadenylation. We propose that a bridging complex forms between the poly(A) tail and the mCRD and ribosome transit disrupts or reorganizes the complex, leading to rapid RNA deadenylation and decay.

Amino Acid Sequence↗
PubMed2005

Cancer in xeroderma pigmentosum and related disorders of DNA repair.

Nucleotide-excision repair diseases exhibit cancer, complex developmental disorders and neurodegeneration. Cancer is the hallmark of xeroderma pigmentosum (XP), and neurodegeneration and developmental disorders are the hallmarks of Cockayne syndrome and trichothiodystrophy. A distinguishing feature is that the DNA-repair or DNA-replication deficiencies of XP involve most of the genome, whereas the defects in CS are confined to actively transcribed genes. Many of the proteins involved in repair are also components of dynamic multiprotein complexes, transcription factors, ubiquitylation cofactors and signal-transduction networks. Complex clinical phenotypes might therefore result from unanticipated effects on other genes and proteins.

Child↗
PubMedDate not supplied

It's all in the timing: linking S phase to chromatin structure and chromosome dynamics.

Many aspects of chromosome biology are fundamentally linked to events that occur during the DNA synthesis (S) phase of the cell cycle. The DNA must be duplicated once, and once exactly, each S phase. Local chromatin structure must also be re-assembled each S phase to incorporate newly replicated sister chromatids. The replication fork is the one complex that potentially interacts with every nucleotide of the genome, providing a mechanism to couple chromatin assembly to S phase passage. Importantly, eukaryotic genomes contain regions of structurally distinct chromatin, such as heterochromatin, defined by distinct patterns of histone modification and specific protein associations. Heterochromatin is generally associated with repeated sequence elements near centromeres, telomeres and other sites. Evidence suggests that heterochromatin assembled during S phase supports the association of multiprotein complexes required for many chromosome transactions, including transcriptional silencing, sister-chromatid cohesion, and kinetochore function. These complexes are in turn essential for regulated gene expression, equal chromosome segregation and genomic stability. Intriguingly, recent studies indicate that these processes are linked to S phase by temporal mechanisms as well as by replication-dependent activities.

Animals↗
PubMed1996

Glucocorticoid repression of the mouse gonadotropin-releasing hormone gene is mediated by promoter elements that are recognized by heteromeric complexes containing glucocorticoid receptor.

We have identified two regions of the mouse gonadotropin-releasing hormone (GnRH) promoter, one between -237 and -201 (distal element) and the other between -184 and -150 (proximal element), which are required for glucocorticoid repression in transiently transfected GT1-7 cells. These sequences show no similarity to known positive or negative glucocorticoid response elements (nGREs) and do not function when placed upstream of heterologous viral promoters. The glucocorticoid receptor (GR) does not bind directly to the distal or proximal promoter elements but may participate in glucocorticoid repression of GnRH gene transcription by virtue of its association within multiprotein complexes at these nGREs. Electrophoretic mobility shift assays with GT1-7 nuclear extract demonstrate the presence of GR-containing protein complexes on GnRH nGREs. One protein that co-occupies the distal nGRE in vitro along with GR is the POU domain transcription factor Oct-1. Thus, the tethering of GR to the GnRH distal nGRE, by virtue of a direct or indirect association with DNA-bound Oct-1, could play a role in hormone-dependent transcriptional repression of the GnRH gene. In contrast, Oct-1 does not appear to be a component of the GR-containing protein complex that is bound to the proximal nGRE.

Animals↗
PubMed1996

XMCM7, a novel member of the Xenopus MCM family, interacts with XMCM3 and colocalizes with it throughout replication.

A minichromosome maintenance (MCM) protein complex has been implicated in restricting DNA replication to once per cell cycle in Xenopus egg extracts, based on the behavior of a single protein, XMCM3. Using a two-hybrid screen with XMCM3, we have identified a novel member of the MCM family in Xenopus that is essential for DNA replication. The protein shows strong homology to Saccharomyces cerevisiae MCM7 (CDC47) and has thus been named XMCM7. XMCM7 is present in a multiprotein complex with other MCM proteins. It binds to chromatin and is displaced from chromatin by the act of replication. XMCM7 does not preferentially colocalize with sites of DNA replication but colocalizes with XMCM3 throughout replication. Immunodepletion of the MCM complex from Xenopus egg extract by anti-XMCM7 antibodies inhibits DNA replication of sperm and permeable HeLa G2 nuclei but not permeable HeLa G1 nuclei. Replication capacity of the Xenopus egg extract immunodepleted of the MCM complex by anti-XMCM7 antibody can be rescued by MCM proteins eluted from anti-XMCM3 antibody. We conclude that both proteins are present in the same complex in Xenopus egg extract throughout the cell cycle, that they remain together after binding to chromatin and during DNA replication, and that they perform similar functions.

Animals↗
PubMed2002

HSP27 modulates agonist-induced association of translocated RhoA and PKC-alpha in muscle cells of the colon.

The recruitment of signal transduction molecules to the membrane is crucial for the efficient coupling of extracellular signals and contractile response. The trafficking is dynamic. We have investigated a possible cross talk between agonist-induced association of translocated RhoA and translocated protein kinase C-alpha (PKC-alpha) and a role for heat shock protein 27 (HSP27) in mediating this interaction. Immunoprecipitation with HSP27 monoclonal antibody followed by immunoblotting with either RhoA antibody or PKC-alpha antibody indicated that acetylcholine induced associations of HSP27-RhoA and HSP27-PKC-alpha in the membrane fraction but not in the cytosolic fraction. Immunoprecipitation with anti-RhoA monoclonal antibody followed by immunoblotting with PKC-alpha antibody indicated that acetylcholine induced a significant complexing of RhoA-PKC-alpha in the membrane fraction but not in the cytosolic fraction. In summary, the data indicate that agonist-induced contraction is associated with 1) association of translocated RhoA with HSP27 on the membrane, 2) association of translocated PKC-alpha with HSP27 on the membrane, and 3) association of PKC-alpha with RhoA on the membrane. The data suggest an important role for HSP27 in modulating a multiprotein complex that includes translocated RhoA and PKC-alpha.

Acetylcholine↗
PubMed1994

Complexes between STE5 and components of the pheromone-responsive mitogen-activated protein kinase module.

We present genetic evidence for complex formation of STE5 and the STE11, STE7, and FUS3 protein kinases, the pheromone-responsive mitogen-activated protein kinase module of Saccharomyces cerevisiae. Interaction between STE5 and STE11 is not dependent on STE7, and interaction between STE5 and STE7 does not require STE11. The N-terminal regulatory domain of STE11 is both necessary and sufficient for interaction with STE5. Interaction between STE7 and STE11 is bridged by STE5, suggesting the formation of a multiprotein complex. We also demonstrate biochemical interaction between STE5 and STE11 by using a combination of bacterially expressed fusion proteins and extracts prepared from yeast. Our results suggest that STE5 is a scaffolding protein that facilitates interactions between components of the pheromone-responsive mitogen-activated protein kinase module. We further propose that such scaffolding proteins serve to inhibit cross-talk between functionally unrelated mitogen-activated protein kinase modules within the same cell.

Adaptor Proteins, Signal Transducing↗
PubMed2002

The Maguk protein, Pals1, functions as an adapter, linking mammalian homologues of Crumbs and Discs Lost.

Membrane-associated guanylate kinase (Maguk) proteins are scaffold proteins that contain PSD-95-Discs Large-zona occludens-1 (PDZ), Src homology 3, and guanylate kinase domains. A subset of Maguk proteins, such as mLin-2 and protein associated with Lin-7 (Pals)1, also contain two L27 domains: an L27C domain that binds mLin-7 and an L27N domain of unknown function. Here, we demonstrate that the L27N domain targets Pals1 to tight junctions by binding to a PDZ domain protein, Pals1-associated tight junction (PATJ) protein, via a unique Maguk recruitment domain. PATJ is a homologue of Drosophila Discs Lost, a protein that is crucial for epithelial polarity and that exists in a complex with the apical polarity determinant, Crumbs. PATJ and a human Crumbs homologue, CRB1, colocalize with Pals1 to tight junctions, and CRB1 interacts with PATJ albeit indirectly via binding the Pals1 PDZ domain. In agreement, we find that a Drosophila homologue of Pals1 participates in identical interactions with Drosophila Crumbs and Discs Lost. This Drosophila Pals1 homologue has been demonstrated recently to represent Stardust, a crucial polarity gene in Drosophila. Thus, our data identifies a new multiprotein complex that appears to be evolutionarily conserved and likely plays an important role in protein targeting and cell polarity.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Drosophila heterochromatin protein 1 (HP1)/origin recognition complex (ORC) protein is associated with HP1 and ORC and functions in heterochromatin-induced silencing.

Heterochromatin protein 1 (HP1) is a conserved component of the highly compact chromatin of higher eukaryotic centromeres and telomeres. Cytogenetic experiments in Drosophila have shown that HP1 localization into this chromatin is perturbed in mutants for the origin recognition complex (ORC) 2 subunit. ORC has a multisubunit DNA-binding activity that binds origins of DNA replication where it is required for origin firing. The DNA-binding activity of ORC is also used in the recruitment of the Sir1 protein to silence nucleation sites flanking silent copies of the mating-type genes in Saccharomyces cerevisiae. A fraction of HP1 in the maternally loaded cytoplasm of the early Drosophila embryo is associated with a multiprotein complex containing Drosophila melanogaster ORC subunits. This complex appears to be poised to function in heterochromatin assembly later in embryonic development. Here we report the identification of a novel component of this complex, the HP1/ORC-associated protein. This protein contains similarity to DNA sequence-specific HMG proteins and is shown to bind specific satellite sequences and the telomere-associated sequence in vitro. The protein is shown to have heterochromatic localization in both diploid interphase and mitotic chromosomes and polytene chromosomes. Moreover, the gene encoding HP1/ORC-associated protein was found to display reciprocal dose-dependent variegation modifier phenotypes, similar to those for mutants in HP1 and the ORC 2 subunit.

Amino Acid Sequence↗
PubMed2004

Nephrin forms a complex with adherens junction proteins and CASK in podocytes and in Madin-Darby canine kidney cells expressing nephrin.

Mutations in the NPHS1 gene encoding nephrin lead to congenital nephrotic syndrome of the Finnish type. Nephrin is a key component of the glomerular slit diaphragms between epithelial foot processes, but its role in the pathogenesis of this disease is poorly understood. To further clarify the molecular mechanisms involved we investigated the interactions between nephrin and other components of the foot processes and filtration slits, especially adherens junction proteins, and searched for novel nephrin interacting proteins. Using co-immunoprecipitation and pull-down assays we show here that nephrin forms a multiprotein complex with cadherins and p120 catenin and with three scaffolding proteins, ZO-1, CD2AP, and CASK, in kidney glomeruli and when expressed in Madin-Darby canine kidney cells. CASK was identified as a novel binding partner of nephrin by mass spectrometry and was localized to podocytes in the glomerulus. CASK is a scaffolding protein that participates in maintenance of polarized epithelial cell architecture by linking membrane proteins and signaling molecules to the actin cytoskeleton. Our results support a model whereby the glomerular slit diaphragms are composed of cell adhesion molecules of the immunoglobulin and cadherin superfamilies that are connected to each other and to the actin cytoskeleton and signaling networks via the cytoplasmic scaffolding proteins CASK, CD2AP, and ZO-1.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Werner syndrome protein participates in a complex with RAD51, RAD54, RAD54B and ATR in response to ICL-induced replication arrest.

Werner syndrome (WS) is a rare genetic disorder characterized by genomic instability caused by defects in the WRN gene encoding a member of the human RecQ helicase family. RecQ helicases are involved in several DNA metabolic pathways including homologous recombination (HR) processes during repair of stalled replication forks. Following introduction of interstrand DNA crosslinks (ICL), WRN relocated from nucleoli to arrested replication forks in the nucleoplasm where it interacted with the HR protein RAD52. In this study, we use fluorescence resonance energy transfer (FRET) and immune-precipitation experiments to demonstrate that WRN participates in a multiprotein complex including RAD51, RAD54, RAD54B and ATR in cells where replication has been arrested by ICL. We verify the WRN-RAD51 and WRN-RAD54B direct interaction in vitro. Our data support a role for WRN also in the recombination step of ICL repair.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2003

Cysteine-rich LIM-only proteins CRP1 and CRP2 are potent smooth muscle differentiation cofactors.

Cysteine-rich LIM-only proteins, CRP1 and CRP2, expressed during cardiovascular development act as bridging molecules that associate with serum response factor and GATA proteins. SRF-CRP-GATA complexes strongly activated smooth muscle gene targets. CRP2 was found in the nucleus during early stages of coronary smooth muscle differentiation from proepicardial cells. A dominant-negative CRP2 mutant blocked proepicardial cells from differentiating into smooth muscle cells. Together with SRF and GATA proteins, CRP1 and CRP2 converted pluripotent 10T1/2 fibroblasts into smooth muscle cells, while muscle LIM protein CRP3 inhibited the conversion. Thus, LIM-only proteins of the CRP family play important roles in organizing multiprotein complexes, both in the cytoplasm, where they participate in cytoskeletal remodeling, and in the nucleus, where they strongly facilitate smooth muscle differentiation.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Structural flexibility in transcription complex formation revealed by protein-DNA photocrosslinking.

The Oct-1 POU domain binds diverse DNA-sequence elements and forms a higher-order regulatory complex with the herpes simplex virus coregulator VP16. The POU domain contains two separate DNA-binding domains joined by a flexible linker. By protein-DNA photocrosslinking we show that the relative positioning of the two POU DNA-binding domains on DNA varies depending on the nature of the DNA target. On a single VP16-responsive element, the POU domain adopts multiple conformations. To determine the structure of the Oct-1 POU domain in a multiprotein complex with VP16, we allowed VP16 to interact with previously crosslinked POU-domain-DNA complexes and found that VP16 can associate with multiple POU-domain conformations. These results reveal the dynamic potential of a DNA-binding domain in directing transcriptional regulatory complex formation.

Binding Sites↗
PubMed2000

Targeting of high mobility group-14/-17 proteins in chromatin is independent of DNA sequence.

Chromosomal proteins high mobility group (HMG)-14 and HMG-17 are nucleosomal-binding proteins that unfold the chromatin fiber and enhance transcription from chromatin templates. Their intracellular organization is dynamic and related to both cell cycle and transcription. Here we examine possible mechanisms for targeting HMG-14/-17 to specific regions in chromatin. Chromatin immunoprecipitation assays indicate that HMG-17 protein is not preferentially associated with chromatin regions containing transcriptionally active genes, or any type of specific DNA. We used a modification of the random amplified polymorphic DNA method to analyze DNA in various HMG-14/-17.nucleosome complexes. We found that although HMG-14 or HMG-17 proteins preferentially associate with core particles in which the DNA has a low frequency of CG dinucleotides, the genome does not contain consensus sequences that serve as specific targeting sites for the binding of either HMG-14 or HMG-17 proteins to nucleosomes. We used size exclusion and ion exchange chromatography to demonstrate that nuclei contain a large portion of HMG-17 associated with other proteins in a multiprotein complex. We suggest that these complexes regulate the dynamic organization of HMG-14/-17 in the nucleus and serve to target the proteins to specific sites in chromatin.

Animals↗
PubMed2000

Signal complex regulation of renal transport proteins: NHERF and regulation of NHE3 by PKA.

The activity of the sodium/hydrogen exchanger 3 (NHE3) isoform of the sodium/hydrogen exchanger in the brush-border membrane of the renal proximal tubule is tightly regulated. Recent biochemical and cellular experiments have established the essential requirement for a new class of regulatory factors, sodium/hydrogen exchanger regulatory factor (NHERF) and NHERF-like proteins, in cAMP-mediated inhibition of NHE3 activity. NHERF is the first PSD-95/Dlg/ZO-1 (PDZ) motif-containing protein localized to apical membranes and appears to facilitate cAMP-dependent protein kinase A (PKA) phosphorylation of NHE3 by interacting with the cytoskeleton to target a multiprotein complex to the brush-border membrane. Other recent experiments have indicated that NHERF also regulates the activity of other renal transport proteins, suggesting that the signal complex model of signal transduction in the kidney may be more common than presently appreciated. This article reviews studies on the regulation of NHE3 by NHERF, PKA, and ezrin and introduces the concept of regulation of renal transporters by signal complexes. Although not the primary focus of this review, recent studies have indicated a role for NHERF in membrane targeting, trafficking, and sorting of transporters, receptors, and signaling proteins. Thus NHERF and related PDZ-containing proteins appear to be essential adapters for regulation of renal transporters in the mammalian kidney that maintain salt and water balance.

Animals↗