Eukaryotic Regulatory Proteins Research Articles

14-3-3 proteins: eukaryotic regulatory proteins with many functions.

The enigmatically named 14-3-3 proteins have been the subject of considerable attention in recent years since they have been implicated in the regulation of diverse physiological processes, in eukaryotes ranging from slime moulds to higher plants. In plants they have roles in the regulation of the plasma membrane H+-ATPase and nitrate reductase, among others. Regulation of target proteins is achieved through binding of 14-3-3 to short, often phosphorylated motifs in the target, resulting either in its activation (e.g. H+-ATPase), inactivation (e.g. nitrate reductase) or translocation (although this function of 14-3-3 proteins has yet to be demonstrated in plants). The native 14-3-3 proteins are homo- or heterodimers and, as each monomer has a binding site, a dimer can potentially bind two targets, promoting their association. Alternatively, target proteins may have more than one 14-3-3-binding site. In this mini review, we present a synthesis of recent results from plant 14-3-3 research and, with reference to known 14-3-3-binding motifs, suggest further subjects for research.

Fusicoccin Effect on the in Vitro Interaction between Plant 14-3-3 Proteins and Plasma Membrane H+-ATPase

A 17-amino acid peptide was selectively cleaved from the highly variant C terminus of the 33-kDa 14-3-3 isoform occurring in fusicoccin receptor preparations from maize and was sequenced. The determined C-terminal sequence was identical to that of the already known maize 14-3-3 homolog GF14-6, thus prompting the use of recombinant GF14-6 in an in vitro protein-protein interaction study. The cDNA of GF14-6 was expressed in Escherichia coli as a 32P-phosphorylatable glutathione S-transferase fusion protein and was used as a probe in overlay experiments with H+-ATPase partially purified from maize roots. The results demonstrated that the recombinant protein specifically bound to H+-ATPase. The binding was dependent on Mg2+ and was strongly increased by fusicoccin. Controlled trypsin digestion of H+-ATPase abolished the association with GF14-6, a finding that was suggestive of an interaction with the C terminus of the enzyme. To confirm this result, the C-terminal domain of H+-ATPase was expressed as a glutathione S-transferase fusion peptide and was used in overlay experiments. GF14-6 was also able to bind to the isolated C terminus, but only in the presence of fusicoccin.

The Aspergillus nidulans sulphur regulatory gene sconB encodes a protein with WD40 repeats and an F-box.

The Aspergillus nidulans gene sconB, one of the four identified genes controlling sulphur metabolite repression, was cloned and analysed. It encodes a polypeptide of 678 amino acids containing seven WD repeats characteristic of the large WD40 family of eukaryotic regulatory proteins. The SCONB protein has nuclear localisation signals and is very similar to the Neurospora crassa SCON2 and Saccharomyces cerevisiae Met30 proteins, both of which are involved in the regulation of sulphur metabolism. The N. crassa scon-2 gene complements the sconB2 mutation. All three proteins also contain a newly identified motif, the F-box, found in a number of eukaryotic regulatory proteins. This motif is responsible, at least in some cases, for ubiquitin-mediated proteolysis. The sconB transcript is derepressed under sulphur limitation conditions and partly repressed by high methionine.

Genetic Analysis of brahma: The Drosophila Homolog of the Yeast Chromatin Remodeling Factor SWI2/SNF2

The Drosophila brahma (brm) gene encodes an activator of homeotic genes related to the yeast chromatin remodeling factor SWI2/SNF2. Here, we report the phenotype of null and dominant-negative brm mutations. Using mosaic analysis, we found that the complete loss of brm function decreases cell viability and causes defects in the peripheral nervous system of the adult. A dominant-negative brm mutation was generated by replacing a conserved lysine in the ATP-binding site of the BRM protein with an arginine. This mutation eliminates brm function in vivo but does not affect assembly of the 2-MD BRM complex. Expression of the dominant-negative BRM protein caused peripheral nervous system defects, homeotic transformations, and decreased viability. Consistent with these findings, the BRM protein is expressed at relatively high levels in nuclei throughout the developing organism. Site-directed mutagenesis was used to investigate the functions of conserved regions of the BRM protein. Domain II is essential for brm function and is required for the assembly or stability of the BRM complex. In spite of its conservation in numerous eukaryotic regulatory proteins, the deletion of the bromodomain of the BRM protein has no discernible phenotype.

A Model for the Cooperative Binding of Eukaryotic Regulatory Proteins to Nucleosomal Target Sites

The mechanism by which gene regulatory proteins gain access to their DNA target sequences in chromatin is not known. We recently showed that nucleosomes are intrinsically dynamic, transiently exposing their DNA to allow sequence-specific protein binding even at buried sites. Here we show that this dynamic behaviour provides a mechanism for cooperativity (synergy) in the binding of two or more proteins to sites on a single nucleosome, even if those proteins do not interact directly with each other in any way. As a consequence of this cooperativity, two proteins binding to the same nucleosome facilitate each other's binding and also control the level of occupancy at each other's sites. This model, with no adjustable parameters, accounts quantitatively for recent reports of cooperative (synergistic) binding to nucleosomes in vitro. We assess the potential importance of this new cooperativity for gene regulation in vivoby comparing its magnitude to free energies of cooperative protein – protein direct contacts having known significance for gene regulation. Possible roles for nucleosome dynamics in eukaryotic gene regulation, and key remaining questions, are discussed.

The fusicoccin receptor of plants is a member of the 14-3-3 superfamily of eukaryotic regulatory proteins

The receptor for the wilt-inducing phytotoxin fusicoccin was purified to homogeneity from plasma membranes of Commelina communis as a complex with the radioligand [ 3H]9′-nor-8′-hydroxyfusicoccin. The preparation consisted of two polypeptides with apparent molecular masses of 30.5 kDa and 31.5 kDa and with isoelectric points of around pH 5.2 and 5.3, respectively. The proteins were N-terminally blocked. Internal amino acid sequences were obtained for both polypeptides of the fusicoccin-binding complex. Sequence information, as well as subsequent immunological analysis, proved that both polypeptides are members of the eukaryotic 14-3-3 family, which comprises structurally conserved regulatory proteins of widespread occurrence and a wide range of functions. 14-3-3 isoform(s) constituting the fusicoccin receptor are distinguishable from other cellular 14-3-3 proteins by their tight association with the plasma membrane. Applying temperature-induced Triton X-114 phase separation experiments, they, as well as the target enzyme of fusicoccin action, the H +-ATPase, partitioned into the phospholipid-rich fraction which contains the most hydrophobic proteins. The results discussed herein provide a basis for the elucidation of the molecular mechanism of fusicoccin action.

LexA repressor and iron uptake regulator from Escherichia coli: new members of the CAP-like DNA binding domain superfamily.

Comparison of structures can reveal surprising connections between protein families and provide new insights into the relationship between sequence, structure and function. The solution structure of LexA repressor from Escherichia coli reveals an unexpected structural similarity to a widespread class of prokaryotic and eukaryotic regulatory proteins, which is typified by catabolite gene activator protein (CAP). The use of combined sequence profiles allows the identification of two new prokaryotic members of the superfamily: listeriolysin regulatory protein (PrfA) and ferric uptake regulatory protein (Fur). LexA, PrfA and Fur are the first examples of prokaryotic regulatory proteins in which DNA recognition is mediated by a variant of the classical helix-turn-helix motif, with an insertion in the turn region.

Human cytomegalovirus IE86 protein interacts with promoter-bound TATA-binding protein via a specific region distinct from the autorepression domain.

The major immediate-early gene of human cytomegalovirus encodes several isoforms of an immediate-early protein which has distinct transcriptional regulatory properties. The IE86 isoform autorepresses the major immediate-early promoter by directly binding the cis repression signal element located between the TATA box and the mRNA cap site. In addition to this activity, IE86 stimulates other viral and cellular promoters. One mechanism by which eukaryotic regulatory proteins are thought to stimulate transcription is by contacting one or more general transcription factors. We show that the IE86 protein physically interacts with the DNA-binding subunit (TATA-binding protein) human transcription factor IID via the TATA-binding protein-contacting domain in the N terminus of IE86. In a mobility shift assay, IE86 was also observed to stabilize the binding of TATA-binding protein to promoter DNA. The domains within IE86 responsible for mediating transactivation and repression functioned independently. These experiments thus demonstrate the elegant ability of human cytomegalovirus to join different protein domains to produce distinct multifunctional proteins.

Genesis of eukaryotic transcriptional activator and repressor proteins by splitting a multidomain anabolic enzyme

The genes necessary for the correctly regulated catabolism of quinate in Aspergillus nidulans and Neurospora crassa are controlled at the level of transcription by a DNA-binding activator protein and a repressor protein that directly interact with one another. The repressor protein is homologous throughout its length with the three C-terminal domains of a pentafunctional enzyme catalysing five consecutive steps in the related anabolic shikimate pathway. We now report that the activator protein is homologous to the two N-terminal domains of the same pentafunctional enzyme and that this proposed structural similarity suggests a molecular mechanism by which the repressor recognises the activator protein. We believe that this is the first report of the genesis of a pair of interacting eukaryotic regulatory proteins by the splitting of a multidomain anabolic enzyme. The recruitment of preformed enzymatically active domains to a regulatory role may represent a general mechanism for the evolution of pathway-specific regulator proteins in dispensible pathways.

Genetic evidence that the Tat proteins of human immunodeficiency virus types 1 and 2 can multimerize in the eukaryotic cell nucleus.

The formation of dimers or higher-order multimers is critical to the biological activity of many eukaryotic regulatory proteins. However, biochemical analyses of the multimerization capacity of the Tat trans activator of human immunodeficiency virus types 1 (HIV-1) and 2 (HIV-2) have yielded contradictory results. We used the two-hybrid genetic assay for protein-protein interactions in the eukaryote Saccharomyces cerevisiae (S. Fields and O.-K. Song, Nature [London] 340:245-246, 1989) to examine the multimerization of Tat in vivo. Both HIV-1 and HIV-2 Tat are shown to form specific homo- but not heteromultimers in the yeast cell nucleus. Mutational analysis indicates a critical role for the essential core motif of Tat in mediating this interaction but demonstrates that efficient Tat multimerization does not require an intact cysteine motif. These data raise the possibility that the multimerization of Tat may be important for Tat function in higher eukaryotes.

Ectopic expression of a conditional GATA-2/estrogen receptor chimera arrests erythroid differentiation in a hormone-dependent manner.

The GATA factors are a family of transcriptional regulatory proteins in eukaryotes that share extensive homology in their DNA-binding domains. One enigmatic aspect of GATA factor expression is that several GATA proteins, which ostensibly share the same DNA-binding site specificity, are coexpressed in erythroid cells. To elucidate the roles of individual GATA factors in erythropoiesis, conditional alleles of GATA-1, GATA-2, and GATA-3 were prepared by fusing each of the factors to the hormone-binding domain of the human estrogen receptor (ER). These GATA/ER chimeric factors were shown to be hormone-inducible trans-activating proteins in transient transfection assays. When stably introduced into primary erythroblasts or conditionally transformed erythroid progenitors cells, exogenous GATA-2/ER promoted proliferation and inhibited terminal differentiation in an estrogen-dependent manner. These phenotypic effects are specifically attributable to the action of ectopically expressed GATA-2/ER because erythroblasts expressing exogenous GATA-2 are constitutively arrested in differentiation and because erythroid progenitors expressing either Gal/ER or GATA-3/ER do not display a hormone-responsive block in differentiation. Thus, the GATA-2 transcription factor appears to play a role in regulating the self-renewal capacity of early erythroid progenitor cells.

Structure, function, and regulation of the kilB locus of promiscuous plasmid RK2

The kil-kor regulon of the self-transmissible, broad-host-range plasmid RK2 is a unique network with eight coregulated operons. Among the genes encoded by the kil-kor regulon are trfA, which encodes the replication initiator, and several kil loci (kilA, kilB, kilC, and kilE), each of which is lethal to the host cell in the absence of appropriate negative regulatory elements encoded by the korA, korB, korC, and korE determinants. We have proposed that the functions of the kil loci are related to RK2 maintenance or host range. Here, we report the nucleotide sequence of a 2.44-kb region that includes the lethal kilB determinant. We identified the first three genes of the kilB operon (designated klbA, klbB, and klbC), and we determined by deletion analysis that the host-lethal phenotype requires klbB. The predicted amino acid sequence of the 34,995-Da klbA product reveals a potential ATP-binding fold. The klbB product is predicted to be a membrane protein with a molecular mass of 15,012 Da with homology to the RK2 KlaC membrane protein encoded by the kilA operon. The amino acid sequence of the 12,085-Da klbC product contains a perfect match to the leucine zipper motif common to eukaryotic regulatory proteins. Primer extension analysis revealed unambiguously that transcription of the kilB operon begins 46 nucleotides upstream of klbA. No transcription was initiated from the sequence previously presumed by other investigators to be the kilB promoter. The abundance of kilB transcripts is reduced in the presence of KorB, consistent with the prediction that KorB acts at the level of transcription. A degenerate KorB-binding site that contains a perfect half-palindrome overlaps the kilB promoter, but this site is insufficient for regulation by KorB. The region containing a KorB-binding site located 183 bp upstream of the transcriptional start is required for regulation by KorB, indicating that KorB acts at a distance to regulate transcription of kilB. Our studies with the mutant plasmid pRP101, a transfer-defective derivative of the RK2-like plasmid RP4, demonstrated that the kilB operon includes the conjugal transfer and surface exclusion genes of the Tra2 region. Nucleotide sequence analysis revealed that the transposon Tn7 insertion in pRP101 is located in the klbC gene, and complementation analysis showed that this mutation has a strong polar effect on the expression of genes for conjugal transfer and surface exclusion located several kilobases downstream. A klbA mutant was constructed and found to be both transfer defective and complementable, thus, demonstrating a requirement was constructed and found to be both transfer defective and complementable, thus demonstrating a requirement for klbA product in plasmid transmissibility. These results have demonstrated a role for the kilB operon in conjugal transfer. The kil-kor regulon of RK2 is the only known example of plasmid-mediated coregulation of replication and transfer.

The HMG domain of lymphoid enhancer factor 1 bends DNA and facilitates assembly of functional nucleoprotein structures

The high mobility group (HMG) domain is a DNA-binding motif that is associated with several eukaryotic regulatory proteins, including the lymphoid enhancer-binding factor LEF-1 and the testis-determining factor SRY. Here, we provide evidence that DNA binding by the HMG domain of LEF-1 involves primarily minor groove contacts and induces a bend of approximately 130° in the DNA helix. Bending was also found to accompany sequence-specific DNA binding by the SRY-HMG domain. Examining possible regulatory roles of HMG domain-induced DNA bends, we found that LEF-1 can function in a manner similar to bacterial integration host factor and facilitate communication between widely separated protein-binding sites in a recombination assay. Together with the previous observation that LEF-1 by itself is unable to augment basal promoter activity, these data suggest that HMG domain proteins can serve as “architectural” elements in the assembly of higher-order nucleoprotein structures.

The DNA binding site of HMG1 protein is composed of two similar segments (HMG boxes), both of which have counterparts in other eukaryotic regulatory proteins.

The mammalian nuclear protein HMG1 contains two segments that show a high sequence similarity to each other. Each of the segments, produced separately from the rest of the protein in Escherichia coli, binds to DNA with high specificity: four-way junction DNA of various sequences is bound efficiently, but linear duplex DNA is not. Both isolated segments exists as dimers in solution, as shown by gel filtration and chemical crosslinking experiments. HMG1-like proteins are present in yeast and in protozoa: they consist of a single repetition of a motif extremely similar to the DNA binding segments of HMG1, suggesting that they too might form dimers with structural specificity in DNA binding. Sequences with recognizable similarity to either of the two DNA binding segments of HMG1, called HMG boxes, also occur in a few eukaryotic regulatory proteins. However, these proteins are reported to bind to specific sequences, suggesting that the HMG box of proteins distantly related to HMG1 might differ significantly from the HMG box of HMG1-like proteins.

Molecular biology of the human immunodeficiency virus type 1

The immunodeficiency virus type 1 is a complex retrovirus. In addition to genes that specify the proteins of the virus particle and the replicative enzymes common to all retroviruses, HIV-1 specifies at least six additional proteins that regulate the virus life cycle. Two of these regulatory genes, tat and rev, specify proteins essential for replication. These proteins bind to specific sequences of newly synthesized virus RNA and profoundly affect virus protein expression. Tat and rev appear to be prototypes of novel eukaryotic regulatory proteins. These two genes may play a central role in regulating the rate of virus replication. Three other viral genes, vif, vpu, and vpr, affect the assembly and replication capacity of newly made virus particles. These genes may play a critical role in spread of the virus from tissue to tissue and from person to person. Our understanding of the contribution of each of the virus structural proteins and regulatory genes to the complex life cycle of the virus in natural infections is incomplete. However, enough insight has been gained into the structure and function of each of these components to provide a firm basis for rational antiviral drug development.

The predicted molecular structure suggests that CTF/NF-I may function as a histone acetylase

The primary structure of nuclear factor-I (CTF/NF-I), a eukaryotic regulatory DNA-binding protein involved in both DNA replication and gene transcription, and the secondary structure predictable from it, are compared here with those of a number of prokaryotic acetylases. Hydropathy and Chou-Fasman analyses reveal that the polypeptide chain of CTF/NF-I is likely to fold to higher order structures similar to those of the acetylases, and significant conservation of functionally important regions of the acetylases is observed in CTF/NF-I. It is therefore suggested that CTF/NF-I may function as a histone acetylase.

Localization of the human HF.10 finger gene on a chromosome region (3p21?22) frequently deleted in human cancers

The finger motif is a tandemly repeated DNA-binding domain recently identified in the primary structure of several eukaryotic transcriptional regulatory proteins. It has been proposed that some members of the finger-gene family are implicated in both normal cell proliferation and differentiation. We isolated several human finger genes by means of hybridization with a finger motif-containing DNA probe. One of these finger genes, HF.10, is expressed at low levels in a variety of human tissues and is down-regulated during the in vitro terminal differentiation of human leukemic myeloid cell lines. By in situ hybridization experiments and analysis of interspecific somatic cell hybrids we mapped the HF.10 gene to 3p21-22, a chromosome region frequently involved in karyotypic rearrangements associated with lung and renal cancer.

Possible finger structure in gene A protein of Microviridae

Microvirid phages α3 and φK encode for A protein which functions in initiation of the viral DNA synthesis. By nucleotide sequencing analysis, we have found that each gene A protein has ‘finger motif structure’ which conserves two cysteine and histidine residues similar to the consensus sequence deduced from more than thirty finger motifs reported from many eukaryotic regulatory proteins. In closely related phages, φX174 and G4, we have detected the same structures in their gene A proteins also.

Eukaryotic transcriptional regulatory proteins.

Eukaryotic transcriptional regulatory proteins.

Eukaryotic Transcriptional Regulatory Proteins

Eukaryotic Transcriptional Regulatory Proteins