The human polo-like kinase, PLK, regulates cdc2/cyclin B through phosphorylation and activation of the cdc25C phosphatase.

Entry into mitosis by mammalian cells is triggered by the activation of the cdc2/cyclin B holoenzyme. This is accomplished by the specific dephosphorylation of key residues by the cdc25C phosphatase. The polo-like kinases are a family of serine/threonine kinases which are also implicated in the control of mitotic events, but their exact regulatory mechanism is not known. Recently, a Xenopus homologue, PLX1, was reported to phosphorylate and activate cdc25, leading to activation of cdc2/cyclin B. Jurkat T leukemia cells were chemically arrested and used to verify that PLK protein expression and its phosphorylation state is regulated with respect to cell cycle phase (i.e., protein is undetectable at G1/S, accumulates at S phase and is modified at G2/M). Herein, we show for the first time that endogenous human PLK protein immunoprecipitated from the G2/M-arrested Jurkat cells directly phosphorylates human cdc25C. In addition, we demonstrate that recombinant human (rh) PLK also phosphorylates rhcdc25C in a time- and concentration-dependent manner. Phosphorylation of endogenous cdc25C and recombinant cdc25C by PLK resulted in the activation of the phosphatase as assessed by dephosphorylation of cdc2/cyclin B. These data are the first to demonstrate that human PLK is capable of phosphorylating and positively regulating human cdc25C activity, allowing cdc25C to dephosphorylate inactive cdc2/cyclin B. As this event is required for cell cycle progression, we define at least one key regulatory mode of action for human PLK in the initiation of mitosis.

Cloning and functional characterization of a novel human CC chemokine that binds to the CCR3 receptor and activates human eosinophils.

Eotaxin has been found to bind exclusively to a single chemokine receptor, CCR3. Using expression sequence tag screening of an activated monocyte library, a second chemokine has been identified; it was expressed and purified from a Drosophila cell culture system and appears to only activate CCR3. Eotaxin-2, MPIF-2, or CKbeta-6, is a human CC chemokine with low amino acid sequence identity to other chemokines. Eotaxin-2 promotes chemotaxis and Ca2+ mobilization in human eosinophils but not in neutrophils or monocytes. Cross-desensitization calcium mobilization experiments using purified eosinophils indicate that eotaxin and MCP-4, but not RANTES, MIP-1alpha, or MCP-3, can completely cross-desensitize the calcium response to eotaxin-2 on these cells, indicating that eotaxin-2 shares the same receptor used by eotaxin and MCP-4. Eotaxin-2 was the most potent eosinophil chemoattractant of all the chemokines tested. Eotaxin-2 also displaced 125I-eotaxin bound to the cloned CCR3 stably expressed in CHO cells (CHO-CCR3) and to freshly isolated human eosinophils with affinities similar to eotaxin and MCP-4. 125I-Eotaxin-2 binds with high affinity to eosinophils and both eotaxin and cold eotaxin-2 displace the ligand with equal affinity. Eotaxin and eotaxin-2 promote a Ca2+ transient in RBL-2H3 cells stably transfected with CCR3 (RBL-2H3-CCR3) and both ligands cross-desensitized the response of the other but not the response to LTD4. The data indicate that eotaxin-2 is a potent eosinophil chemotactic chemokine exerting its activity solely through the CCR3 receptor.

Characterization of Streptomyces promoter sequences using the Escherichia coli galactokinase gene.

A gene fusion system that uses the Escherichia coli galK gene has been developed to characterize Streptomyces transcriptional regulatory sequences. The system consists of galK-deficient Streptomyces lividans mutants and plasmids containing the E. coli galK gene with its natural ribosome-binding site and sites upstream of galK for insertion of transcription signals. Expression of the E. coli galK gene in S. lividans can be quantitated by either an enzymatic or immunoblot assay or detected by genetic complementation of an S. lividans galK- mutant. The utility of the plasmid to select, detect and assess promoter function was examined using the S. lividans XP55 and S. fradiae aph gene promoters. The potential use of the galK fusion system to isolate and characterize Streptomyces transcription signals is discussed.

Soluble forms of the human T cell receptor CD4 are efficiently expressed by Streptomyces lividans.

We have developed a new gene expression and secretion system for Streptomyces lividans and used it to produce soluble forms of a human T-cell receptor CD4 at levels greater than 300 mg/l. The system uses the transcription, translation and secretion signals of the serine protease inhibitor gene STI-II which is naturally produced by S. longisporus. Using these signals, soluble derivatives of CD4 were secreted directly into the culture supernatant as correctly processed soluble, biologically active proteins. High level expression of the CD4 proteins depended on the transcription initiation signal, the amino acid sequence surrounding the signal peptide cleavage site and temporally controlled protease activities. We discuss these results in the context of the potential of this system for producing other eukaryotic proteins in Streptomyces.

Native complex formation between apolipoprotein E isoforms and the Alzheimer's disease peptide A beta.

To explore whether the genetic linkage between apolipoprotein E (ApoE) alleles and susceptibility to Alzheimer's disease might be attributable to a direct molecular interaction between ApoE and the amyloid peptide A beta, we have produced ApoE variants in Escherichia coli and studied their interactions with A beta under native conditions. When incubated with A beta at 20-40 microM concentrations, all three isoforms of ApoE (2, 3, and 4) readily form complexes with A beta which can be isolated by gel filtration in native buffer. Freshly mixed ApoE and A beta generate a complex that co-migrates in gel filtration with the main A280 peak, which migrates identically to the ApoE tetramer alone. After several hours incubation, an additional, high molecular weight, soluble aggregate appears which also contains both ApoE and A beta. Neither ApoE nor A beta incubated by themselves produces high molecular weight aggregates under these conditions. Incubation of A beta with control proteins bovine serum albumin and immunoglobulin generates negligable binding in the gel filtration assay. Similar results were obtained whether A beta (1-40) or A beta (1-42) was used, and plasma-derived ApoE gave similar results to E. coli-produced material. The data are consistent with a role for ApoE-A beta interactions in modulating the development of AD. Since no major differences were observed in the behavior of the three ApoE isotypes, however, the molecular basis of the genetic trend between ApoE alleles and AD cannot be attributed to specific activity differences between the molecular forms of ApoE characterized in this study.

Gene organization and structure of the Streptomyces lividans gal operon.

We present the gene organization and DNA sequence of the Streptomyces lividans galactose utilization genes. Complementation of Escherichia coli galE, galT, or galK mutants and DNA sequence analysis were used to demonstrate that the galactose utilization genes are organized within an operon with the gene order galT, galE, and galK. Comparison of the inferred protein sequences for the S. lividans gal gene products to the corresponding E. coli and Saccharomyces carlbergensis sequences identified regions of structural homology within each of the galactose utilization enzymes. Finally, we discuss a potential relationship between the gene organization of the operon and the functional roles of the gal enzymes in cellular metabolism.

Two promoters, one inducible and one constitutive, control transcription of the Streptomyces lividans galactose operon.

Galactose utilization in Streptomyces lividans was shown to be controlled by an operon that is induced in the presence of galactose and repressed by glucose. Two promoters, galP1 and galP2, which direct transcription of two distinct polycistronic transcripts, have been identified. galP1 is located immediately upstream of the operon and is induced in the presence of galactose. This promoter directs transcription of the galT, galE, and galK genes. The second promoter, galP2, is located within the operon just upstream of the galE gene. This promoter is responsible for constitutive transcription of the galE and galK genes. Comparison of the S. lividans gal operon to the Escherichia coli gal operon indicates the presence of a constitutive promoter positioned upstream of galE in both operons. We suggest that coupling the operon's constitutive promoter to the galE gene fulfills a physiological requirement for constitutive UDPgalactose 4-epimerase expression in Streptomyces.

Molecular cloning of developmentally regulated, low-abundance mRNA sequences from embryonic muscle.

To study the role of low-abundance, embryonic muscle-specific gene transcripts, we have developed a method to screen cDNA clones from embryonic muscle for such sequences. The protocol involves two stages: first, partial enrichment for cDNA clones carrying possible embryo-specific sequences by selecting clones of low-abundance sequences; and second, determination, by hybridization to RNA attached to diazobenzyloxymethyl-paper, which sequences from this category are regulated in an embryonic muscle-specific manner during development. At least three different clones were obtained which hybridized to sequences present in early muscle development but absent, or present at relatively low levels, at late embryonic and adult muscle stages. Two of these clones were not muscle-specific because they hybridized to poly(A)+RNA from liver or brain or both. The third clone, 106A4, did not detectably hybridize to total poly(A)+RNA at any stage of brain or liver development tested. This sequence also was not detectable in poly(A)+RNA from embryonic muscle progenitor cells. Thus, the 106A4 sequence is a likely candidate for an embryonic muscle-specific sequence. We have demonstrated that the 106A4 sequence is a mRNA, although the specific identity and function of the translated product is unknown. The method used to identify embryonic muscle-specific cDNA clones should be generally applicable for obtaining clones for low abundance transcripts regulated in a tissue-specific or developmental stage-specific manner.

Structure and developmental expression of the chick alpha-actin gene.

Recombinant DNA clones containing chick alpha-actin mRNA sequence have been isolated and used as probes to analyze the structure and developmental expression of the chick alpha-actin gene. The full length, 2000 nucleotide alpha-actin mRNA is detected in poly(A) RNA at early and late stages of in vivo leg muscle development. As expected, the alpha-actin mRNA is present at very low levels at early myogenic stages but is a high abundance species in terminally differentiated muscle. However, most of the alpha-actin mRNA from fused leg muscle is shorter than 2000 nucleotides, and occurs in relatively discrete size classes. An alpha-actin-like mRNA can be detected in poly(A) RNA from early embryonic brain, indicating that transcription of the alpha-actin gene may not be strictly muscle-specific at all stages of development. We have identified at least 3, very short (< 100 base pairs) intervening sequences in the alpha-actin gene which was isolated from a chick genomic library. The structure of the chick alpha-actin gene differs, therefore, from the structures of actin genes from yeast and Drosophila, both of which contain a single, relatively long, intervening sequence.

The complete nucleotide sequence of the chick a-actin gene and its evolutionary relationship to the actin gene family.

The nucleotide sequence of the chick a-actin gene reveals that the gene is comprised of 7 exons separated by six very short intervening sequences (IVS). The first IVS interrupts the 73 nucleotide 5' untranslated segment between nucleotides 61 and 62. The remaining IVS interrupt the translated region at codons 41/42, 150, 204, 267, and 327/328. The 272 nucleotide 3' untranslated segment is not interrupted by IVS. The amino acid sequence derived from the nucleotide sequence is identical to the published sequence for chick a-actin except for the presence of a met-cys dipeptide at the amino-terminus. The IVS positions in the chick a-actin gene are identical to those of the rat a-actin gene. While there is partial coincidence of the IVS in the a-actin genes with the vertebrate b-actin genes and 2 sea urchin actin genes, there is no coincidence with actin genes from any other source except soybean where one IVS position is shared. This discordance in IVS positions makes the actin gene family unique among the eucaryotic genes analyzed to date.

Two novel Streptomyces protein protease inhibitors. Purification, activity, cloning, and expression.

In contrast to the Gram-negative bacteria, Gram-positive bacteria such as Streptomyces lack a mucopolysaccharide cell wall which allows them to produce and secrete a variety of proteins directly into their environment. In an effort to understand and eventually exploit the synthesis and secretion of proteins by Streptomyces, we identified and characterized two naturally occurring abundantly produced proteins in culture supernatants of Streptomyces lividans and Streptomyces longisporus. We purified these 10-kDa proteins and obtained partial amino acid sequence information which was then used to design oligonucleotide probes in order to clone their genes. Analysis of the sequence data indicated that these proteins were related to each other and to several other previously characterized Streptomyces protein protease inhibitors. We demonstrate that both proteins are protein protease inhibitors with specificity for trypsin-like enzymes. The presumptive signal peptidase cleavage sites and subsequent aminopeptidase products of each protein are characterized. Finally, we show that the cloned genes contain all of the information necessary to direct synthesis and secretion of the proteins by Streptomyces spp. or Escherichia coli.

Identification of the residues in human CD4 critical for the binding of HIV.

The CD4 molecule is a T cell surface glycoprotein that interacts with high affinity with the envelope glycoprotein of the human immunodeficiency virus, HIV, thus serving as a cellular receptor for this virus. To define the sites on CD4 essential for binding to gp120, we produced several truncated, soluble derivatives of CD4 and a series of 26 substitution mutants. Quantitative binding analyses with the truncated proteins demonstrate that the determinants for high affinity binding lie solely with the first 106 amino acids of CD4 (the V1 domain), a region having significant sequence homology to immunoglobulin variable regions. Analysis of the substitution mutants further defines a discrete binding site within this domain that overlaps a region structurally homologous to the second complementarity-determining region of antibody variable domains. Finally, we demonstrate that the inhibition of virus infection and virus-mediated cell fusion by soluble CD4 proteins depends on their association with gp120 at this binding site.

Identification of a truncated form of the CC chemokine CK beta-8 demonstrating greatly enhanced biological activity.

A new CC chemokine, designated CKbeta-8 or myeloid progenitor inhibitor factor-1, was recently identified in a large scale sequencing effort and was cloned from a human aortic endothelial library. CKbeta-8 cDNA encodes a signal sequence of 21 amino acids, followed by a 99-amino acid predicted mature form. CKbeta-8 was expressed and purified from a baculovirus insect cell expression system, which resulted in the identification of different N-terminal variants of the secreted chemokine. The three major forms (containing amino acids 1-99, 24-99, and 25-99 of the secreted chemokine) showed a large variation in potency. CKbeta-8 activated both monocytes and eosinophils to mobilize intracellular calcium; however, the shortest form of CKbeta-8 (25-99) was >2 orders of magnitude more potent than the longest form. Cross-desensitization experiments in both monocytes and eosinophils suggested that the CCR1 receptor was probably the predominant receptor that mediates this chemokine's physiologic response. However, incomplete desensitization was encountered in both cell systems, suggesting involvement of an additional receptor(s). Interestingly, the short form of CKbeta-8 was the most potent chemotactic chemokine that we have ever evaluated in the monocyte system (EC50 = 54 pM). However, in contrast to its action on monocytes, CKbeta-8 was a very poor chemotactic factor for eosinophils.

Inhibition of transforming growth factor (TGF)-beta1-induced extracellular matrix with a novel inhibitor of the TGF-beta type I receptor kinase activity: SB-431542.

Transforming growth factor beta1 (TGF-beta1) is a potent fibrotic factor responsible for the synthesis of extracellular matrix. TGF-beta1 acts through the TGF-beta type I and type II receptors to activate intracellular mediators, such as Smad proteins, the p38 mitogen-activated protein kinase (MAPK), and the extracellular signal-regulated kinase pathway. We expressed the kinase domain of the TGF-beta type I receptor [activin receptor-like kinase (ALK)5] and the substrate, Smad3, and determined that SB-431542 is a selective inhibitor of Smad3 phosphorylation with an IC50 of 94 nM. It inhibited TGF-beta1-induced nuclear Smad3 localization. The p38 mitogen-activated protein kinase inhibitors SB-203580 and SB-202190 also inhibit phosphorylation of Smad3 by ALK5 with IC50 values of 6 and 3 microM, respectively. This suggests that these p38 MAPK inhibitors must be used at concentrations of less than 10 microM to selectively address p38 MAPK mechanisms. However, the p38 MAPK inhibitor SB-242235 did not inhibit ALK5. To evaluate the relative contribution of Smad signaling and p38 MAPK signaling in TGF-beta1-induced matrix production, the effect of SB-431542 was compared with that of SB-242235 in renal epithelial carcinoma A498 cells. All compounds inhibited TGF-beta1-induced fibronectin (FN) mRNA, indicating that FN synthesis is mediated in part via the p38 MAPK pathway. In contrast, SB-431542, but not the selective p38 MAPK inhibitor SB-242235, inhibited TGF-beta1-induced collagen Ialpha1 (col Ialpha1). These data indicate that some matrix markers that are stimulated by TGF-beta1 are mediated via the p38 MAPK pathway (i.e., FN), whereas others seem to be activated via ALK5 signaling independent of the p38 MAPK pathway (i.e., col Ialpha1).

Identification of potent, selective non-peptide CC chemokine receptor-3 antagonist that inhibits eotaxin-, eotaxin-2-, and monocyte chemotactic protein-4-induced eosinophil migration.

Eosinophils have been implicated in the pathogenesis of asthma and other allergic diseases. Several CC chemokines including eotaxin (CCL-11), eotaxin-2 (CCL-24), RANTES (CCL-5), and monocyte chemotactic protein-3 (MCP-3, CCL-7) and 4 (MCP-4, CCL-13) are potent eosinophil chemotactic and activating peptides acting through CC chemokine receptor-3 (CCR3). Thus, antagonism of CCR3 could have a therapeutic role in asthma and other eosinophil-mediated diseases. A high throughput, cellular functional screen was configured using RBL-2H3 cells stably expressing CCR3 (RBL-2H3-CCR3) to identify non-peptide receptor antagonists. A small molecule CCR3 antagonist was identified, SK&F 45523, and chemical optimization led to the generation of a number of highly potent, selective CCR3 antagonists including SB-297006 and SB-328437. These compounds were further characterized in vitro and demonstrated high affinity, competitive inhibition of (125)I-eotaxin and (125)I-MCP-4 binding to human eosinophils. The compounds were potent inhibitors of eotaxin- and MCP-4-induced Ca(2+) mobilization in RBL-2H3-CCR3 cells and eosinophils. Additionally, SB-328437 inhibited eosinophil chemotaxis induced by three ligands that activate CCR3 with similar potencies. Selectivity was affirmed using a panel of 10 seven-transmembrane receptors. This is the first description of a non-peptide CCR3 antagonist, which should be useful in further elucidating the pathophysiological role of CCR3 in allergic inflammatory diseases.