Mouse Erk-1 gene product is a serine/threonine protein kinase that has the potential to phosphorylate tyrosine.

Bacterial expression of mouse gene Erk-1 yielded an active kinase with the same substrate specificity shown for ERK1 protein purified from rat cells. Although rat gene ERK1 is believed to encode a serine/threonine kinase based on sequence data and known ERK1 substrate phosphorylation sites, bacterially-produced mouse Erk-1 (bt-Erk-1) autophosphorylated on tyrosine in addition to serine and threonine residues. The bt-Erk-1 protein also had the capacity to reactivate the ribosomal protein S6 kinase (S6KII). Furthermore, treatment of bt-Erk-1 with either serine/threonine-specific phosphatase 2A or tyrosine-specific phosphatase 1B significantly decreased its kinase activity. These findings predict that autophosphorylation may play an important role in Erk-1/ERK1 regulation.     

Expression of a murine homologue of the inhibitor of apoptosis protein is related to cell proliferation

The inhibitor of apoptosis (IAP) proteins form a highly conserved gene family that prevents cell death in response to a variety of stimuli. Herein we describe a newly defined murine IAP, designated Tiap, that proved to be a murine homologue of human survivin based on sequence comparison. TIAP has one baculovirus IAP repeat and lacks a C-terminal RING finger motif. TIAP interacted with the processed form of caspase 3 and inhibited caspase-induced cell death. Histological examinations revealed that TIAP is expressed in growing tissues such as thymus, testis, and intestine of adult mice and many tissues of embryos. In in vitro studies, TIAP was induced in splenic T cells activated with anti-CD3 antibody or Con A, and the expression of TIAP was up-regulated in synchronized NIH 3T3 cells at S to G₂/M phase of the cell cycle. We propose that during cell proliferation, cellular protective activity may be augmented with inducible IAPs such as TIAP.     

Identification and cloning of a protein kinase-encoding mouse gene, Plk, related to the polo gene of Drosophila.

We have determined the nucleotide sequence of a cDNA encoding a protein kinase that is closely related to the enzyme encoded by the Drosophila melanogaster mutant polo and that we have designated Plk (polo-like kinase). Plk is also related to the products of the Saccharomyces cerevisiae cell cycle gene MSD2 (CDC5) and the recently described early growth response gene Snk. Together, Plk, polo, Snk, and MSD2 define a subfamily of serine/threonine protein kinases. Plk is expressed at high levels in a number of fetal and newborn mouse tissues but is not expressed in the corresponding adult organs. With the exception of adult hemopoietic tissues, the only adult tissues in which we could detect Plk expression were ovaries and testes. Taken together, the patterns of Plk expression suggest an association with proliferating cells. Since polo is required for mitosis in Drosophila it is possible that Plk is involved in some aspect of cell cycle regulation in mammalian cells.     

AKT2, a putative oncogene encoding a member of a subfamily of protein-serine/threonine kinases, is amplified in human ovarian carcinomas.

We isolated cDNA clones containing the entire coding region of the putative oncogene AKT2. Sequence analysis and in vitro translation demonstrated that AKT2 encodes a 56-kDa protein with homology to serine/threonine kinases; moreover, this protein contains a Src homology 2-like domain. AKT2 was shown to be amplified and overexpressed in 2 of 8 ovarian carcinoma cell lines and 2 of 15 primary ovarian tumors. AKT2 was mapped to chromosome region 19q13.1-q13.2 by fluorescence in situ hybridization. In the two ovarian carcinoma cell lines exhibiting amplification of AKT2, the amplified sequences were localized within homogeneously staining regions. We conclude that AKT2 belongs to a distinct subfamily of protein-serine/threonine kinases containing Src homology 2-like domains and that alterations of AKT2 may contribute to the pathogenesis of ovarian carcinomas.     

TTYH2, a human homologue of the Drosophila melanogaster gene tweety, is up-regulated in colon carcinoma and involved in cell proliferation and cell aggregation

AIM: To investigate the expression patterns of TTYH2 in the human colon cancer and colon cancer cell lines and to evaluate the inhibitory effect of small interfering RNA (siRNA) on the expression of TTYH2 in colon cancer cell lines.METHODS: We investigated the expression patterns of TTYH2 in colon cancer, adjacent non-tumorous colon mucosa, and cancer cell lines (DLD-1, caco-2, and Lovo) by RT-PCR. Furthermore, a siRNA plasmid expression vector against TTYH2 was constructed and transfected into DLD-1 and Caco-2 with LipofectamineTM 2000. The down regulation of TTYH2 expression was detected by RT-PCR and the role of siRNA in inducing cell proliferation and cell aggregation was evaluated by MTT and aggregation assay.RESULTS: TTYH2 gene expression in colon cancer tissue was significantly up-regulated compared with normal colonic mucosa (1.23 ± 0.404 vs 0.655 ± 0.373, P = 0.0103). Colon cancer derived cell lines including DLD-1, Caco-2, and Lovo also expressed high levels of TTYH2. In contrast, transfection with siRNA-TTYH2 signifi cantly inhibited both proliferation and scattering of these cancer cell lines.CONCLUSION: The present work demonstrates, for the fi rst time, that the TTYH2 gene expression is signifi cantly up-regulated in colon cancer. The TTYH2 gene may play an important role in regulating both proliferating andmetastatic potentials of colorectal cancer.     

Evidence that a 90-kDa phosphoprotein, an associated kinase, and a specific phosphatase are involved in the regulation of Cloudman melanoma cell proliferation by insulin.

Proliferation of wild-type Cloudman S91 melanoma cells is inhibited when insulin is included in the culture medium. Using growth inhibition as a selective marker, we isolated variant cell lines that are either resistant to insulin or dependent upon insulin for growth. We have studied the effects of insulin on proliferation by using combined genetic and biochemical approaches. Through a series of genetic hybridization analyses, we have identified three complementation groups and determined that, in general, insulin-sensitivity is dominant to insulin-resistance. Through analyses of in vitro protein phosphorylation reactions, we have identified a protein of approximately 90 kDa (pp90) whose phosphorylation is a function of at least one of the complementation groups. Although pp90 is not phosphorylated in extracts of insulin-resistant variants, it is phosphorylated in extracts of insulin-sensitive hybrids formed between complementing resistant variants. Insulin itself exhibits little or no regulation over the phosphorylation of pp90; rather, the ability to phosphorylate pp90 correlates with the ability of cells to respond to insulin. Migration in NaDodSO4/polyacrylamide gels, solubility characteristics, and divalent cation requirements indicate that pp90 is distinct from the 95-kDa beta-subunit of the insulin receptor. Both pp90 and its associated phosphoprotein kinase are found in 30,000 X g pellets of sonicated cell lysates, whereas a specific pp90 phosphoprotein phosphatase activity is found in 30,000 X g supernatant fractions. Phosphorylation of pp90 occurs at tyrosine and serine residues. Our evidence indicates that the state of phosphorylation of pp90 is an important determinant in the regulation of cellular proliferation by insulin.     

Purification of a murine protein-tyrosine/threonine kinase that phosphorylates and activates the Erk-1 gene product: relationship to the fission yeast byr1 gene product.

We report the purification to near homogeneity of a 45-kDa phorbol ester-stimulated protein kinase that phosphorylates and activates the Erk-1 gene product. This kinase, which we provisionally denote MEK for MAPK/Erk kinase, phosphorylated kinase-inactive Erk-1 protein primarily on a tyrosine residue and, to a lesser extent, on a threonine. We extend our previous results and show that two forms of purified MEK activated the myelin basic protein kinase encoded by Erk-1. MEK was inactivated by the serine/threonine phosphatase 2A but not by the protein-tyrosine phosphatase 1B. Sequence analysis of peptides generated by trypsin digestion of MEK revealed similarity to the proteins encoded by the Schizosaccharomyces pombe byr1 and Saccharomyces cerevisiae STE7 genes. These data are discussed with regard to a possible signal transduction mechanism.     

Human cell surface glycoprotein related to cell proliferation is the receptor for transferrin.

A cell surface glycoprotein antigen with an apparent molecular weight of about 100,000 that is selectively expressed on proliferating cells was purified from deoxycholate-solubilized membranes of a cultured human leukemic thymus-derived (T) cell line by affinity chromatography on a monoclonal antibody-Sepharose column. A conventional xenoantiserum prepared by immunization with the affinity-purified glycoprotein was found to contain antibodies against a serum component that bound tightly to cultured cells. This molecule was shown to be specifically associated with the cell surface glycoprotein purified by immunoprecipitation from lysates of cells. We have identified the serum component as transferrin and conclude that the membrane glycoprotein is the cell surface transferrin receptor.     

Identification and characterization of a membrane-bound cytotoxin of murine cytolytic lymphocytes that is related to tumor necrosis factor/cachectin.

Cytotoxic T lymphocytes (CTLs) kill their targets by a contact-dependent mechanism. We investigated the possibility that the CTL membranes themselves could exert direct cytotoxic activity. Murine CTLs that had been fixed with paraformaldehyde retained a slow cytotoxic activity toward various target cells that are also sensitive to another cytokine, tumor necrosis factor (TNF)/cachectin. This cytotoxic activity was neutralized by antibodies specific for TNF. Membrane fractions obtained from CTLs were cytotoxic to TNF-sensitive targets but not to several TNF-resistant cell lines. Immunoblot analysis revealed a membrane protein band of 50-60 kDa from CTLs that reacts with anti-TNF antibodies. The surface localization of this cytokine was further ascertained by flow cytometry, indirect immunofluorescence, and immunoelectron microscopy studies using TNF-specific antibodies. Radioiodination of CTL surface proteins followed by immunoprecipitation with anti-TNF antibodies confirmed the presence of a TNF-related cytokine in the plasma membranes of CTLs that migrated with an apparent molecular mass of 50-60 kDa under disulfide-reducing conditions. This cytokine can be removed from membranes by treatment with detergents but not with high-salt buffers, suggesting that it may be an integral membrane protein.     

Phorbol ester stimulates a protein-tyrosine/threonine kinase that phosphorylates and activates the Erk-1 gene product.

The regulation of the Erk (extracellular-signal-regulated kinase) gene-encoded protein kinase activity by reversible phosphorylation has been reported to involve either an activator of autophosphorylation or an upstream protein kinase. In this communication we describe assays utilizing the Erk-1 protein fused to glutathione S-transferase that permit the identification of protein kinase(s) that phosphorylate and activate the myelin basic protein kinase activity encoded by the Erk-1 gene. A phorbol ester-stimulated protein kinase activity was identified that phosphorylated a kinase-negative Erk-1 gene product on tyrosine and threonine. The protein kinase phosphorylated and activated wild-type protein expressed in bacteria from 20- to 50-fold. The activation of the Erk-1-encoded myelin basic protein kinase required ATP and correlated directly with the degree of phosphorylation on the same amino acid residues previously shown to be phosphorylated in vivo. Conversion of the tyrosine site of phosphorylation to phenylalanine yielded an Erk-1 gene product that could not be activated. Similar results were obtained when the threonine site was mutated to valine. It is likely that the phorbol ester-stimulated protein-tyrosine/threonine kinase(s) is an up-stream target for multiple extracellular signals.     

Molecular cloning of a Drosophila diacylglycerol kinase gene that is expressed in the nervous system and muscle.

We have isolated a Drosophila melanogaster diacylglycerol kinase (DGK, EC 2.7.1.107) homologue by using a porcine DGK cDNA probe and we have characterized its structure and expression. The DGK cDNA has a single open reading frame that encodes 791 amino acids. The Drosophila and porcine DGKs share a similar carboxyl-terminal region, a putative catalytic domain, which is divided into two separate domains in Drosophila. The DGK gene was mapped to the cytogenetic position 43F1, and its DGK mRNA is abundant both in embryo and in adult fly. By in situ hybridization to sections of adult flies, we demonstrated that the mRNA is present predominantly in the nervous system and muscles, including compound eyes, brain cortex, fibrillar muscle, and tubular muscle. In a 10- to 11-hr embryo, the DGK gene is expressed abundantly in a limited number of cells in the procephalic region and in the ventral nerve cord. The pattern of temporal and spatial expression suggests that the DGK protein has an important function in the adult nervous system and muscle and during the development of the embryonic nervous system.     

A type 1 serine/threonine kinase receptor that can dorsalize mesoderm in Xenopus.

We have cloned a type I serine/threonine kinase receptor, XTrR-I, from Xenopus. XTrR-I (Xenopus transforming growth factor beta-related receptor type I) is expressed in all regions of embryos throughout early development. Overexpression of this receptor does not affect ectoderm or endoderm but dorsalizes the mesoderm such that muscle appears in ventral mesoderm and notochord appears in lateral mesoderm normally fated to become muscle. In addition, overexpression of XTrR-I in UV-treated embryos is able to cause formation of a partial dorsal axis. These results suggest that XTrR-I encodes a receptor which responds in normal development to a transforming growth factor beta-like ligand so as to promote dorsalization. Its function would therefore be to direct mesodermalized tissue into muscle or notochord.     

The rate of proliferation among African trypanosomes is a stable trait that is directly related to virulence

The relationship between the growth rate of Trypanosoma brucei gambiense and its virulence was investigated. A cloned, monomorphic, slow growing, and relatively avirulent line of T. b. gambiense was serially passaged at 3- to 5-day intervals through immunosuppressed mice. The growth rate measured within the first 2 patent days of infection did not vary significantly through the first 25 passages but by passage 50 had decreased significantly from 11.9 +/- 1.1 hr to 9.0 +/- 0.7 hr. A clone from passage 50 and three different second peak heterologous variants all had statistically similar growth rates, indicating that the rate of proliferation was a stable trait. With the faster rate of proliferation there was a corresponding increase in virulence. The inoculum necessary to kill 50% of normal outbred mice in the first peak of parasitemia (LD50) dropped significantly from 3 X 10(6) first passage parasites to 4 X 10(5) passage 50 parasites. The lethal load for both fast and slow growing organisms was the same (greater than 2 X 10(9) trypanosomes/ml of blood). To further link virulence and growth rate, a strong correlation (r = 0.89) was measured when generation times of 10 closely related lines of T. b. gambiense, and 2 lines of pleomorphic T. b. rhodesiense were compared to their LD50 values. While the rate of trypanosomal proliferation was similar between the day of inoculation through the second patent day, it slowed to 64% of that level once parasitemias exceeded 3 X 10(8) organisms/ml of host blood.(ABSTRACT TRUNCATED AT 250 WORDS)     

COS1, a two-component histidine kinase that is involved in hyphal development in the opportunistic pathogen Candida albicans

Two-component histidine kinases recently have been found in eukaryotic organisms including fungi, slime molds, and plants. We describe the identification of a gene, COS1, from the opportunistic pathogen Candida albicans by using a PCR-based screening strategy. The sequence of COS1 indicates that it encodes a homolog of the histidine kinase Nik-1 from the filamentous fungus Neurospora crassa. COS1 is also identical to a gene called CaNIK1 identified in C. albicans by low stringency hybridization using CaSLN1 as a probe [Nagahashi, S., Mio, T., Yamada-Okabe, T., Arisawa, M., Bussey, H. & Yamada-Okabe, H. (1998) Microbiol. 44, 425–432]. We assess the function of COS1/CaNIK1 by constructing a diploid deletion mutant. Mutants lacking both copies of COS1 appear normal when grown as yeast cells; however, they exhibit defective hyphal formation when placed on solid agar media, either in response to nutrient deprivation or serum. In constrast to the Δnik-1 mutant, the Δcos1/Δcos1 mutant does not demonstrate deleterious effects when grown in media of high osmolarity; however both Δnik-1 and Δcos1/Δcos1 mutants show defective hyphal formation. Thus, as predicted for Nik-1, Cos1p may be involved in some aspect of hyphal morphogenesis and may play a role in virulence properties of the organism.     

Extracellular signal-regulated protein kinase, but not c-Jun N-terminal kinase, is activated by type II gonadotropin-releasing hormone involved in the inhibition of ovarian cancer cell proliferation

Although a novel second form of GnRH (GnRH-II) has been reported to have an antiproliferative effect on gynecologic cancer cells, its biological mechanism remains to be elucidated. We have previously demonstrated that GnRH-II activates p38 MAPK. There is accumulating evidence that activation of MAPKs by GnRH-I and -II is important for cell proliferation, differentiation, and apoptosis. In the present study, we further investigated the involvement of GnRH-II in the inhibition of cell proliferation and activation of ERK1/2 and c-Jun N-terminal protein kinase/stress-activated protein kinase (JNK/SAPK) in ovarian cancer cells, OVCAR-3. The [(3)H]thymidine incorporation and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays revealed that treatment with GnRH-II suppresses cell proliferation of ovarian cancer cells. Western blot analysis demonstrated that ERK1/2 was activated by GnRH-II (100 nm). Moreover, PD98059 (10 mum), an inhibitor of a MAPK/ERK kinase, reversed the activation of ERK1/2 induced by GnRH-II. The activation of ERK1/2 by GnRH-II subsequently phosphorylated Elk-1 as a downstream pathway, which was blocked by PD98059. On the other hand, it is not likely that GnRH-II activates the JNK/SAPK pathway. Taken together, these results indicate that the ERK1/2 pathway is involved in the effect of GnRH-II on antiproliferation and may be an important target for ovarian cancer therapy.     

Evidence that stem cell factor is involved in the rebound thrombocytosis that follows 5-fluorouracil treatment.

The mechanisms responsible for 5-fluorouracil (5FU)-induced rebound thrombocytosis are not completely understood. SI/SI(d) mice, which do not undergo rebound thrombocytosis in response to 5FU, provide a genetic approach to the study of this phenomenon. Recent reports by several groups that the SI locus encodes a protein known variably as stem cell factor (SCF), mast cell growth factor, or kit ligand, suggests the possibility that the lack of wild-type SCF in SI/SI(d) mice is responsible for their defective response to 5FU-induced thrombocytopenia. It is shown in this report that SCF-treated SI/SI(d) mice are as capable as their wild-type littermates in undergoing rebound thrombocytosis. W/Wv mice, mutated at the locus encoding the SCF receptor, also do not undergo rebound thrombocytosis, but are not responsive to SCF treatment. In normal mice, it is shown by RNA solution hybridization that SCF mRNA expression is increased during the 5FU-induced platelet nadir period. It is also shown by autoradiography that maturing megakaryocytes express SCF receptors, and that in vivo administration of SCF significantly raises the numbers of megakaryocytes, as well as circulating platelet counts. Taken together, these data indicate that SCF may be an important regulator of platelet production under both normal and physiologically disturbed situations.     

Insulin-stimulated microtubule-associated protein kinase is phosphorylated on tyrosine and threonine in vivo.

Exposure of 3T3-L1 cells to insulin stimulates a soluble, serine(threonine)-specific protein kinase that phosphorylates microtubule-associated protein 2 (MAP-2) in vitro. The enzyme, termed MAP kinase, was isolated from insulin-treated or control cells radiolabeled with 32Pi. A 40-kDa phosphoprotein was found to elute in exact correspondence with enzymatic activity during hydrophobic interaction and gel filtration chromatography of extracts from cells stimulated with insulin. Both MAP kinase activity and the phosphoprotein were absent in fractions prepared from untreated cells. The 32P incorporated into the 40-kDa protein was stable during treatment with alkali. Phospho amino acid analysis confirmed that the radiolabel was primarily incorporated into phosphotyrosine and to a lesser extent phosphothreonine. In addition, MAP kinase was incompletely but specifically adsorbed by antibodies to phosphotyrosine. We conclude, based on these data and additional studies from this laboratory, that MAP kinase is phosphorylated on tyrosine in vivo. The data are consistent with the possibility that MAP kinase may be a substrate for the insulin receptor or another insulin-regulated tyrosine kinase.