Imidazo[5,1-f][1,2,4]triazin-2-amines as novel inhibitors of polo-like kinase 1.

The synthesis and biological activities of imidazo[5,1-f][1,2,4]triazin-2-amines (imidazotriazines) as novel polo-like kinase 1 inhibitors are reported.

Expression and purification of active recombinant ATM protein from transiently transfected mammalian cells.

The gene mutated in the human disease ataxia telangiectasia (AT), termed ATM, encodes a large protein kinase involved in DNA repair and cell cycle control. Biochemical characterization of ATM function has been somewhat difficult because of its large size (approximately 370 kDa) and relatively low level of expression in several systems. The majority of studies have used immunoprecipitated ATM or purified ATM obtained through relatively complex procedures. Here, we describe an efficient method for the expression and purification of FLAG-epitope-tagged recombinant human ATM protein (F-ATM). This method utilizes the expression of F-ATM in transiently transfected 293T cells followed by anti-FLAG-agarose affinity chromatography. The transfection procedure has been optimized for large (225-cm(2)) culture flasks and F-ATM can be purified to near homogeneity as judged by SDS-PAGE. This procedure yields approximately 1 microg of catalytically active F-ATM protein/225-cm(2) flask that can be used for biochemical studies.

Monoclonal antibodies generated against recombinant ATM support kinase activity.

We report on the rapid generation of two monoclonal antibodies, ATM A16.35 and ATM D16.11, that bind to the kinase domain of mutated ataxia telangiectasia (ATM). These antibodies were generated against E. coli-expressed recombinant protein using the RIMMS strategy. We show that ATM A16.35 binds ATM by Western blot analysis, and ATM D16.11 forms immune complexes with native ATM in immunoprecipitations without neutralizing kinase activity.

Mutational activation of pp60(c-src) leads to a tumorigenic phenotype in a preneoplastic Syrian hamster embryo cell line.

Previous studies indicated that overexpression of wild-type avian c-src cannot induce neoplastic transformation of NIH 3T3 cells. In this study, we isolated and characterized novel spontaneously derived transforming mutants of avian pp60(c-src) from a Syrian hamster embryo-derived cell line, 10W, transfected with the avian c-src gene. Seventeen independently derived transfected 10W cell clones were injected into athymic nude mice. After a latency period, tumors eventually arose and were established in culture. The tumorigenic phenotype was always accompanied by the presence of the avian c-src DNA and functional expression of pp60(c-src). However, most of the tumor-derived cell lines expressed an electrophoretically altered form of pp60(c-src), suggesting mutations in src. Consistent with this hypothesis, DNAs isolated from the tumor-derived lines, but not the parental 10W cell lines, morphologically transformed NIH 3T3 cells in a focus-forming assay. We characterized pp60(c-src) in detail from three of the tumor-derived lines: 4AT, 4BT, and E2T. Two of these lines contained mutations within the exogenous c-src coding region. Line 4AT has an internal repeat of 29 amino acids immediately following Gln-513, which disrupts the spacing between the end of the kinase domain and Tyr-527, the negative regulatory site in pp60(c-src). Line 4BT has a 5-bp deletion following Phe-520, which results in loss of Tyr-527. However, the DNA sequence of the coding region of pp60(c-src) from a third line, E2T, was completely wild type. Cyanogen bromide cleavage analyses of the altered pp60(c-src) from lines 4AT and 4BT showed that Tyr-527, the site of negative regulation of c-src, is not phosphorylated, but Tyr-416, the site of in vitro autophosphorylation, is phosphorylated. However, in line E2T, Tyr-527 was phosphorylated, and Tyr-416 was phosphorylated to a lesser extent. Additionally, two proteins that indicate activation of src, p85 cortactin and p120(cas), are phosphorylated in at least six of the tumor-derived cell lines, although to a lesser extent in line E2T. These results suggest that dephosphorylation of Tyr-527 and phosphorylation of Tyr-416 correlate with activation of pp60(c-src) in the tumor-derived lines 4AT and 4BT, respectively. However, in line E2T, the high levels of pp60(c-src), in combination with a partial activation of the pp60(c-src) protein as indicated by phosphorylation of Tyr-416, appear to be involved in the neoplastic process, rather than mutation.

Separate pathways for p53 induction by ionizing radiation and N-(phosphonoacetyl)-L-aspartate.

The tumor suppressor gene product, p53, appears to be a significant participant in signaling pathways that mediate cellular responses to cytotoxic stresses. In particular, p53 appears to be a critical determinant of whether the cell lives or dies and how it progresses through the cell cycle after the cytotoxic exposure. Many of the molecular details for these signaling pathways remain to be elucidated, and whether all cytotoxic signals utilize the same pathway to increase p53 expression is not clear. Here, we demonstrate the existence of cell types in which the induction of p53 and associated G1 arrest by the antimetabolite, N-(phosphonoacetyl)-L-aspartate (PALA), is defective, whereas p53 induction and G1 arrest induced by ionizing radiation are intact. These observations demonstrate the existence of genetic defects that can alter p53 induction and associated cellular outcomes after some, but not all, cytotoxic insults and suggest distinct pathways of p53 induction by PALA and ionizing radiation.

Role of c-Src tyrosine kinase in G protein-coupled receptor- and Gbetagamma subunit-mediated activation of mitogen-activated protein kinases.

Several G protein-coupled receptors that interact with pertussis toxin-sensitive heterotrimeric G proteins mediate Ras-dependent activation of mitogen-activated protein (MAP) kinases. The mechanism involves Gbetagamma subunit-mediated increases in tyrosine phosphorylation of the Shc adapter protein, Shc*Grb2 complex formation, and recruitment of Ras guanine nucleotide exchange factor activity. We have investigated the role of the ubiquitous nonreceptor tyrosine kinase c-Src in activation of the MAP kinase pathway via endogenous G protein-coupled lysophosphatidic acid (LPA) receptors or by transient expression of Gbetagamma subunits in COS-7 cells. In vitro kinase assays of Shc immunoprecipitates following LPA stimulation demonstrated rapid, transient recruitment of tyrosine kinase activity into Shc immune complexes. Recruitment of tyrosine kinase activity was pertussis toxin-sensitive and mimicked by cellular expression of Gbetagamma subunits. Immunoblots for coprecipitated proteins in Shc immunoprecipitates revealed a transient association of Shc and c-Src following LPA stimulation, which coincided with increases in Shc-associated tyrosine kinase activity and Shc tyrosine phosphorylation. LPA stimulation or expression of Gbetagamma subunits resulted in c-Src activation, as assessed by increased c-Src autophosphorylation. Overexpression of wild-type or constitutively active mutant c-Src, but not kinase inactive mutant c-Src, lead to increased tyrosine kinase activity in Shc immunoprecipitates, increased Shc tyrosine phosphorylation, and Shc.Grb2 complex formation. MAP kinase activation resulting from LPA receptor stimulation, expression of Gbetagamma subunits, or expression of c-Src was sensitive to dominant negatives of mSos, Ras, and Raf. Coexpression of Csk, which inactivates Src family kinases by phosphorylating the regulatory C-terminal tyrosine residue, inhibited LPA stimulation of Shc tyrosine phosphorylation, Shc.Grb2 complex formation, and MAP kinase activation. These data suggest that Gbetagamma subunit-mediated formation of Shc.c-Src complexes and c-Src kinase activation are early events in Ras-dependent activation of MAP kinase via pertussis toxin-sensitive G protein-coupled receptors.

The p53 signal transduction pathway is intact in human neuroblastoma despite cytoplasmic localization.

Mutations of the p53 tumor suppressor gene are rarely found in neuroblastoma. Though typically a nuclear protein, a number of tumor cell types have recently been reported to exhibit cytoplasmic p53 immunostaining, and it has been suggested that altered cellular localization is another mechanism of inhibiting p53 function. We examined p53 protein expression, localization, and function in neuroblastoma cell lines with wild-type p53 genes. Basal p53 levels were largely confined to the cytoplasmic compartment in these cells. However, after irradiation, p53 protein levels increased predominately in the nucleus. Transcriptional activity of p53 was intact in these cells because "downstream" proteins, p21WAF1 and MDM2, were induced by irradiation. In contrast to a neuroblastoma cell line harboring a mutant p53 gene, the neuroblastoma cells with wild-type protein were associated with an intact G1 arrest after DNA damage. The induced nuclear protein in these neuroblastoma cells also appeared functional as measured by its capacity to bind to a DNA oligomer containing a p53-consensus sequence. We have concluded that although p53 expression in neuroblastoma cells is primarily localized to the cytosol, ionizing radiation induces a functional p53 protein in the nucleus and that this cytoplasmic sequestration of p53 in human neuroblastoma is not a mechanism of inactivating p53 function.

Characterization of human Gadd45, a p53-regulated protein.

GADD45 (growth arrest and DNA damage) is a DNA-damage-inducible gene regulated in part by the tumor suppressor p53. A role in negative growth control has recently been suggested based on significant (more than 75%) reduction of colony formation following over expression of Gadd45. To better understand the role of Gadd45, we have developed specific rabbit and murine antibodies raised against the human recombinant protein. Using these antibodies, we have found that in ML-1 cells Gadd45 is predominantly a nuclear protein. MyD118, a protein induced by terminal differentiation sharing 57% homology with Gadd45, does not cross-react with any of the antibodies produced. As expected, the induction of Gadd45 protein by ionizing radiation (IR) was also found to be dependent on a wild type p53 phenotype. Interestingly, WI-L2-NS, a human lymphoid cell line, showed very high basal levels of Gadd45 mRNA and protein in addition to a high constitutive level of a mutated p53 protein. In this cell line, the high levels of GADD45 did not inhibit cellular growth in spite of the fact that no mutations were found in GADD45 sequence. These results indicate that some cell line(s) can tolerate high levels of Gadd45 and abrogate its growth suppression function.

Involvement of pp60c-src with two major signaling pathways in human breast cancer.

The phosphotyrosine residues of receptor tyrosine kinases serve as unique binding sites for proteins involved in intracellular signaling, which contain SRC homology 2 (SH2) domains. Since overexpression or activation of the pp60c-src kinase has been reported in a number of human tumors, including primary human breast carcinomas, we examined the interactions of the SH2 and SH3 domains of human SRC with target proteins in human carcinoma cell lines. Glutathione S-transferase fusion proteins containing either the SH2, SH3, or the entire SH3/SH2 region of human SRC were used to affinity purify tyrosine-phosphorylated proteins from human breast carcinoma cell lines. We show here that in human breast carcinoma cell lines, the SRC SH2 domain binds to activated epidermal growth factor receptor (EGFR) and p185HER2/neu. SRC SH2 binding to EGFR was also observed in a nontumorigenic cell line after hormone stimulation. Endogenous pp60c-src was found to tightly associate with tyrosine-phosphorylated EGFR. Association of the SRC SH2 with the EGFR was blocked by tyrosyl phosphopeptides containing the sequences surrounding tyrosine-530, the regulatory site in the SRC C terminus, or sequences surrounding the major sites of autophosphorylation in the EGFR. These results raise the possibility that association of pp60c-src with these receptor tyrosine kinases is an integral part of the signaling events mediated by these receptors and may contribute to malignant transformation.

The protooncogene c-sea encodes a transmembrane protein-tyrosine kinase related to the Met/hepatocyte growth factor/scatter factor receptor.

c-sea is the cellular homologue of the avian erythroblastosis virus S13-encoded oncogene v-sea. We have isolated and determined the nucleotide sequence of overlapping chicken cDNAs that encode the putative c-sea protooncogene product. The predicted reading frame encoded a 1404-aa polypeptide that had the structure of a receptor-like protein-tyrosine kinase and exhibited the highest degree of sequence similarity with the Met/hepatocyte growth factor/scatter factor receptor. Analysis of steady-state RNA expression revealed that c-sea mRNA levels were elevated approximately 5-fold in chicken embryo cells transformed by activated variants of the src nonreceptor protein-tyrosine kinase gene but not in cells transformed by the nuclear oncogenes v-myc or v-rel. A survey of c-sea expression in a variety of chicken tissues indicated that the highest levels of mRNA were located in peripheral white blood cell populations and in the intestine.

Activation of pp60c-src transforming potential by mutations altering the structure of an amino terminal domain containing residues 90-95.

The overexpression of the c-src gene product, pp60c-src, in avian and rodent embryo cells is not sufficient to induce cellular transformation. In this study we report that structural alterations within an amino terminal domain of pp60c-src, the exon 3 domain (residues 84-115) activate the oncogenic potential of the c-src gene product. Site-directed mutagenesis of the c-src gene was used to generate c-src variants encoding pp60c-src proteins with the following amino acid alterations: tyr 90 to phe (pm90F); tyr 92 to phe (pm92F); arg 95 to either trp, lys, glu or gln (pm95W, 95K, 95E and 95Q, respectively), and deletion of residues 92-95 (dl92). C-src variants encoding proteins with the alteration of arg 95 to trp, glu, or lys, or containing the deletion of residues 92-95, induced alterations in cell morphology and promoted growth in soft agar as well as changes in glucose transport and in vivo tyrosine phosphorylation of cellular proteins (including calpactin I heavy chain, p36). Analysis of in vivo phosphorylation of the transforming variant src proteins revealed little detectable alteration in the phosphorylation of tyr 527, a putative site of tyrosine kinase regulation. Our results suggest that structural alterations within a domain distal to the catalytic (kinase) domain activate pp60c-src kinase activity and, concomitantly, oncogenic potential. Furthermore, we suggest that the exon 3 domain of pp60c-src may contribute to the regulation and/or substrate specificity of the c-src protein.

Activation of the oncogenic potential of the avian cellular src protein by specific structural alteration of the carboxy terminus.

The role of tyrosine phosphorylation in the regulation of tyrosine protein kinase activity was investigated using site-directed mutagenesis to alter the structure and environment of the three tyrosine residues present in the C terminus of avian pp60c-src. Mutations that change Tyr 527 to Phe or Ser activate in vivo tyrosine protein kinase activity and induce cellular transformation of chicken cells in culture. In contrast, alterations of tyrosine residues present at positions 511 or 519 in c-src do not induce transformation or in vivo tyrosine protein kinase activity. Amber mutations, which alter the structure of the pp60c-src C terminus by inducing premature termination of the c-src protein at either residue 518 or 523 also induce morphological transformation and increase in vivo tyrosine phosphorylation, whereas removal of the last four residues of c-src by chain termination at residue 530 does not alter the kinase activity or the biological activity of the resultant c-src protein. We conclude from these studies that C-terminal alterations which either remove or replace Tyr 527 serve to activate the c-src protein resulting in cellular transformation and increased in vivo tyrosine protein kinase activity.

Oral ultrastructure and oral development of the misaligned undulating membrane mutant of Tetrahymena thermophila.

The misaligned undulating membrane (mum) mutant of Tetrahymena thermophila is a non-conditional, single gene recessive mutation. The major effect of the mum mutation is the production of multiple undulating membrane (UM) fragments in the oral apparatus (OA). The ultrastructure of the UM fragments of mum OAs is identical to that of the single UM of wild-type OAs. Analysis of OA development at midbody using a combination of light microscopy of protargol-stained cells and SEM of demembranated whole cells showed that the phenotypic effect of the mum mutation first becomes evident during mid to late stage 4 and is fully manifested in early stage 5. The effect of the mutation involves a proliferation of excess basal bodies in the UM field. Subsequent events in the development of the mum OA from mid to late stage 5 are identical to those in wild-type OAs. This study suggests that the mum mutation establishes conditions that allow the production of multiple UMs and thus reveals that the UM field is competent for the complete and coordinated development of several adjacent UMs. This level of regional control is not clearly evident when a single UM is present. The comparison of development of wild-type and mum OAs required an extensive reanalysis of stages 4 and 5 of normal oral development. On the basis of current and previous observations, we propose a new and more subdivided staging system for oral development in Tetrahymena.