The Abl-1 Kinase is Dispensable for NK Cell Inhibitory Signalling and is not Involved in Murine NK Cell Education.

Natural killer (NK) cell responsiveness in the mouse is determined in an education process guided by inhibitory Ly49 and NKG2A receptors binding to MHC class I molecules. It has been proposed that inhibitory signalling in human NK cells involves Abl-1 (c-Abl)-mediated phosphorylation of Crk, lowering NK cell function via disruption of a signalling complex including C3G and c-Cbl, suggesting that NK cell education might involve c-Abl. Mice deficient in c-Abl expression specifically in murine NK cells displayed normal inhibitory and activating receptor repertoires. Furthermore, c-Abl-deficient NK cells fluxed Ca2+ normally after triggering of ITAM receptors, killed YAC-1 tumour cells efficiently and showed normal, or even slightly elevated, capacity to produce IFN-γ after activating receptor stimulation. Consistent with these results, c-Abl deficiency in NK cells did not affect NK cell inhibition via the receptors Ly49G2, Ly49A and NKG2A. We conclude that signalling downstream of murine inhibitory receptors does not involve c-Abl and that c-Abl plays no major role in NK cell education in the mouse.

Arg/Abl2 promotes invasion and attenuates proliferation of breast cancer in vivo.

Tumor progression is a complex, multistep process involving accumulation of genetic aberrations and alterations in gene expression patterns leading to uncontrolled cell division, invasion into surrounding tissue and finally dissemination and metastasis. We have previously shown that the Arg/Abl2 non-receptor tyrosine kinase acts downstream of the EGF receptor and Src tyrosine kinases to promote invadopodium function in breast cancer cells, thereby promoting their invasiveness. However, whether and how Arg contributes to tumor development and dissemination in vivo has never been investigated. Using a mouse xenograft model, we show that knocking down Arg in breast cancer cells leads to increased tumor cell proliferation and significantly enlarged tumor size. Despite having larger tumors, the Arg-knockdown (Arg KD) tumor-bearing mice exhibit significant reductions in tumor cell invasion, intravasation into blood vessels and spontaneous metastasis to lungs. Interestingly, we found that proliferation-associated genes in the Ras-MAPK (mitogen-activated protein kinase) pathway are upregulated in Arg KD breast cancer cells, as is Ras-MAPK signaling, while invasion-associated genes are significantly downregulated. These data suggest that Arg promotes tumor cell invasion and dissemination, while simultaneously inhibiting tumor growth. We propose that Arg acts as a switch in metastatic cancer cells that governs the decision to 'grow or go' (divide or invade).

The Abl-related gene (Arg) nonreceptor tyrosine kinase uses two F-actin-binding domains to bundle F-actin.

Abl family nonreceptor tyrosine kinases regulate cellular morphogenesis and motility through functional interactions with the actin cytoskeleton. Although Abl family kinases are known to contain filamentous (F)-actin-binding domains at their C termini, it is unclear how Abl family kinases regulate the structure and/or function of the actin cytoskeleton. We show here that the Abl-related kinase Arg binds with positive cooperativity to F-actin in vitro with binding saturating at a ratio of one Arg/two actin molecules. Measurements of the F-actin-binding properties of Arg deletion mutants led to the identification of a second, previously uncharacterized internal F-actin-binding domain in Arg. Purified Arg can bundle F-actin in vitro, and this bundling activity requires both F-actin-binding domains. An Arg-yellow fluorescent protein fusion protein can induce the formation of actin-rich structures at the lamellipodia of Swiss 3T3 fibroblasts. Both of Arg's F-actin-binding domains are necessary and sufficient for the formation of these actin-rich structures. Together, our data suggest that Arg can use its F-actin-bundling activity to directly regulate actin cytoskeletal structure in vivo.

The ARG tyrosine kinase interacts with Siva-1 in the apoptotic response to oxidative stress.

The Abl family of mammalian nonreceptor tyrosine kinases consists of c-Abl and ARG (Abl-related gene). Certain insights are available regarding the involvement c-Abl in the response of cells to stress. ARG, however, has no known function in cell signaling. The present studies demonstrate that ARG associates with the proapoptotic Siva-1 protein. The functional significance of the ARG-Siva-1 interaction is supported by the finding that ARG is activated by oxidative stress and that this response involves ARG-mediated phosphorylation of Siva-1 on Tyr(48). The proapoptotic effects of Siva-1 are accentuated in cells stably expressing ARG and are inhibited in ARG-deficient cells. Moreover, the proapoptotic effects of Siva-1 are abrogated by mutation of the Tyr(48) site. We also show that the apoptotic response to oxidative stress is attenuated in ARG-deficient cells and that this defect is corrected by reconstituting ARG expression. These findings support a model in which the activation of ARG by oxidative stress induces apoptosis by a Siva-1-dependent mechanism.

Essential roles for the Abl and Arg tyrosine kinases in neurulation.

The Abl and Arg tyrosine kinases play fundamental roles in the development and function of the central nervous system. Arg is most abundant in adult mouse brain, especially in synapse-rich regions. arg(-/-) mice develop normally but exhibit multiple behavioral abnormalities, suggesting that arg(-/-) brains suffer from defects in neuronal function. Embryos deficient in both Abl and Arg suffer from defects in neurulation and die before 11 days postcoitum (dpc). Although they divide normally, abl(-/-)arg(-/-) neuroepithelial cells display gross alterations in their actin cytoskeleton. We find that Abl and Arg colocalize with each other and with actin microfilaments at the apical surface of the developing neuroepithelium. Thus, Abl and Arg play essential roles in neurulation and can regulate the structure of the actin cytoskeleton.

Association of an activator with an RNA polymerase II holoenzyme.

RNA polymerase II holoenzymes have been described that consist of RNA polymerase II, a subset of general transcription factors, and four SRB proteins. The SRB proteins, which were identified through a selection for genes involved in transcription initiation by RNA polymerase II in vivo, are a hallmark of the holoenzyme. We report here the isolation and characterization of additional SRB genes. We show that the products of all nine SRB genes identified thus far are components of the RNA polymerase II holoenzyme and are associated with a holoenzyme subcomplex termed the mediator of activation. The holoenzyme is capable of responding to a transcriptional activator, suggesting a model in which activators function, in part, through direct interactions with the holoenzyme. Immunoprecipitation experiments with anti-SRB5 antibodies demonstrate that the acidic activating domain of VP16 specifically binds to the holoenzyme. Furthermore, the holoenzyme and the mediator subcomplex bind to a VP16 affinity column. These results provide a more complete description of the RNA polymerase II holoenzyme and suggest that this form of the transcription apparatus can be recruited to promoters via direct interactions with activators.

A kinase-cyclin pair in the RNA polymerase II holoenzyme.

The RNA polymerase II holoenzyme consists of RNA polymerase II, a subset of general transcription factors, and regulatory proteins known as SRB proteins. The genes encoding SRB proteins were isolated as suppressors of mutations in the RNA polymerase II carboxy-terminal domain (CTD). The CTD and SRB proteins have been implicated in the response to transcriptional regulators. We report here the isolation of two new SRB genes, SRB10 and SRB11, which encode kinase- and cyclin-like proteins, respectively. Genetic and biochemical evidence indicates that the SRB10 and SRB11 proteins form a kinase-cyclin pair in the holoenzyme. The SRB10/11 kinase is essential for a normal transcriptional response to galactose induction in vivo. Holoenzymes lacking SRB10/11 kinase function are strikingly deficient in CTD phosphorylation. Although defects in the kinase substantially affect transcription in vivo, purified holoenzymes lacking SRB10/11 kinase function do not show defects in defined in vitro transcription systems, suggesting that the factors necessary to elicit the regulatory role of the SRB10/11 kinase are missing in these systems. These results indicate that the SRB10/11 kinase is involved in CTD phosphorylation and suggest that this modification has a role in the response to transcriptional regulators in vivo.

The RNA polymerase II holoenzyme and its implications for gene regulation.

The RNA polymerase II (Pol II) transcription initiation apparatus consists of several multisubunit complexes, including Pol II, general transcription factors and suppressor of RNA polymerase B (SRB) proteins. Recent evidence indicates that many of these components assemble into a large complex, called the RNA polymerase holoenzyme, the SRB components of which participate in the response to transcriptional regulators. We discuss these results and their implications for the regulation of gene expression.

Reduction of caveolin and caveolae in oncogenically transformed cells.

Caveolae are flask-shaped non-clathrin-coated invaginations of the plasma membrane. In addition to the demonstrated roles for caveolae in potocytosis and transcytosis, caveolae may regulate the transduction of signals from the plasma membrane. Transformation of NIH 3T3 cells by various oncogenes leads to reductions in cellular levels of caveolin, a principal component of the protein coat of caveolae. The reduction in caveolin correlates very well with the size of colonies formed by these transformed cells when grown in soft agar. Electron microscopy reveals that caveolae are morphologically absent from these transformed cell lines. These observations suggest that functional alterations in caveolae may play a critical role in oncogenic transformation, perhaps by disrupting contact inhibition in transformed cells.

Caveolae, transmembrane signalling and cellular transformation.

Caveolae are approximately 50-100 nm membrane micro-invaginations associated with the plasma membrane of a wide variety of cells. Although they were first identified in transmission electron micrographs approximately 40 years ago, their exact function(s) has remained controversial. Two well-established functions include: (1) the transcytosis of both large and small molecules across capillary endothelial cells and (2) the utilization of GPI-linked proteins to concentrate small molecules in caveolae for translocation to the cytoplasm (termed potocytosis). Recently, interest in a 'third' proposed caveolar function, namely transmembrane signalling, has been revived by the identification of caveolin--a transformation-dependent v-Src substrate and caveolar marker protein--and the isolation of caveolin-rich membrane domains from cultured cells. Here we will discuss existing evidence that suggests a role for caveolae in signalling events.

An RNA polymerase II holoenzyme responsive to activators.

RNA polymerase II requires multiple general transcription factors to initiate site-specific transcription. These proteins can assemble in an ordered fashion onto promoter DNA in vitro, and such ordered assembly may occur in vivo (Fig. 1a). Some general transcription factors can interact with RNA polymerase II in the absence of DNA, however, suggesting that RNA polymerase II may also assemble into a multi-component complex containing a subset of initiation factors before binding to promoter DNA (Fig. 1b). Here we present evidence from the yeast Saccharomyces cerevisiae for such an RNA polymerase II holoenzyme, a multi-subunit complex containing roughly equimolar amounts of RNA polymerase II, a subset of general transcription factors, and SRB regulatory proteins. Transcription by this holoenzyme is stimulated by the activator protein GAL4-VP16, a feature not observed with purified RNA polymerase II and general transcription factors alone. We propose that the holoenzyme is a form of RNA polymerase II readily recruited to promoters in vivo.

A multisubunit complex associated with the RNA polymerase II CTD and TATA-binding protein in yeast.

We report genetic and biochemical evidence that the RNA polymerase II carboxy-terminal domain (CTD) interacts with a large multisubunit complex that contains TATA-binding protein (TBP) and is an integral part of the transcription initiation complex. The isolation and characterization of extragenic suppressors of S. cerevisiae RNA polymerase II CTD truncation mutations led us to identify SRB2, SRB4, SRB5, and SRB6 as genes involved in CTD function in vivo. SRB2 was previously isolated and shown to encode a 23 kd TBP-binding protein. The four SRB proteins and a portion of cellular TBP are components of a high molecular weight multisubunit complex that is tightly bound to RNA polymerase II. This SRB-TBP complex binds specifically to recombinant CTD protein. In vitro transcription and template commitment assays confirm that SRB2 and SRB5 are components of a functional preinitiation complex and are required for efficient transcription initiation.

Quantitative comparison of initiation and mutation phenotypes in hepatocytes of the analbuminemic rat.

The potential relationship between mutagenesis and carcinogenesis has been examined in the Nagase analbuminemic rat treated with a single dose of benzo[a]pyrene, an incomplete liver carcinogen. The apparent mutation rate at the albumin locus was calculated by determining the number of hepatocytes expressing a cross-reactive product of albumin in analbuminemic rats treated with benzo[a]pyrene. The rate of initiation, the first stage in carcinogenesis, was determined by assessing the number of hepatocytes expressing the placental isozyme of glutathione S-transferase (PGST) after administration of benzo[a]pyrene. Since the expression of PGST may represent hepatocellular changes independent of initiation, promotion with phenobarbital was employed to clonally expand those putatively initiated hepatocytes expressing PGST. With immunohistochemical measures to assess changes in albumin expression, a threefold increase in the number of hepatocytes expressing albumin was detected after administration of benzo[a]pyrene in Nagase analbuminemic rats. A more than five-fold increase in altered hepatic foci (AHF) exhibiting increased PGST expression was observed in animals given benzo[a]pyrene treatment followed by phenobarbital, compared with those given benzo[a]pyrene alone. The number of albumin-expressing single hepatocytes detected was of the same order of magnitude as the number of individual hepatocytes and AHF expressing PGST, suggesting that similar events may be involved in their formation. Since 3 x 10(6) single hepatocytes expressing albumin were found in the analbuminemic rat liver after a single administration of benzo[a]pyrene, while less than 2 x 10(4) AHF expressing PGST were observed, formation of individual hepatocytes expressing albumin was a far more frequent event than clonal expansion of initiated hepatocytes in the Nagase analbuminemic rat. However, the number of loci of PGST expression including AHF and single hepatocytes is comparable to that of single hepatocytes expressing albumin.

A novel transcription factor reveals a functional link between the RNA polymerase II CTD and TFIID.

The RNA polymerase II large subunit carboxy-terminal domain (CTD) plays a role in transcription initiation, but its mechanism of action is not well understood. We have investigated the function of the SRB2 gene, which was isolated as a dominant suppressor of CTD truncation mutations. The allele specificity of this suppressor indicates that SRB2 and the CTD are involved in the same function. Indeed, cells lacking SRB2 and cells lacking a large portion of the CTD exhibit the same set of conditional growth phenotypes and exhibit very similar defects in gene expression in vivo. The SRB2 protein is a novel transcription factor that has an important role in basal and activated transcription in vitro and is essential for efficient establishment of the transcription initiation apparatus. Template commitment experiments suggest that SRB2 becomes physically associated with the transcription initiation complex. We find that SRB2 binds specifically to TFIID. As SRB2 and the RNA polymerase II CTD are involved in the same function, these results reveal a functional link between the CTD and the TATA-binding factor. This study implicates the CTD in recruitment of RNA polymerase II to the transcription initiation complex.

Phenobarbital-inducible aldehyde dehydrogenase in the rat. cDNA sequence and regulation of the mRNA by phenobarbital in responsive rats.

In the rat, a cytosolic isozyme of aldehyde dehydrogenase, designated ALDH-PB, can be induced in the liver by administration of phenobarbital (PB). ALDH-PB activity and mRNA are induced in Long-Evans rats that possess a responsive (R) allele but are not induced in homozygous nonresponsive rats (rr), although the rr genotype is competent to induce other PB-responsive mRNAs. ALDH-PB mRNA is expressed in the basal state (without PB administration) in hepatic tissue in both RR and rr genotypes. We report the complete nucleotide sequence of the rat ALDH-PB mRNA. The protein encoded by the ALDH-PB mRNA is 501 amino acids in length and has a predicted molecular mass of 54,540 daltons. The amino acid sequence predicted from the mRNA demonstrates a strong conservation between the rat ALDH-PB and the human cytosolic aldehyde dehydrogenase hALDH-1. We demonstrate the ALDH-PB, cytochrome P-450b, cytochrome P-450e, and glutathione S-transferase Ya subunit mRNA levels in the liver are altered noncoordinately by administration of PB in RR and rr genotypes. The strikingly different responses to PB administration between the various mRNA species in each of the genotypes suggest that the regulation of specific gene expression by PB may involve multiple pathways.