Cas ligand with multiple Src homology (SH) 3 domains (CMS) is an ubiquitously expressed signal transduction molecule that interacts with the focal adhesion protein p130 Cas . CMS contains three SH3 in its NH 2 terminus and proline-rich sequences in its center region. The latter sequences mediate the binding to the SH3 domains of p130 Cas , Src-family kinases, p85 subunit of phosphatidylinositol 3-kinase, and Grb2. The COOH-terminal region contains putative actin binding sites and a coiled-coil domain that mediates homodimerization of CMS. CMS is a cytoplasmic protein that colocalizes with F-actin and p130 Cas to membrane ruff les and leading edges of cells. Ectopic expression of CMS in COS-7 cells resulted in alteration in arrangement of the actin cytoskeleton. We observed a diffuse distribution of actin in small dots and less actin fiber formation. Altogether, these features suggest that CMS functions as a scaffolding molecule with a specialized role in regulation of the actin cytoskeleton.Tumor cells are characterized by their acquired capacity of uncontrolled proliferation and an increased ability to migrate and invade healthy tissue. Transformation of normal mammalian cells into an invasive tumor results in alterations of cell matrix responses by integrins and loss of polarity (reviewed in refs. 1 and 2). Most integrins are transmembrane linkers, which mediate the interaction between the cytoskeleton and the extracellular matrix. Their activation leads to the assembly of signaling complexes, known as focal adhesions and focal contacts, and subsequently to the activation of intracellular signaling cascades (3, 4).p130 Cas , a docking protein that is tyrosine-phosphorylated in response to a variety of extracellular stimuli, such as growth factors, cell-cell interaction, and cell-matrix interaction, appears to play a critical role in the integrin-linked formation of focal complexes (5-8). In p130 Cas -deficient mice, a prominent feature is heart failure caused by the disruption of the Z discs in cardiomyocytes (9). Z discs are structures similar to focal adhesions, and their role is to organize the actin cytoskeleton (10). Moreover, recent work has indicated a role for phosphorylated p130 Cas in cell migration (11). p130Cas is composed of an NH 2 -terminal Src homology (SH) 3 domain and regions with SH3 and SH2-binding motifs. It initially was identified as highly phosphorylated protein in v-src and v-crk transformed fibroblasts (12-14), and a role in transformation has been suggested (15). In these cells, the constitutive phosphorylation of p130 Cas results in the permanent recruitment of SH2 containing signaling proteins into focal complexes (16,17). The consequence is the constitutive propagation of downstream signals, resulting in changes in the cytoskeleton, induction of migration, and enhanced invasive potential of carcinoma cells (11,18 Cas suggests a function as scaffolding protein involved in the dynamic regulation of the actin cytoskeleton. MATERIALS AND METHODSCells and Reagents. Human 293T k...

PVR, the Drosophila homolog of the PDGF/VEGF receptor, has been implicated in border cell migration during oogenesis and hemocyte migration during embryogenesis. It was earlier shown that Mbc, a CDM family protein, and its effector, Rac, transduced the guidance signal from PVR during border cell migration. Here we demonstrate that PVR is also required for the morphogenetic process, thorax closure, during metamorphosis. The results of genetic and biochemical experiments indicate that PVR activates the JNK pathway. We present evidence showing Crk (an adaptor molecule), Mbc, ELMO (a homolog of Caenorhabditis elegans CED-12 and mammalian ELMO), and Rac to be mediators of JNK activation by PVR. In addition, we suppose that not only Rac but also Cdc42 is activated and involved in JNK activation downstream of PVR

The cellular signal transduction pathways by which C3G, a RAS family guanine nucleotide exchange factor, mediates v-crk transformation are not well understood. Here we report the identification of Drosophila C3G, which, like its human cognate, specifically binds to CRK but not DRK/GRB2 adaptor molecules. During Drosophila development, constitutive membrane binding of C3G, which also occurs during v-crk transformation, results in cell fate changes and overproliferation, mimicking overactivity of the RAS-MAPK pathway. The effects of C3G overactivity can be suppressed by reducing the gene dose of components of the RAS-MAPK pathway and of RAP1. These findings provide the first in vivo evidence that membrane localization of C3G can trigger activation of RAP1 and RAS resulting in the activation of MAPK, one of the hallmarks of v-crk transformation previously thought to be mediated through activation of SOS.

The nucleotide sequence of the internal region of a Drosophila retrotransposon. 412, was determined. The genome of 412 was found to consist of two long open-reading frames (ORFs 1 and 2), an unusually long putative leader region and long terminal repeats (LTRs). As with 17.6, 297 and gypsy, ORFs 1 and 2 slightly overlap each other and are out of phase by + 2 . ORF2 includes the nucleotide sequences coding for the putative protease, reverse transcriptase and integrase, and is similar in entire organization to the pol gene of Moloney murine leukaemia virus. In spite of the difference in insertion specificity, integrase, an enzyme presumably responsible for insertion, was found to be similar in amino acid sequence to the counterparts of 17.6, 297 and gypsy. There is no ORF in 412 which corresponds to retroviral env or ORF3s of 17.6 and 297. Analysis of 412 transcripts suggested that 412 LTR is composed of U3, R and U5. The gene for a potential primer tRNA for putative reverse transcription of 412 was also surveyed and the 3'-terminal 15 nucleotides of a putative arginine tRNA were found to be exactly complementary to the putative primer-binding site of 412.Drosophila copia-like elements or retrotransposons are formed from the largest group of transposable genetic elements in Drosophila melanogaster. They are intimately related in evolution to retroviral proviruses in vertebrates In our previous experiments the complete nucleotide sequences of three Drosophila retrotransposons 17.6 [15], 297 [16] and copia [17] were determined. 17.6 and 297 contained three open reading frames (ORFs) similar in size and location to retroviral gag, pol and env. The second ORFs of these two elements were further shown each to include the nucleotide sequence coding for a reverse-transcriptase-like enzyme and be, in all respects of their organization, very similar to the pol gene of Moloney murine leukaemia virus (Mo-MuLV) [15,16, 181. In contrast, the genome of copia contained a single long ORF [17, 191 including genes for the major capsid protein of copia-related virus-like particles and reverse-transcriptase-like enzyme much less similar to the reverse transcriptase of MoMuLV [17,19]. These results suggest the genome organization in Drosophila retrotransposons to be very much diversified.To confirm and determine the further implications of this notion, a determination was made of the complete nucleotide sequence of another Drosophila retrotransposon, 412, having terminal dinucleotides identical to those of copia and retrovirus proviruses but different from those of 17.6, 297 and gypsy [4, 15-17, 20, 211. The genome of 412 was found to contain two long ORFs (ORFs 1 and 2) and an unusually long putative leader region with two short ORFs. As with 17.6 [15], 297 [16] and gypsy (unpublished), ORFs 3 and 2 slightly overlap each other and are out of phase by t 2 . The entire structure of ORF2 is of the 17.6 type and the putative integrase encoded by a portion of ORF2 is similar in amino acid sequence to the counterparts of 17.6, 297 and gy...

Aberrant expression of the full-length isoform of DUX4 (DUX4-FL) appears to underlie pathogenesis in facioscapulohumeral muscular dystrophy (FSHD). DUX4-FL is a transcription factor and ectopic expression of DUX4-FL is toxic to most cells. Previous studies showed that DUX4-FL-induced pathology requires intact homeodomains and that transcriptional activation required the C-terminal region. In this study, we further examined the functional domains of DUX4 by generating mutant, deletion, and fusion variants of DUX4. We compared each construct to DUX4-FL for (i) activation of a DUX4 promoter reporter, (ii) expression of the DUX4-FL target gene ZSCAN4, (iii) effect on cell viability, (iv) activation of endogenous caspases, and (v) level of protein ubiquitination. Each construct produced a similarly sized effect (or lack of effect) in each assay. Thus, the ability to activate transcription determined the extent of change in multiple molecular and cellular properties that may be relevant to FSHD pathology. Transcriptional activity was mediated by the C-terminal 80 amino acids of DUX4-FL, with most activity located in the C-terminal 20 amino acids. We also found that non-toxic constructs with both homeodomains intact could act as inhibitors of DUX4-FL transcriptional activation, likely due to competition for promoter sites.

Paxillin is a protein containing four LIM domains, and functions in integrin signaling. We report here that two transcripts are generated from the paxillin gene locus in Drosophila; one encodes a protein homolog of the vertebrate Paxillin (DPxn37), and the other a protein with only three LIM domains, partly encoded by its own specific exon (PDLP). At the myotendinous junctions of Drosophila embryos where integrins play important roles, both DPxn37 and PDLP are highly expressed with different patterns; DPxn37 is predominantly concentrated at the center of the junctions, whereas PDLP is highly enriched at neighboring sides of the junction centers, primarily expressed in the mesodermal myotubes. Northern blot analysis revealed that DPxn37 is ubiquitously expressed throughout the life cycle, whereas PDLP expression exhibits a biphasic pattern during development, largely concomitant with muscle generation and remodeling. Our results collectively reveal that a unique system exists in Drosophila for the generation of a novel type of LIM-only protein, highly expressed in the embryonic musclature, largely utilizing the Paxillin LIM domains.

Human diploid fibroblasts (HDF) immortalized by hTERT and simian virus 40 (SV40) early region (ER) exhibit a limited degree of transformation upon the expression of activated H-RAS (H-RAShuman diploid fibroblast ͉ mitogen-activated protein kinase kinase͞extracellular signal-regulated kinase ͉ simian virus 40 early region ͉ telomerase ͉ rat embryonic fibroblasts I n contrast to rodent cells, human cells have long been very difficult to be transformed in vitro. Normal (nonestablished) rodent cells can be readily transformed by the concomitant expression of two oncogenes, whereas normal human cells have proven to be resistant to the transformation induced by the same combinations of oncogenes, indicating the fundamental differences in cellular requirements for oncogenic transformation between these species. In 1999, Weinberg and colleagues (1) reported that ectopic expression of the catalytic subunit of human telomerase (hTERT) made normal human foreskin fibroblasts and kidney epithelial cells susceptible to transformation by the combined expression of simian virus 40 (SV40) early region (SV40 ER) and activated H-Ras (H-Ras V12). They further succeeded in transforming several different types of normal human cells by the same combinations of genetic elements. Based on these results, they proposed that the telomere maintenance by hTERT, inactivation of p53 and retinoblastoma (Rb) by SV40 large T antigen, suppression of protein phosphatase PP2A activity by SV40 small t antigen, and constitutive activation of mitogenic signaling by activated Ras are sufficient to transform normal human cells (2). However, we recently demonstrated that, even when all these requirements are fulfilled, the transformed phenotypes of human fibroblasts are much less malignant than those of rat fibroblasts in terms of morphological changes, anchorage independence, and tumorigenicity in nude mice (3, 4). Our results strongly suggest that normal human cells have still undefined intrinsic mechanisms rendering them resistant to oncogenic transformation. In the present study, we found that the expression of FRA1, a member of the family of AP-1 transcription factors, was differentially regulated by RAS in human and rat fibroblasts and concluded that this transcription factor is one of the determinant factors for species-specific susceptibility to RAS-induced transformation. Results FRA1 Is Highly Induced by RAS in Rodent Fibroblasts but Not in HumanFibroblasts. To explore the molecular basis underlying the difference in RAS-induced transformation phenotypes between rodent and human fibroblasts, we performed gene expression profiling by using a CodeLink microarray. Rat embryonic fibroblasts (REF), mouse embryonic fibroblasts (MEF) and a strain of human diploid fibroblasts (HDF), TIG3 ectopically expressing hTERT (TIG3͞T), were analyzed. We made comparisons between cells expressing SV40 ER (S) alone and cells coexpressing SV40 ER and H-Ras V12 (SR), because the drastic phenotypic differences between rodent and human fibroblasts were observed in respo...

Using synthetic oligonucleotides corresponding to the amino acid sequences best conserved among retroviral reverse transcriptases, we developed a rapid method to detect cloned DNA fragments with the genes for reverse transcriptases. By this technique followed by nucleotide sequence determination, nucleotide sequences coding for reverse transcriptase-like enzymes were identified in two Drosophila retrotransposons, gypsy and 412. Our sequence analysis suggested that there are at least two major groups of retrotransposons in Drosophila with respect to putative reverse transcriptases and both gypsy and 412 belong to a category of retrotransposons which have putative reverse transcriptases very similar in amino acid sequence to the counterpart of Moloney murine leukaemia virus, a typical mammalian retrovirus.