Zyxin, a regulator of actin filament assembly, targets the mitotic apparatus by interacting with h-warts/LATS1 tumor suppressor

The mitotic apparatus plays a pivotal role in dividing cells to ensure each daughter cell receives a full set of chromosomes and complement of cytoplasm during mitosis. A human homologue of the Drosophila warts tumor suppressor, h-warts/LATS1, is an evolutionarily conserved serine/threonine kinase and a dynamic component of the mitotic apparatus. We have identified an interaction of h-warts/LATS1 with zyxin, a regulator of actin filament assembly. Zyxin is a component of focal adhesion, however, during mitosis a fraction of cytoplasmic-dispersed zyxin becomes associated with h-warts/LATS1 on the mitotic apparatus. We found that zyxin is phosphorylated specifically during mitosis, most likely by Cdc2 kinase, and that the phosphorylation regulates association with h-warts/LATS1. Furthermore, microinjection of truncated h-warts/LATS1 protein, including the zyxin-binding portion, interfered with localization of zyxin to mitotic apparatus, and the duration of mitosis of these injected cells was significantly longer than that of control cells. These findings suggest that h-warts/LATS1 and zyxin play a crucial role in controlling mitosis progression by forming a regulatory complex on mitotic apparatus.     

The discs-large tumor suppressor gene of Drosophila encodes a guanylate kinase homolog localized at septate junctions.

Mutations of the lethal(1)discs large-1 (dlg) tumor suppressor gene of Drosophila cause neoplastic overgrowth of the imaginal discs. Sequencing of a near full-length cDNA predicts a protein containing a domain homologous to yeast guanylate kinase and a region homologous to SH3, a putative regulatory motif in nonreceptor protein tyrosine kinases and other signal transduction proteins. Immunofluorescence analysis using antibodies directed against fusion peptides shows that the dlg gene product is localized in an apical belt of the lateral cell membrane, at the position of the septate junction. The results suggest that a signal transduction process involving guanine nucleotides occurs at the septate junction and is necessary for cell proliferation control in Drosophila epithelia.     

Inhibition of cell growth by conditional expression of kpm,a human homologue of Drosophila warts/lats tumor suppressor

kpm is a human serine/threonine kinase that is homologous to Drosophila tumor suppressor warts/lats and its mammalian homologue LATS1. In order to define the biological function of kpm, we generated stable transfectants of wild-type kpm (kpm-wt), a kinase-dead mutant of kpm (kpm-kd), and luciferase in HeLa Tet-Off cells under the tetracycline-responsive promoter. Western blot analysis showed that high levels of expression of kpm-wt as well as kpm-kd with an apparent mass of 150 kDa were induced after the removal of doxycycline. Induction of kpm-wt expression resulted in a marked decline in viable cell number measured by both trypan blue dye exclusion and MTT assay, whereas that of kpm-kd or luciferase had no effect. We then analyzed the cell cycle progression and apoptosis upon induction of kpm expression. 2-3 days after removal of doxycycline, cells underwent G(2)/M arrest, demonstrated by flow cytometric analysis of propidium iodide incorporation and MPM-2 reactivity. In vitro kinase assay showed that induction of kpm-wt led to down-regulation of kinase activity of the Cdc2-cyclin B complex, which was accompanied by an increase in the hyperphosphorylated form of Cdc2 and a change of phosphorylation status of Cdc25C. Furthermore, both DAPI staining and TUNEL assay showed that the proportion of apoptotic cells increased as kpm expression was induced. Taken together, these results indicate that kpm negatively regulates cell growth by inducing G(2)/M arrest and apoptotic cell death through its kinase activity.     

Binding of the human homolog of the Drosophila discs large tumor suppressor protein to the mitochondrial ribosomal protein MRP-S34

The human homolog of the Drosophila discs large tumor suppressor protein (hDLG) functions as a scaffolding protein that facilitates the transmission of diverse downstream signals. Here we show that hDLG interacts through its PDZ domains with the carboxy-terminal S/TXV motif of the mitochondrial ribosomal protein S-34 (MRP-S34). Our results suggest that hDLG interacts with MRP-S34 prior to entry of MRP-S34 into the mitochondria and may be involved in the trafficking of MRP-S34.     

Drosophila p53 is a structural and functional homolog of the tumor suppressor p53

The importance of p53 in carcinogenesis stems from its central role in inducing cell cycle arrest or apoptosis in response to cellular stresses. We have identified a Drosophila homolog of p53 ("Dmp53"). Like mammalian p53, Dmp53 binds specifically to human p53 binding sites, and overexpression of Dmp53 induces apoptosis. Importantly, inhibition of Dmp53 function renders cells resistant to X ray-induced apoptosis, suggesting that Dmp53 is required for the apoptotic response to DNA damage. Unlike mammalian p53, Dmp53 appears unable to induce a G1 cell cycle block when overexpressed, and inhibition of Dmp53 activity does not affect X ray-induced cell cycle arrest. These data reveal an ancestral proapoptotic function for p53 and identify Drosophila as an ideal model system for elucidating the p53 apoptotic pathway(s) induced by DNA damage.     

Binding of the mammalian homolog of the Drosophila discs large tumor suppressor protein to the ribosome receptor

DLG, the mammalian homolog of the Drosophila Discs Large suppressor protein, functions as a scaffolding protein that facilitates the transmission of diverse downstream signals. In the present study, we attempted to identify partner proteins for DLG, and found that DLG interacts through its PDZ domains with the ribosome receptor. The ribosome receptor is an integral endoplasmic reticulum protein that has been suggested to be involved in secretion. Our finding raises the possibility that DLG plays a role in the regulation of secretion by interacting with the ribosome receptor.     

Oncogenic function for the Dlg1 mammalian homolog of the Drosophila discs-large tumor suppressor

The fact that several different human virus oncoproteins, including adenovirus type 9 E4-ORF1, evolved to target the Dlg1 mammalian homolog of the membrane-associated Drosophila discs-large tumor suppressor has implicated this cellular factor in human cancer. Despite a general belief that such interactions function solely to inactivate this suspected human tumor suppressor protein, we demonstrate here that E4-ORF1 specifically requires endogenous Dlg1 to provoke oncogenic activation of phosphatidylinositol 3-kinase (PI3K) in cells. Based on our results, we propose a model wherein E4-ORF1 binding to Dlg1 triggers the resulting complex to translocate to the plasma membrane and, at this site, to promote Ras-mediated PI3K activation. These findings establish the first known function for Dlg1 in virus-mediated cellular transformation and also surprisingly expose a previously unrecognized oncogenic activity encoded by this suspected cellular tumor suppressor gene.     

The rat brain postsynaptic density fraction contains a homolog of the Drosophila discs-large tumor suppressor protein.

In CNS synapses, the synaptic junctional complex with associated postsynaptic density is presumed to contain proteins responsible for adhesion between pre- and postsynaptic membranes and for postsynaptic signal transduction. We have found that a prominent, brain-specific protein (PSD-95) enriched in the postsynaptic density fraction from rat brain is highly similar to the Drosophila lethal(1)discs-large-1 (dlg) tumor suppressor protein. The dlg protein is associated with septate junctions in developing flies and contains a guanylate kinase domain that is required for normal control of cell division. The sequence similarity between dlg and PSD-95 suggests that molecular mechanisms critical for growth control in developing organisms may also regulate synapse formation, stabilization, or function in the adult brain.     

mMaspin: the mouse homolog of a human tumor suppressor gene inhibits mammary tumor invasion and motility.

BACKGROUND: The human maspin gene encodes a protein in the serine proteinase inhibitor (serpin) family with tumor-suppressing functions in cell culture and in nude mice. In order to examine the role of maspin in an intact mammal, we cloned and sequenced the cDNA of mouse maspin. The recombinant protein was produced and its activity in cell culture was assessed. MATERIALS AND METHODS: Mouse maspin (mMaspin) was cloned by screening a mouse mammary gland cDNA library with the human maspin cDNA probe. Northern blot analysis was used to examine the expression patterns in mouse tissues, mammary epithelial cells, and carcinomas. Recombinant mMaspin protein was produced in E. coli. Invasion and motility assays were used to assess the biological function of mMaspin. RESULTS: mMaspin is 89% homologous with human maspin at the amino acid level. Like its human homolog, mMaspin is expressed in normal mouse mammary epithelial cells and down-regulated in mouse breast tumor cell lines. The expression is altered at different developmental stages in mammary gland. Addition of the recombinant mMaspin protein to mouse tumor cells was shown to inhibit invasion in a dose-dependent manner. As with the human protein, recombinant mMaspin protein also inhibited mouse mammary tumor motility. Deletion in the putative mMaspin reactive site loop (RSL) region resulted in the loss of its inhibitory functions. CONCLUSIONS: mMaspin is the mouse homolog of a human tumor suppressor gene. The expression of mMaspin is down-regulated in tumor cells and is altered at different developmental stages of mammary gland. mMaspin has inhibitory properties similar to those of human maspin in cell culture, suggesting that the homologous proteins play similar physiological roles in vivo.     

The Drosophila tumor suppressor expanded regulates growth, apoptosis, and patterning during development

The Drosophila expanded (ex) gene encodes a protein thought to play a role in signaling at apical junctions of epithelial cells. Previous studies have characterized this gene as a tumor suppressor involved in regulating the growth of a subset of Drosophila imaginal discs (Boedigheimer, M., Laughon, A., 1993. expanded: a gene involved in the control of cell proliferation in imaginal discs, Development 118, 1291-1301); although ex negatively regulates cell proliferation in the developing wing, it appeared to have a conflicting role in the eye. In contrast, our analysis of the loss-of-function phenotype indicates that ex does, in fact, regulate growth in the eye. We also show that this gene plays a role in patterning of the eye, mainly at the level of planar polarity. Our studies further demonstrate that, contrary to what was expected based on loss-of-function data, the tissue reduction phenotypes resulting from Ex overexpression are attributable to the induction of apoptotic cell death. Taken together, our data suggest that Ex is a versatile molecule that plays a role in most of the processes that govern disc development.     

The tumor suppressor Lats2 is pivotal in Aurora A and Aurora B signaling during mitosis

Accurate coordination between chromosome segregation and cytokinesis by various mitotic kinases, such as Aurora, prevent tetraploidization and subsequent tumorigensis. The tumor suppressors Lats1 and Lats2 are serine/threonine kinases that localize to the centrosome and regulate cell cycle progression and apoptosis. In the present study, Aurora A was demonstrated to phosphorylate Lats2 on serine 380 (S380) during mitosis. Immunocytochemical observations revealed that the subcellular localization of Lats2 was distinct during the cell cycle and depended on which site was phosphorylated. Interestingly, the S380-phosphorylated Lats2 protein (pS380) colocalized at the central spindle with Aurora B. Physical interactions were observed between Aurora A, Lats2, Lats1 and Aurora B. The Lats1 kinase was shown to phosphorylate Aurora B. Cells expressing a nonphosphorylated mutant (S380A) of Lats2 caused chromosome missegregation and cytokinesis failure, similar to cells with aberrantly expressed Aurora B. Together, the results suggest that the Aurora A-Lats1/2-Aurora B axis might be a novel pathway that regulates accurate mitotic progression by ensuring the proper mitotic localization of Lats2.     

Structure, expression, and chromosome mapping of LATS2, a mammalian homologue of the Drosophila tumor suppressor gene lats/warts

We have cloned and characterized LATS2, a novel mammalian homologue of the Drosophila tumor suppressor gene lats/warts. Northern blot analysis showed ubiquitous expression of mouse LATS2 (MmLATS2) mRNA, whereas expression of human LATS2 (HsLATS2) mRNA was enhanced in skeletal muscle and heart. Immunoblotting analysis of fractionated cell lysates showed HsLats2 to be a nuclear protein. We mapped the MmLATS2 gene to mouse chromosome 14 by interspecific backcross analysis. We also mapped the HsLATS2 gene (by fluorescence in situ hybridization) to the 13q11-q12 region, in which a loss of heterozygosity has been frequently observed in many primary cancers and to which the tumor suppressor genes RB and BRCA2 have also been mapped.     

Htid-1, the human homolog of the Drosophila melanogaster l(2)tid tumor suppressor, defines a novel physiological role of APC

Htid-1, the human counterpart of the Drosophila tumor suppressor gene lethal(2)tumorous imaginal discs (l(2)tid) encodes three splice forms translated into three cytosolic - Tid50, Tid48 and Tid46 - and three mitochondrial - Tid43, Tid40 and Tid38 - proteins. Here we provide evidence for the association of the endogenous Tid50/Tid48 proteins with the adenomatous polyposis coli (APC) tumor suppressor in normal colon epithelium, colorectal cancer cells and mouse NIH3T3 fibroblasts. Using the Glutathione S-transferase binding assay we show that the N-terminal region including the Armadillo domain (ARM) of APC is sufficient to bind the Tid molecules. Using immunoprecipitation and confocal microscopy we show that the two molecular partners complex at defined areas of the cells with further proteins such as Hsp70, Hsc70, Actin, Dvl and Axin. Our data implicate that the formation of the complex is not associated with APC's involvement in beta-Catenin degradation. Furthermore, though it is linked to Actin it is neither associated with regulation of Actin cytoskeleton due to APC's binding to Asef nor to Tid's binding to Ras-GAP. We suggest that the novel complex acts in maintaining APC's availability for its distinct roles in the Wnt signaling important for the cell to take the right decision, either to switch the cascade OFF or ON, thus, to regulate the onset of proliferation of the cells.     

Molecular mechanism of hTid-1, the human homolog of Drosophila tumor suppressor l(2)Tid, in the regulation of NF-kappaB activity and suppression of tumor growth

hTid-1, a human homolog of the Drosophila tumor suppressor l(2)Tid and a novel DnaJ protein, regulates the activity of nuclear factor kappaB (NF-kappaB), but its mechanism is not established. We report here that hTid-1 strongly associated with the cytoplasmic protein complex of NF-kappaB-IkappaB through direct interaction with IkappaBalpha/beta and the IKKalpha/beta subunits of the IkappaB kinase complex. These interactions resulted in suppression of the IKK activity in a J-domain-dependent fashion and led to the cytoplasmic retention and enhanced stability of IkappaB. Overexpression of hTid-1 by using recombinant baculovirus or adenovirus led to inhibition of cell proliferation and induction of apoptosis of human osteosarcoma cells regardless of the p53 expression status. Adherent cultured cells transduced with Ad.hTid-1 detached from the dish surface. Morphological changes consistent with apoptosis and cell death were evident 48 h after Ad.EGFP-hTid-1 transduction. In contrast, cells transduced with Ad.EGFP or Ad.EGFP-hTd-1DeltaN100, a mutant that has the N-terminal J domain deletion and that lost suppressive activity on IKK, continued to proliferate. Similar data were obtained with A375 human melanoma cells. Ad.EGFP or Ad.EGFP-hTd-1DeltaN100 ex vivo-transduced A375 cells injected subcutaneously into nude mice produced growing tumors, whereas Ad.EGFP-hTid-1-transduced cells did not. Collectively, the data suggest that hTid-1 represses the activity of NF-kappaB through physical and functional interactions with the IKK complex and IkappaB and, in doing so, it modulates cell growth and death.