Microfilament reorganization during apoptosis: the role of Gas2, a possible substrate for ICE-like proteases.

Gas2, a component of the microfilament system, belongs to the class of gas genes whose expression is induced at growth arrest. After serum or growth factor addition to quiescent NIH 3T3 cells, Gas2 is hyperphosphorylated and relocalized at the membrane ruffles. By overexpressing gas2wt and a series of deletion mutants of the C-terminal region, we have analysed its role in the organization of the actin cytoskeleton in different cell lines. Overexpression of Gas2 deleted at its C-terminal region (delta 276-314 and delta 236-314), but not its wild-type form, induces dramatic changes in the actin cytoskeleton and cell morphology. These effects are not due to interference of the deleted forms with the endogenous Gas2wt function but could be ascribed to a gain of function. We demonstrate that during apoptosis the C-terminal domain of Gas2 is removed by proteolytic cleavage, resulting in a protein that is similar in size to the described delta 276-314. Moreover, by using in vitro mutagenesis, we also demonstrate that the proteolytic processing of Gas2 during apoptosis is dependent on an aspartic acid residue at position 279. The evidence accumulated here could thus represent a first example of a mechanism linking apoptosis with the co-ordinated microfilament-dependent cell shape changes, as possibly mediated by an interleukin-1 beta-converting enzyme (ICE)-like dependent proteolytic cleavage of the Gas2 protein.     

Interaction of Rab3B with microtubule-binding protein Gas8 in NIH 3T3 cells

Rab3 subfamily small G proteins (Rab3A, Rab3B, Rab3C, and Rab3D) control the regulated exocytosis in neuronal/secretory cells. Rab3B is also detected and upregulated in non-neuronal/non-secretory cells, whereas its function remains elusive. In the present study, we identified growth-arrest-specific gene 8 (Gas8), an evolutionally conserved microtubule-binding protein that is upregulated in growth-arrested NIH 3T3 cells and involved in the dynein motor regulation in flagellar/ciliary axoneme, as a novel Rab3B-binding protein using a yeast two-hybrid system. Rab3B as well as Gas8 was upregulated in growth-arrested NIH 3T3 cells and enriched in testis and lung with well-developed flagella/cilia. Gas8 was specifically interacted with the GTP-bound form of Rab3B and co-localized with Rab3B at the Golgi in NIH 3T3 cells. Furthermore, Rab3B was relocated upon expression of the Rab3B-binding domain of Gas8. These results suggest that Gas8 links Rab3B to microtubules in NIH 3T3 cells.     

Association of the growth-arrest-specific protein Gas7 with F-actin induces reorganization of microfilaments and promotes membrane outgrowth.

The growth-arrest-specific gene, Gas7, is required for neurite outgrowth in cerebellar neurons. Here we report that Gas7 can induce the formation of extended cellular processes in NIH3T3 cells by interacting with actin and mediating reorganization of microfilaments. The Gas 7 protein, which increased markedly during growth arrest of NIH3T3 cells and persisted transiently at high levels upon reentry of cells into the cell cycle, localized near the plasma membrane and selectively colocalized with microfilaments in membrane ruffles. Process extensions induced by ectopic overexpression of Gas7 were blocked by the actin-depolymerizing agent cytochalasin D, suggesting that membrane extensions produced by Gas7 require actin polymerization. Association of endogenous Gas7 protein with microfilaments was verified by F-actin affinity chromatography; direct binding of purified His-Gas7 to actin also was demonstrated and shown to be mediated by the Gas7 C-terminal domain. Similarly, localization of Gas7 in membrane ruffles was mediated by the C-terminal domain, although neither this region nor the N-terminal domain was individually sufficient to induce process formation. Biochemical studies and electron microscopy showed that both full-length Gas7 protein and its C-terminal region can promote actin assembly as well as the crosslinking of actin filaments. We propose that Gas7 localized near the plasma membrane induces the assembly of actin and the membrane outgrowth.     

The protein encoded by a growth arrest-specific gene (gas6) is a new member of the vitamin K-dependent proteins related to protein S, a negative coregulator in the blood coagulation cascade.

A set of growth arrest-specific genes (gas) whose expression is negatively regulated after serum induction has previously been described (C. Schneider, R. M. King, and L. Philipson, Cell 54:787-793, 1988). The detailed analysis of one of them, gas6, is reported here, gas6 mRNA (2.6 kb) is abundantly expressed in serum-starved (48 h in 0.5% fetal calf serum) NIH 3T3 cells but decreases dramatically after fetal calf serum or basic fibroblast growth factor stimulation. The human homolog of gas6 was also cloned and sequenced, revealing a high degree of homology and a similar pattern of expression in IMR90 human fibroblasts. Computer analysis of the protein encoded by murine and human gas6 cDNAs showed significant homology (43 and 44% amino acid identity, respectively) to human protein S, a negative coregulator in the blood coagulation pathway. By using an anti-human Gas6 monospecific affinity-purified antibody, we show that the biosynthetic level of human Gas6 fully reflects mRNA expression in IMR90 human fibroblasts. This finding thus defines a new member of vitamin K-dependent proteins that is expressed in many human and mouse tissues and may be involved in the regulation of a protease cascade relevant in growth regulation.     

Requirement of phosphatidylinositol 3-kinase-dependent pathway and Src for Gas6-Axl mitogenic and survival activities in NIH 3T3 fibroblasts.

Gas6 is a secreted protein previously identified as the ligand of the Axl receptor tyrosine kinase. We have shown that Gas6 is able to induce cell cycle reentry of serum-starved NIH 3T3 cells and to efficiently prevent apoptosis after complete growth factor removal, a survival effect uncoupled from Gas6-induced mitogenesis. Here we report that the mitogenic effect of Gas6 requires phosphatidylinositol 3-kinase (PI3K) activity since it is abrogated both by the specific inhibitor wortmannin and by overexpression of the dominant negative P13K p85 subunit. Consistently, Gas6 activates the P13K downstream targets S6K and Akt, whose activation is abrogated by addition of wortmannin. Moreover, rapamycin treatment blocks Gas6-induced entry into the S phase of serum-starved NIH 3T3 cells. We also demonstrate the requirement of Src tyrosine kinase for Gas6 signalling since stable or transient expression of a catalytically inactive form of Src significantly inhibited Gas6-stimulated entry into the S phase. Accordingly, Gas6 addition to serum-starved NIH 3T3 cells causes activation of the intrinsic Src kinase activity. When specifically analyzed in a survival assay, these elements were found to be required for the survival effect of Gas6. Taken together, the evidence presented here identifies elements involved in the Gas6 transduction pathway that are responsible for its antiapoptotic effect and suggests that Src is involved in the events regulating cell survival.     

DEF6, a novel PH-DH-like domain protein, is an upstream activator of the Rho GTPases Rac1, Cdc42, and RhoA

In this paper, we describe the characterization of DEF6, a novel PH-DH-like protein related to SWAP-70 that functions as an upstream activator of Rho GTPases. In NIH 3T3 cells, stimulation of the PI 3-kinase signaling pathway with either H2O2 or platelet-derived growth factor (PDGF) resulted in the translocation of an overexpressed DEF6-GFP fusion protein to the cell membrane and induced the formation of filopodia and lamellipodia. In contrast to full-length DEF6, expression of the DH-like (DHL) domain as a GFP fusion protein potently induced actin polymerization, including stress fiber formation in COS-7 cells, in the absence of PI 3-kinase signaling, indicating that it was constitutively active. The GTP-loading of Cdc42 was strongly enhanced in NIH 3T3 cells expressing the DH domain while filopodia formation, membrane ruffling, and stress fiber formation could be inhibited by the co-expression of the DH domain with dominant negative mutants of either N17Rac1, N17Cdc42, or N19RhoA, respectively. This indicated that DEF6 acts upstream of the Rho GTPases resulting in the activation of the Cdc42, Rac1, and RhoA signaling pathways. In vitro, DEF6 specifically interacted with Rac1, Rac2, Cdc42, and RhoA, suggesting a direct role for DEF6 in the activation of Rho GTPases. The ability of DEF6 to both stimulate actin polymerization and bind to filamentous actin suggests a role for DEF6 in regulating cell shape, polarity, and movement.     

MAP, a protein interacting with a tumor suppressor, merlin, through the run domain

Merlin (or schwannomin) is a tumor suppressor encoded by the neurofibromatosis type 2 gene. Many studies have suggested that merlin is involved in the regulation of cell growth and proliferation through interactions with various cellular proteins. To better understand the function of merlin, we tried to identify the proteins that bind to merlin using the yeast two-hybrid screening. Characterization of the positive clones revealed a protein of 749 amino acids named merlin-associated protein (MAP), which showed wide tissue distribution in Northern blot analysis. Sequence analysis revealed that MAP is a potential homologue of a yeast check-point protein, BUB2, and contains TBC, SH3, and RUN domains, thereby implicating its role in the Ras-like GTPase signal pathways. MAP and merlin were directly associated in vitro and in vivo, and colocalized in NIH3T3 cells. The RUN domain of MAP and the C-terminus of merlin appeared to be responsible for their interaction. MAP decreased the AP-1-dependent promoter activity additively with merlin in NIH3T3 cells. In addition, merlin and MAP synergistically reduced the colony formation of NIH3T3 cells. These results suggest that MAP may play a cooperative role in the merlin-mediated growth suppression of cells.     

Phosphorylation of the growth arrest-specific protein Gas2 is coupled to actin rearrangements during Go-->G1 transition in NIH 3T3 cells

Growth arrest-specific (Gas2) protein has been shown to be a component of the microfilament system, that is highly expressed in growth arrested mouse and human fibroblasts and is hyperphosphorylated upon serum stimulation of quiescent cells. (Brancolini, C., S. Bottega, and C. Schneider. 1992. J. Cell Biol. 117:1251-1261). In this study we demonstrate that the kinetics of Gas2 phosphorylation, during Go-->G1 transition, as induced by addition of 20% FCS to serum starved NIH 3T3 cells, is temporally coupled to the reorganization of actin cytoskeleton. To better dissect the relationship between Gas2 phosphorylation and the modification of the microfilament architecture we used specific stimuli for both membrane ruffling (PDGF and PMA) and stress fiber formation (L-alpha-lysophosphatidic acid LPA) (Ridley, A. J., and A. Hall. 1992. Cell. 70:389-399). All of them, similarly to 20% FCS, are able to downregulate Gas2 biosynthesis. PDGF and PMA induce Gas2 hyperphosphorylation that is temporally coupled with the appearance of membrane ruffling where Gas2 localizes. On the other hand LPA, a specific stimulus for stress fiber formation, fails to induce a detectable Gas2 hyperphosphorylation. Thus, Gas2 hyperphosphorylation is specifically correlated with the formation of membrane ruffling possibly implying a role of Gas2 in this process.     

Enhancement of cell growth by intracellularly expressed herpes simplex virus thymidine kinase

A recombinant cell line (NIH3T3:pLtkSN) was made by infecting parental cells (NIH3T3) with a recombinant retrovirus (pLtkSN) encoding herpes simplex virus thymidine kinase (HSVtk) gene. The cells expressing HSVtk (NIH3T3:pLtkSN) grew 2.3 times more than the parental cells (NIH3T3) in Dulbecco's Modified Eagles Media containing 10% (v/v) horse serum. The NIH3T3:pLtkSN cells also showed a significant enhancement in the maximal cell concentration and the specific growth rate even at 2.5% serum concentration. The specific O2 uptake rate of NIH3T3 was 2.1 times greater than that of NIH3T3:pLtkSN. Under both O2-limited and O2-unlimited conditions, it appears that HSVtk plays an important role in enhancing the growth characteristics of animal cells.     

MRGD, a MAS-related G-protein coupled receptor, promotes tumorigenisis and is highly expressed in lung cancer

To elucidate the function of MAS-related GPCR, member D (MRGD) in cancers, we investigated the in vitro and in vivo oncogenic function of MRGD using murine fibroblast cell line NIH3T3 in which MRGD is stably expressed. The expression pattern of MRGD in clinical samples was also analyzed. We found that overexpression of MRGD in NIH3T3 induced focus formation and multi-cellular spheroid formation, and promoted tumors in nude mice. In other words, overexpression of MRGD in NIH3T3 induced the loss of contact inhibition, anchorage-independent growth and in vivo tumorigenesis. Furthermore, it was found that the ligand of MRGD, beta-alanine, enhanced spheroid formation in MRGD-expressing NIH3T3 cells. From investigation of clinical cancer tissues, we found high expression of MRGD in several lung cancers by immunohistochemistry as well as real time PCR. Based on these results, MRGD could be involved in tumorigenesis and could also be a novel anticancer drug target.     

A specific protein, p92, detected in flat revertants derived from NIH/3T3 transformed by human activated c-Ha-ras oncogene

Total proteins from a mouse embryo fibroblast cell line NIH/3T3, NIH/3T3 cells transformed by human activated c-Ha-ras (EJ-ras) oncogene (EJ-NIH/3T3), and the two flat revertant cell lines, R1 and R2, were analyzed by two-dimensional gel electrophoresis (IEF and NEPHGE). Several hundred polypeptides were resolved as seen by silver staining. Common alterations in four polypeptide spots were observed in the revertants when compared with NIH/3T3 and EJ-NIH/3T3 cells. In these alterations, a new polypeptide spot p92-5.7 (designated by molecular weight x 10(-3) and pI) was detected only in the revertants and not in NIH/3T3 and EJ-NIH/3T3 cells. Furthermore, the expression level of p92-5.7 seemed to be associated with the flat morphology and the reduced tumorigenicity of the revertants. Polypeptide p92-5.7 was also not detected in the total proteins extracted from BALB/3T3 cells, NIH Swiss mouse primary embryo fibroblasts, NRK (normal rat kidney) cells, and L6 (rat myoblast). Subcellular fractionation of total protein from R1 cells revealed that the p92-5.7 was present in the cytosol. Western blot analysis using an anti-gelsolin antibody demonstrated that the p92-5.7 might be a variant form of gelsolin which is thought to be an actin regulatory protein or a gelsolin-like polypeptide. These results may suggest that the expression of p92-5.7 detected only in the revertants is associated, at least in part, with the reversion. This may be the first demonstration of specific protein expression in the flat revertants.     

FGF3 attached to a phosholipid membrane anchor gains a high transforming capacity. Implications of microdomains for FGF3 cell transformation

NIH3T3 cells transformed by mouse FGF3-cDNA (DMI cells) selected for their ability to grow as anchorage-independent colonies in soft agar and in defined medium lacking growth factors exhibit a highly transformed phenotype. We have used dominant negative (DN) fibroblast growth factor (FGF) receptor 2 (FGFR2) isoforms to block the FGF response in DMI cells. When the DN-FGFR was expressed in DMI cells, their transformed phenotype can be reverted. The truncated FGFR2(IIIb), the high affinity FGFR for FGF3, is significantly more efficient at reverting the transformed phenotype as the IIIc isoform, reaffirming the notion that the affinity of the ligand to the DN-FGFR2 isoform determines the effect. Heparin or heparan sulfate displaces FGF3 from binding sites on the cell surface inhibiting the growth of DMI cells and reverts the transformed phenotype (). However, the presence of heparin is necessary to induce a mitogenic response in NIH3T3 cells when stimulated with soluble purified mouse FGF3. We have investigated the importance of cell surface binding of FGF3 for its ability to transform NIH3T3 cells by creating an FGF3 mutant anchored to the membrane via glycosylphosphatidylinositol (GPI). The GPI anchor renders the cell surface association of FGF3 independent from binding to heparan sulfate-proteoglycan of the cell surface membrane. Attachment of a GPI anchor to FGF3 also confers a much higher transforming potential to the growth factor. Even more, the purified GPI-attached FGF3 is as much transforming as the secreted protein acting in an autocrine mode. Because NIH3T3 cells do not express the high affinity tyrosine kinase FGF receptors for FGF3, these findings suggest that FGF3 attached to GPI-linked heparan sulfate-proteoglycan may have a broader biological activity as when bound to transmembrane or soluble heparan sulfate-proteoglycan.     

Effect of cyclin E overexpression on lovastatin-induced G1 arrest and RhoA inactivation in NIH3T3 cells.

The HMG-CoA reductase inhibitor, lovastatin, blocks targeting of the Rho and Ras families of small GTPases to their active sites by inhibiting protein prenylation. Control NIH3T3 cells, and those overexpressing human cyclin E protein were treated with lovastatin for 24 h to determine the effects of cyclin E overexpression on lovastatin-induced growth arrest and cell rounding. Lovastatin treatment (10 microM) of control 3T3 cells resulted in growth arrest at G1 accompanied by actin stress fiber disassembly, cell rounding, and decreased active RhoA from the membranous protein fraction. By contrast, in NIH3T3 cells overexpressing cyclin E, lovastatin did not cause loss of RhoA from the membrane (active) protein fraction, actin stress fiber disassembly, cell rounding or growth arrest within 24 h. Analysis of cell cycle proteins showed that 24 h of lovastatin treatment in the control cells caused an elevation in the levels of the cyclin-dependent kinase inhibitor p27(kip1), inhibition of both cyclin E- and cyclin A-dependent kinase activity, and decreased levels of hyperphosphorylated retinoblastoma protein (pRb). By contrast, lovastatin treatment of the cyclin E overexpressors did not suppress either cyclin E- or cyclin A-dependent kinase activity, nor did it alter the level of maximally phosphorylated pRb, despite increased levels of p27(kip1). However, by 72 h, the cyclin E overexpressors rounded up but remained attached to the substratum, indicating a delayed response to lovastatin. In contrast with lovastatin, inactivation of membrane-bound Rho proteins (i.e., GTP-bound RhoA, RhoB, RhoC) with botulinum C3 transferase caused cell rounding and G1 growth arrest in both cell types but did not inhibit cyclin E-dependent histone kinase activity in the cyclin E overexpressors. In addition, 24 h of cycloheximide treatment caused depletion of RhoA from the membrane (active) fraction in neo cells, but in the cells overexpressing cyclin E, RhoA remained in the active (membrane-associated) fraction. Our observations suggest that (1) RhoA activation occurs downstream of cyclin E-dependent kinase activation, and (2) overexpression of cyclin E decreased the turnover rate of active RhoA.     

The murine homeo box gene product, Hox 1.1 protein, is growth-controlled and associated with chromatin

In order to gain insight into the function of the Hox 1.1 gene, we studied the expression of the murine homeo box gene product, the Hox 1.1 protein. Monoclonal antibodies were raised against synthetic peptides of the Hox 1.1 protein to study the localization and expression pattern of this protein under various culture conditions. By means of indirect immunofluorescence we localized the Hox 1.1 protein to the nucleus in differentiated F9 and NIH 3T3 cells. During mitosis the protein was found to be associated with chromatin. Confluent NIH 3T3 cells harbored little if any Hox 1.1 protein. After "wounding" the cells in this confluent monolayer, we observed an induction of the expression of the Hox 1.1 protein. However, addition of insulin to F9 and contact-inhibited NIH 3T3 cells led to an increase of the Hox 1.1 RNA and protein expression. Thus, the induction of the Hox 1.1 protein is associated not only with the differentiation of embryonal carcinoma (EC) cells, but may also correlate with stages of cell growth.     

Phosphorylation-dephosphorylation of retinoblastoma protein not necessary for passage through the mammalian cell division cycle

Phosphorylation of the retinoblastoma protein (Rb) during the G1-phase of the mammalian cell division cycle is currently believed to be a controlling element regulating the passage of cells into S-phase. We find, however, that the suspension-grown cell lines U937, L1210, and MOLT-4 contain exclusively hyperphosphorylated Rb. Furthermore, when adherent NIH3T3 cells are grown at very low densities to avoid overgrowth and contact inhibition, they also contain only hyperphosphorylated Rb. NIH3T3 cells exhibit hypophosphorylation when the cells are grown at moderate to high cell densities. We propose that cultures of adherent cells such as NIH3T3, when grown to moderate cell densities, are made up of two populations of cells: (a) cells that are relatively isolated and therefore growing exponentially without contact inhibition, and (b) cells that are growth-inhibited by local cell density or contact inhibition. The common observation in adherent cell lines, that Rb is both hyper- and hypophosphorylated in the G1-phase and only hyperphosphorylated in the S- and G2-phases, is explained by the effects of cell density and contact inhibition. Thus, phosphorylation-dephosphorylation of Rb protein during the G1 phase is not a necessary process during the NIH3T3, L1210, MOLT-4, and U937 division cycles. We propose that phosphorylation-dephosphorylation of Rb is independent of the division cycle and is primarily determined by growth conditions throughout the division cycle.     

Isolation and characterization of NIH 3T3 cells expressing polyomavirus small T antigen.

The polyomavirus small T-antigen gene, together with the polyomavirus promoter, was inserted into a retrovirus vector pGV16 which contains the Moloney sarcoma virus long terminal repeat and neomycin resistance gene driven by the simian virus 40 promoter. This expression vector, pGVST, was packaged into retrovirus particles by transfection of psi 2 cells which harbor packaging-defective murine retrovirus genome. NIH 3T3 cells were infected by this replication-defective retrovirus containing pGVST. Of the 15 G418-resistant cell clones, 8 express small T antigen at various levels as revealed by immunoprecipitation. A cellular protein with an apparent molecular weight of about 32,000 coprecipitates with small T antigen. Immunofluorescent staining shows that small T antigen is mainly present in the nuclei. Morphologically, cells expressing small T antigen are indistinguishable from parental NIH 3T3 cells and have a microfilament pattern similar to that in parental NIH 3T3 cells. Cells expressing small T antigen form a flat monolayer but continue to grow beyond the saturation density observed for parental NIH 3T3 cells and eventually come off the culture plate as a result of overconfluency. There is some correlation between the level of expression of small T antigen and the growth rate of the cells. Small T-antigen-expressing cells form small colonies in soft agar. However, the proportion of cells which form these small colonies is rather small. A clone of these cells tested did not form tumors in nude mice within 3 months after inoculation of 10(6) cells per animal. Thus, present studies establish that the small T antigen of polyomavirus is a second nucleus-localized transforming gene product of the virus (the first one being large T antigen) and by itself has a function which is to stimulate the growth of NIH 3T3 cells beyond their saturation density in monolayer culture.