Dependence of Dbl and Dbs transformation on MEK and NF-kappaB activation.

Dbs was identified initially as a transforming protein and is a member of the Dbl family of proteins (>20 mammalian members). Here we show that Dbs, like its rat homolog Ost and the closely related Dbl, exhibited guanine nucleotide exchange activity for the Rho family members RhoA and Cdc42, but not Rac1, in vitro. Dbs transforming activity was blocked by specific inhibitors of RhoA and Cdc42 function, demonstrating the importance of these small GTPases in Dbs-mediated growth deregulation. Although Dbs transformation was dependent upon the structural integrity of its pleckstrin homology (PH) domain, replacement of the PH domain with a membrane localization signal restored transforming activity. Thus, the PH domain of Dbs (but not Dbl) may be important in modulating association with the plasma membrane, where its GTPase substrates reside. Both Dbs and Dbl activate multiple signaling pathways that include activation of the Elk-1, Jun, and NF-kappaB transcription factors and stimulation of transcription from the cyclin D1 promoter. We found that Elk-1 and NF-kappaB, but not Jun, activation was necessary for Dbl and Dbs transformation. Finally, we have observed that Dbl and Dbs regulated transcription from the cyclin D1 promoter in a NF-kappaB-dependent manner. Previous studies have dissociated actin cytoskeletal activity from the transforming potential of RhoA and Cdc42. These observations, when taken together with those of the present study, suggest that altered gene expression, and not actin reorganization, is the critical mediator of Dbl and Rho family protein transformation.

Effects of inhibitors of tumoricidal activity upon schistosomulum killing by activated macrophages.

Larvae of the helminth parasite Schistosoma mansoni are efficiently killed in vitro by lymphokine-activated macrophages, leading to the hypothesis that these cells may participate in the effector mechanism of protective immunity against schistosomiasis. Larvacidal activity has also been demonstrated in the IC-21 macrophage cell line in the absence of a demonstrable respiratory burst, indicating that macrophages possess nonoxidative mechanisms of schistosomulum killing. In this study, we demonstrated that IC-21 larval killing was most effective when contact was allowed between cells and target. Nonoxidative larvacidal activity was prevented by protein synthesis inhibitors, by the inhibition of microtubule polymerization, and by tosyllysylchloromethylketone but not by other inhibitors or substrates of tryptic or chymotryptic protease activity. The addition of excess iron to the culture also prevented IC-21-mediated larval killing, suggesting that the production of an iron-binding molecule may be involved. In contrast, the addition of excess thymidine or arginine did not reverse macrophage larvacidal activity, nor did lysosomotropic agents that depress the activity of acid hydrolases. Under appropriate conditions of activation and surface membrane stimulation, IC-21 cells could be induced to release soluble cytotoxic factors retaining larvacidal activity. These observations provide insight into the mechanism of macrophage-mediated schistosome killing, in comparison to the cytotoxic mechanisms described in the better-studied tumoricidal models, and supply a basis for further biochemical investigation of macrophage function against a multicellular target.

The Dbl-related protein, Lfc, localizes to microtubules and mediates the activation of Rac signaling pathways in cells.

The possibility that the Dbl family member Lfc can activate Rac1 in cells is investigated in this study. Previously, we demonstrated that both Lfc and Lsc, like their closest relative Lbc, can act catalytically in stimulating the guanine nucleotide exchange activity of RhoA in vitro. Neither Lfc nor Lsc stimulated the in vitro exchange activity of Cdc42 or Rac1; however, Lfc was capable of forming a tight complex with Rac1 in vitro. We show here that Lfc stimulates c-Jun kinase (JNK) activity in COS-7 cells. This stimulation was blocked by a dominant negative mutant of Rac1 and somewhat less effectively by dominant negative RhoA, but not by dominant negative Cdc42. Overexpression of Lfc in NIH 3T3 cells induced the formation of actin stress fibers and membrane ruffles, consistent with the activation of both RhoA and Rac1 signaling pathways, whereas overexpression of Lsc led exclusively to well developed stress fibers. Using a recently developed assay for measuring the cellular activation of Rac, we did not find that expression of Lfc increased the levels of GTP-bound Rac1. However, an examination of the cellular localization of Lfc showed that it was localized to microtubules, similar to what has been reported for activated Rac1, the mixed lineage kinase (MLK) and JNK. Moreover, we have found that the Pleckstrin homology (PH) domain of Lfc specifically associates with tubulin. Taken together, these findings suggest a model where the PH domain-mediated localization of Lfc to microtubules enables the recruitment of Rac to a site proximal to its signaling targets, resulting in JNK activation and actin cytoskeletal changes.

Lfc and Lsc oncoproteins represent two new guanine nucleotide exchange factors for the Rho GTP-binding protein.

Lfc and Lsc are two recently identified oncoproteins that contain a Dbl homology domain in tandem with a pleckstrin homology domain and thus share sequence similarity with a number of other growth regulatory proteins including Dbl, Tiam-1, and Lbc. We show here that Lfc and Lsc, like their closest relative Lbc, are highly specific guanine nucleotide exchange factors (GEFs) for Rho, causing a >10-fold stimulation of [3H]GDP dissociation from Rho and a marked stimulation of GDP-[35S]GTPgammas (guanosine 5'-O-(3-thiotriphosphate) exchange. All three proteins (Lbc, Lfc, and Lsc) are able to act catalytically in stimulating the guanine nucleotide exchange activity, such that a single molecule of each of these oncoproteins can activate a number of molecules of Rho. Neither Lfc nor Lsc shows any ability to stimulate GDP dissociation from other related GTP-binding proteins such as Rac, Cdc42, or Ras. Thus Lbc, Lfc, and Lsc appear to represent a subgroup of Dbl-related proteins that function as highly specific GEFs toward Rho and can be distinguished from Dbl, Ost, and Dbs which are less specific and show GEF activity toward both Rho and Cdc42. Consistent with these results, Lbc, Lfc, and Lsc each form tight complexes with the guanine nucleotide-depleted form of Rho and bind weakly to the GDP- and GTPgammaS-bound states. None of these oncoproteins are able to form complexes with Cdc42 or Ras. However, Lfc (but not Lbc nor Lsc) can bind to Rac, and this binding occurs equally well when Rac is nucleotide-depleted or is in the GDP- or GTPgammaS-bound state. These findings raise the possibility that in addition to acting directly as a GEF for Rho, Lfc may play other roles that influence the signaling activities of Rac and/or coordinate the activities of the Rac and Rho proteins.

Phosphatidylinositol 4,5-bisphosphate provides an alternative to guanine nucleotide exchange factors by stimulating the dissociation of GDP from Cdc42Hs.

Members of the Rho subfamily of Ras-related GTP-binding proteins play important roles in the organization of the actin cytoskeleton and in the regulation of cell growth. We have shown previously that the dbl oncogene product, which represents a prototype for a family of growth regulatory proteins, activates Rho subfamily GTP-binding proteins by catalyzing the dissociation of GDP from their nucleotide binding site. In the present study, we demonstrate that the acidic phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2), provides an alternative mechanism for the activation of Cdc42Hs. Among a variety of lipids tested, only PIP2 was able to stimulate GDP release from Cdc42Hs in a dose-dependent manner, with a half-maximum effect at approximately 50 microM. Unlike the Dbl oncoprotein, which requires the presence of (free) guanine nucleotide in the medium to replace the GDP bound to Cdc42Hs, PIP2 stimulates GDP release from Cdc42Hs in the absence of free guanine nucleotide. PIP2, when incorporated into phosphatidylcholine carrier vesicles, binds tightly to the guanine nucleotide-depleted form of Cdc42Hs and weakly to the GDP-bound form of the GTP-binding protein but does not bind to GTP-bound Cdc42Hs, similar to what was observed for the Dbl oncoprotein. However, mutational analysis of Cdc42Hs indicates that the site that is essential for the functional interaction between PIP2 and Cdc42Hs is distinct from the Dbl-binding site and is located at the positively charged carboxyl-terminal end of the GTP-binding protein. The GDP-releasing activity of PIP2 is highly effective toward Cdc42Hs and Rho (and is similar to the reported effects of PIP2 on Arf (Terui, T., Kahn, R. A., and Randazzo, P. A., (1994) J. Biol. Chem. 269, 28130-28135)), is less effective with Rac, and is not observed with Ras, Rap1a, or Ran. The ability of PIP2 to activate Cdc42Hs (or Rho) and Arf provides a possible point of convergence for the biological pathways regulated by these different GTP-binding proteins and may be related to the synergism observed between Arf and Rho-subtype proteins in the stimulation of phospholipase D activity (Singer, W. D., Brown, H. A., Bokoch, G. M., and Sternweis, P. C. (1995) J. Biol. Chem. 270, 14944-14950).