Patricia A. Solski

Activation of Rac1, RhoA, and Mitogen-Activated Protein Kinases Is Required for Ras Transformation

Although substantial evidence supports a critical role for the activation of Raf-1 and mitogen-activated protein kinases (MAPKs) in oncogenic Ras-mediated transformation, recent evidence suggests that Ras may activate a second signaling pathway which involves the Ras-related proteins Rac1 and RhoA. Consequently, we used three complementary approaches to determine the contribution of Rac1 and RhoA function to oncogenic Ras-mediated transformation. First, whereas constitutively activated mutants of Rac1 and RhoA showed very weak transforming activity when transfected alone, their coexpression with a weakly transforming Raf-1 mutant caused a greater than 35-fold enhancement of transforming activity. Second, we observed that coexpression of dominant negative mutants of Rac1 and RhoA reduced oncogenic Ras transforming activity. Third, activated Rac1 and RhoA further enhanced oncogenic Ras-triggered morphologic transformation, as well as growth in soft agar and cell motility. Finally, we also observed that kinase-deficient MAPKs inhibited Ras transformation. Taken together, these data support the possibility that oncogenic Ras activation of Rac1 and RhoA, coupled with activation of the Raf/MAPK pathway, is required to trigger the full morphogenic and mitogenic consequences of oncogenic Ras transformation.

Oncogenic ras activation of Raf/mitogen-activated protein kinase-independent pathways is sufficient to cause tumorigenic transformation

Substantial evidence supports a critical role for the activation of the Raf-1/MEK/mitogen-activated protein kinase pathway in oncogenic Ras-mediated transformation. For example, dominant negative mutants of Raf-1, MEK, and mitogen-activated protein kinase all inhibit Ras transformation. Furthermore, the observation that plasma membrane-localized Raf-1 exhibits the same transforming potency as oncogenic Ras suggests that Raf-1 activation alone is sufficient to mediate full Ras transforming activity. However, the recent identification of other candidate Ras effectors (e.g., RalGDS and phosphatidylinositol-3 kinase) suggests that activation of other downstream effector-mediated signaling pathways may also mediate Ras transforming activity. In support of this, two H-Ras effector domain mutants, H-Ras(12V, 37G) and H-Ras(12V, 40C), which are defective for Raf binding and activation, induced potent tumorigenic transformation of some strains of NIH 3T3 fibroblasts. These Raf-binding defective mutants of H-Ras induced a transformed morphology that was indistinguishable from that induced by activated members of Rho family proteins. Furthermore, the transforming activities of both of these mutants were synergistically enhanced by activated Raf-1 and inhibited by the dominant negative RhoA(19N) mutant, indicating that Ras may cause transformation that occurs via coordinate activation of Raf-dependent and -independent pathways that involves Rho family proteins. Finally, cotransfection of H-Ras(12V, 37G) and H-Ras(12V, 40C) resulted in synergistic cooperation of their focus-forming activities, indicating that Ras activates at least two Raf-independent, Ras effector-mediated signaling events.

Dbl and Vav mediate transformation via mitogen-activated protein kinase pathways that are distinct from those activated by oncogenic Ras.

Vav and Dbl are members of a novel class of oncogene proteins that share significant sequence identity in a approximately 250-amino-acid domain, designated the Dbl homology domain. Although Dbl functions as a guanine nucleotide exchange factor (GEF) and activator of Rho family proteins, recent evidence has demonstrated that Vav functions as a GEF for Ras proteins. Thus, transformation by Vav and Dbl may be a consequence of constitutive activation of Ras and Rho proteins, respectively. To address this possibility, we have compared the transforming activities of Vav and Dbl with that of the Ras GEF, GRF/CDC25. As expected, GRF-transformed cells exhibited the same reduction in actin stress fibers and focal adhesions as Ras-transformed cells. In contrast, Vav- and Dbl-transformed cells showed the same well-developed stress fibers and focal adhesions observed in normal or RhoA(63L)-transformed NIH 3T3 cells. Furthermore, neither Vav- or Dbl-transformed cells exhibited the elevated levels of Ras-GTP (60%) observed with GRF-transformed cells. Finally, GRF, but not Vav or Dbl, induced transcriptional activation from Ras-responsive DNA elements (ets/AP-1, fos promoter, and kappa B). However, like Ras- and GRF-transformed cells, both Vav- and Dbl-transformed cells exhibited constitutively activated mitogen-activated protein kinases (MAPKs) (primarily p42MAPK/ERK2). Since kinase-deficient forms of p42MAPK/ERK2 and p44MAPK/ERK1 inhibited Dbl transformation, MAPK activation may be an important component of its transforming activity. Taken together, our observations indicate that Vav and Dbl transformation is not a consequence of Ras activation and instead may involve the constitutive activation of MAPKs.

Cellular functions of TC10, a Rho family GTPase: regulation of morphology, signal transduction and cell growth.

The small Ras-related GTPase, TC10, has been classified on the basis of sequence homology to be a member of the Rho family. This family, which includes the Rho, Rac and CDC42 subfamilies, has been shown to regulate a variety of apparently diverse cellular processes such as actin cytoskeletal organization, mitogen-activated protein kinase (MAPK) cascades, cell cycle progression and transformation. In order to begin a study of TC10 biological function, we expressed wild type and various mutant forms of this protein in mammalian cells and investigated both the intracellular localization of the expressed proteins and their abilities to stimulate known Rho family-associated processes. Wild type TC10 was located predominantly in the cell membrane (apparently in the same regions as actin filaments), GTPase defective (75L) and GTP-binding defective (31N) mutants were located predominantly in cytoplasmic perinuclear regions, and a deletion mutant lacking the carboxyl terminal residues required for post-translational prenylation was located predominantly in the nucleus. The GTPase defective (constitutively active) TC10 mutant: (1) stimulated the formation of long filopodia; (2) activated c-Jun amino terminal kinase (JNK); (3) activated serum response factor (SRF)-dependent transcription; (4) activated NF-κB-dependent transcription; and (5) synergized with an activated Raf-kinase (Raf-CAAX) to transform NIH3T3 cells. In addition, wild type TC10 function is required for full H-Ras transforming potential. We demonstrate that an intact effector domain and carboxyl terminal prenylation signal are required for proper TC10 function and that TC10 signals to at least two separable downstream target pathways. In addition, TC10 interacted with the actin-binding and filament-forming protein, profilin, in both a two-hybrid cDNA library screen, and an in vitro binding assay. Taken together, these data support a classification of TC10 as a member of the Rho family, and in particular, suggest that TC10 functions to regulate cellular signaling to the actin cytoskeleton and processes associated with cell growth.

A non-farnesylated Ha-Ras protein can be palmitoylated and trigger potent differentiation and transformation

Ha-Ras undergoes post-translational modifications (including attachment of farnesyl and palmitate) that culminate in localization of the protein to the plasma membrane. Because palmitate is not attached without prior farnesyl addition, the distinct contributions of the two lipid modifications to membrane attachment or biological activity have been difficult to examine. To test if palmitate is able to support these crucial functions on its own, novel C-terminal mutants of Ha-Ras were constructed, retaining the natural sites for palmitoylation, but replacing the C-terminal residue of the CAAX signal for prenylation with six lysines. Both the Ext61L and ExtWT proteins were modified in a dynamic fashion by palmitate, without being farnesylated; bound to membranes modestly (40% as well as native Ha-Ras); and retained appropriate GTP binding properties. Ext61L caused potent transformation of NIH 3T3 cells and, unexpectedly, an exaggerated differentiation of PC12 cells. Ext61L with the six lysines but lacking palmitates was inactive. Thus, farnesyl is not needed as a signal for palmitate attachment or removal, and a combination of transient palmitate modification and basic residues can support Ha-Ras membrane binding and two quite different biological functions. The roles of palmitate can therefore be independent of and distinct from those of farnesyl. Reciprocally, if membrane association can be sustained largely through palmitates, farnesyl is freed to interact with other proteins.

Citron Kinase, a RhoA Effector, Enhances HIV‐1 Virion Production by Modulating Exocytosis

RhoGTPases play important roles in the regulation of protein transport and membrane recycling. Little is known, however, about how RhoGTPases affect HIV‐1 virion production, which is dependent on the endosomal sorting pathway. We report that ectopic expression of citron kinase (citron‐K), a RhoA effector, preferentially enhances HIV‐1 virion production. Depletion of endogenous citron‐K inhibits HIV‐1 virion production. Citron‐N, which lacks the kinase domain, also enhances HIV‐1 virion production. The leucine zipper, Rho‐binding and zinc finger domains of citron‐N are necessary for the enhancement activity. Citron‐K also enhances murine leukemia virion production and the HIV‐1 late domain is not required for the citron‐K‐mediated enhancement. Ectopic expression of citron‐K leads to the formation of cytoplasmic structures containing citron‐K and HIV‐1 Gag proteins. HIV‐1 and citron‐K cooperatively enhance acidic endosome and lysosome compartments. Finally, citron‐K promotes exocytosis of microvesicles or exosomes that co‐purify with HIV‐1 virions. We conclude that citron‐K enhances HIV‐1 virion production by stimulating the endosomal compartments and exocytosis.

Modulation of HIV-1 Replication by a Novel RhoA Effector Activity

The RhoA GTPase is involved in regulating actin cytoskeletal organization, gene expression, cell proliferation, and survival. We report here that p115-RhoGEF, a specific guanine nucleotide exchange factor (GEF) and activator of RhoA, modulates HIV-1 replication. Ectopic expression of p115-RhoGEF or Gα13, which activates p115-RhoGEF activity, leads to inhibition of HIV-1 replication. RhoA activation is required and the inhibition affects HIV-1 gene expression. The RhoA effector activity in inhibiting HIV-1 replication is genetically separable from its activities in transformation of NIH3T3 cells, activation of serum response factor, and actin stress fiber formation. These findings reveal that the RhoA signal transduction pathway regulates HIV-1 replication and suggest that RhoA inhibits HIV-1 replication via a novel effector activity.

Targeting proteins to membranes, using signal sequences for lipid modifications

Changing an existing lipid or appending a lipid to a cytosolic protein has emerged as an important technique for targeting proteins to membranes and for constitutively activating the membrane-bound protein. The potential for more precise or regulated interactions of lipidated proteins in membrane subdomains suggests that this method for membrane targeting will be of increasing usefulness.

The Ect2 Rho Guanine Nucleotide Exchange Factor Is Essential for Early Mouse Development and Normal Cell Cytokinesis and Migration

Ect2 is a member of the human Dbl family of guanine nucleotide exchange factors (RhoGEFs) that serve as activators of Rho family small GTPases. Although Ect2 is one of at least 25 RhoGEFs that can activate the RhoA small GTPase, cell culture studies using established cell lines determined that Ect2 is essential for mammalian cell cytokinesis and proliferation. To address the function of Ect2 in normal mammalian development, we performed gene targeting to generate Ect2 knockout mice. The heterozygous Ect2(+/-) mice showed normal development and life span, indicating that Ect2 haplodeficiency was not deleterious for development or growth. In contrast, Ect2(-/-) embryos were not found at birth or postimplantation stages. Ect2(-/-) blastocysts were recovered at embryonic day 3.5 but did not give rise to viable outgrowths in culture, indicating that Ect2 is required for peri-implantation development. To further assess the importance of Ect2 in normal cell physiology, we isolated primary fibroblasts from Ect2(fl/fl) embryos (MEFs) and ablated Ect2 using adenoviral delivery of Cre recombinase. We observed a significant increase in multinucleated cells and accumulation of cells in G2/M phase, consistent with a role for Ect2 in cytokinesis. Ect2 deficiency also caused enlargement of the cytoplasm and impaired cell migration. Finally, although Ect2-dependent activation of RhoA has been implicated in cytokinesis, Ect2 can also activate Rac1 and Cdc42 to cause growth transformation. Surprisingly, ectopic expression of constitutively activated RhoA, Rac1, or Cdc42, known substrates of Ect2, failed to phenocopy Ect2 and did not rescue the defect in cytokinesis caused by loss of Ect2. In summary, our results establish the unique role of Ect2 in development and normal cell proliferation.