Xosé R. Bustelo

S. typhimurium Encodes an Activator of Rho GTPases that Induces Membrane Ruffling and Nuclear Responses in Host Cells

S. typhimurium stimulates signaling pathways leading to membrane ruffling, actin cytoskeleton rearrangements, and nuclear responses. The stimulation requires a protein secretion system (type III) that translocates bacterial proteins into the host cell. We show that SopE, a substrate of this secretion system, stimulates cytoskeletal reorganization and JNK activation in a CDC42- and Rac-1-dependent manner. A lambda gt11 cDNA library screen for proteins that interact with SopE identified Rac-1 and CDC42. Furthermore, purified SopE was shown to stimulate GDP/GTP nucleotide exchange in several Rho GTPases in vitro, including Rac-1 and CDC42. These findings establish a paradigm for microbial stimulation of cellular responses in which the pathogen induces signaling events by directly engaging the signaling machinery within the host cell.

Regulatory and Signaling Properties of the Vav Family

X.R.B. is a Sinsheimer Scholar for Cancer Research whose own work is supported by the National Cancer Institute (CA7373501), the Baldwin Foundation for Breast Cancer Research, and the Association for International Cancer Research.

Phosphorylation-dependent and constitutive activation of Rho proteins by wild-type and oncogenic Vav-2

We show here that Vav‐2, a member of the Vav family of oncoproteins, acts as a guanosine nucleotide exchange factor (GEF) for RhoG and RhoA‐like GTPases in a phosphotyrosine‐dependent manner. Moreover, we show that Vav‐2 oncogenic activation correlates with the acquisition of phosphorylation‐independent exchange activity. In vivo, wild‐type Vav‐2 is activated oncogenically by tyrosine kinases, an effect enhanced further by co‐expression of RhoA. Likewise, the Vav‐2 oncoprotein synergizes with RhoA and RhoB proteins in cellular transformation. Transient transfection assays in NIH‐3T3 cells show that phosphorylated wild‐type Vav‐2 and the Vav‐2 oncoprotein induce cytoskeletal changes resembling those observed by the activation of the RhoG pathway. In contrast, the constitutive expression of the Vav‐2 oncoprotein in rodent fibroblasts leads to major alterations in cell morphology and to highly enlarged cells in which karyokinesis and cytokinesis frequently are uncoupled. These results identify a regulated GEF for the RhoA subfamily, provide a biochemical explanation for vav family oncogenicity, and establish a new signaling model in which specific Vav‐like proteins couple tyrosine kinase signals with the activation of distinct subsets of the Rho/Rac family of GTPases.

Biological and Regulatory Properties of Vav-3, a New Member of the Vav Family of Oncoproteins

ABSTRACT We report here the identification and characterization of a novel Vav family member, Vav-3. Signaling experiments demonstrate that Vav-3 participates in pathways activated by protein tyrosine kinases. Vav-3 promotes the exchange of nucleotides on RhoA, on RhoG and, to a lesser extent, on Rac-1. During this reaction, Vav-3 binds physically to the nucleotide-free states of those GTPases. These functions are stimulated by tyrosine phosphorylation in wild-type Vav-3 and become constitutively activated upon deletion of the entire calponin-homology region. Expression of truncated versions of Vav-3 leads to drastic actin relocalization and to the induction of stress fibers, lamellipodia, and membrane ruffles. Moreover, expression of Vav-3 alters cytokinesis, resulting in the formation of binucleated cells. All of these responses need only the expression of the central region of Vav-3 encompassing the Dbl homology (DH), pleckstrin homology (PH), and zinc finger (ZF) domains but do not require the presence of the C-terminal SH3-SH2-SH3 regions. Studies conducted with Vav-3 proteins containing loss-of-function mutations in the DH, PH, and ZF regions indicate that only the DH and ZF regions are essential for Vav-3 biological activity. Finally, we show that one of the functions of the Vav-3 ZF region is to work coordinately with the catalytic DH region to promote both the binding to GTP-hydrolases and their GDP-GTP nucleotide exchange. These results highlight the role of Vav-3 in signaling and cytoskeletal pathways and identify a novel functional cross-talk between the DH and ZF domains of Vav proteins that is imperative for the binding to, and activation of, Rho GTP-binding proteins.

Vav proteins, adaptors and cell signaling.

The Vav family is a group of signal transduction molecules with oncogenic potential that play important roles in development and cell signaling. Members of this family are distributed in all animal metazoans but not in unicellular organisms. Recent genomic studies suggest that the function of Vav proteins co-evolved with tyrosine kinase pathways, probably to assure the optimal conversion of extracellular signals into biological responses coupled to the cytoskeleton and gene transcription. To date, the best-known function of Vav proteins is their role as GDP/GTP exchange factors for Rho/Rac molecules, a function strictly controlled by tyrosine phosphorylation. Recent publications indicate that this function is highly dependent on the interaction of adaptor proteins that aid in the proper phosphorylation of Vav proteins, their interaction with other signaling molecules, and in modulating the strength of their signal outputs. In addition to the function of Vav proteins as exchange factors, there is increasing evidence suggesting that Vav proteins can mediate other cellular functions independently of their exchange activities, probably by working themselves as adaptor molecules. In this review, we will give a summary of the recent advances in this field, placing special emphasis on the non-catalytic roles of Vav and its interaction with other adaptor molecules.

Tyrosine Phosphorylation Mediates Both Activation and Downmodulation of the Biological Activity of Vav

ABSTRACT Vav works as a GDP/GTP exchange factor for Rac GTPases, thereby facilitating the transition of these proteins from the inactive (GDP-bound) into the active (GTP-bound) state. The stimulation of Vav exchange activity during cell signaling is mediated by tyrosine phosphorylation. To understand the roles of phosphorylation in the regulation of Vav activity, we have initiated the characterization of the residues of Vav that are phosphorylated during signal transduction. Here we show that a Y-to-F mutation in one of these residues, Y174, leads to the oncogenic activation of Vav and to the enhancement of other Vav-mediated signals such as those for cytoskeletal reorganization, JNK activation, and stimulation of the nuclear factor of activated T cells. The effect induced by the Y174F mutation is further accentuated by mutations in residue Y142 or Y160. The Y174F mutation has no effect on the exchange activity of Vav in vitro but results in higher levels of phosphorylation in vivo. Using a phosphospecific antibody, we found that Y174 is phosphorylated following stimulation of mitogenic and antigenic receptors. This phosphorylation event is conserved in Vav-2 and Vav-3, the other two members of the Vav family. These results identify a previously unknown mechanism for the oncogenic activation of Vav and suggest that the activity of this exchange factor is modulated by two antagonistic phosphorylation events, one involved in Vav activation and a second one implicated in Vav inactivation.

Rac1 mediates STAT3 activation by autocrine IL-6

The activity of the small GTPase, Rac1, plays a role in various cellular processes including cytoskeletal rearrangement, gene transcription, and malignant transformation. In this report constitutively active Rac1 (Rac V12) is shown to stimulate the activation of STAT3, a member of the family of signal transducers and activators of transcription (STATs). The activity of Rac1 leads to STAT3 translocation to the nucleus coincident with STAT3-dependent gene expression. The expression of Vav (Δ1–187), a constitutively active guanine nucleotide exchange factor for the Rho GTPases, or activated forms of Ras or Rho family members, leads to STAT3-specific activation. The activation of STAT3 requires tyrosine phosphorylation at residue 705, but is not dependent on phosphorylation of Ser-727. Our studies indicate that Rac1 induces STAT3 activation through an indirect mechanism that involves the autocrine production and action of IL-6, a known mediator of STAT3 response. Rac V12 expression results in the induction of the IL-6 and IL-6 receptor genes and neutralizing antibodies directed against the IL-6 receptor block Rac1-induced STAT3 activation. Furthermore, inhibition of the nuclear factor-κB activation or disruption of IL-6-mediated signaling through the expression of IκBα S32AS36A and suppressor of cytokine signaling 3 , respectively, blocks Rac1-induced STAT3 activation. These findings elucidate a mechanism dependent on the induction of an autocrine IL-6 activation loop through which Rac1 mediates STAT3 activation establishing a link between oncogenic GTPase activity and Janus kinase/STAT signaling.

The 90S preribosome is a multimodular structure that is assembled through a hierarchical mechanism.

ABSTRACT The 90S preribosomal particle is required for the production of the 18S rRNA from a pre-rRNA precursor. Despite the identification of the protein components of this particle, its mechanism of assembly and structural design remain unknown. In this work, we have combined biochemical studies, proteomic techniques, and bioinformatic analyses to shed light into the rules of assembly of the yeast 90S preribosome. Our results indicate that several protein subcomplexes work as discrete assembly subunits that bind in defined steps to the 35S pre-rRNA. The assembly of the t-UTP subunit is an essential step for the engagement of at least five additional subunits in two separate, and mutually independent, assembling routes. One of these routes leads to the formation of an assembly intermediate composed of the U3 snoRNP, the Pwp2p/UTP-B, subunit and the Mpp10p complex. The other assembly route involves the stepwise binding of Rrp5p and the UTP-C subunit. We also report the use of a bioinformatic approach that provides a model for the topological arrangement of protein components within the fully assembled particle. Together, our data identify the mechanism of assembly of the 90S preribosome and offer novel information about its internal architecture.

Specific Phosphorylation of p120-Catenin Regulatory Domain Differently Modulates Its Binding to RhoA

ABSTRACT p120-catenin is an adherens junction-associated protein that controls E-cadherin function and stability. p120-catenin also binds intracellular proteins, such as the small GTPase RhoA. In this paper, we identify the p120-catenin N-terminal regulatory domain as the docking site for RhoA. Moreover, we demonstrate that the binding of RhoA to p120-catenin is tightly controlled by the Src family-dependent phosphorylation of p120-catenin on tyrosine residues. The phosphorylation induced by Src and Fyn tyrosine kinases on p120-catenin induces opposite effects on RhoA binding. Fyn, by phosphorylating a residue located in the regulatory domain of p120-catenin (Tyr112), inhibits the interaction of this protein with RhoA. By contrast, the phosphorylation of Tyr217 and Tyr228 by Src promotes a better affinity of p120-catenin towards RhoA. In agreement with these biochemical data, results obtained in cell lines support the important role of these phosphorylation sites in the regulation of RhoA activity by p120-catenin. Taken together, these observations uncover a new regulatory mechanism acting on p120-catenin that contributes to the fine-tuned regulation of the RhoA pathways during specific signaling events.

Cbl-b, a member of the Sli-1/c-Cbl protein family, inhibits Vav-mediated c-Jun N-terminal kinase activation.

We have used the yeast two-hybrid system to identify proteins that interact with Vav, a GDP/GTP exchange factor for the Rac-1 GTPase that plays an important role in cell signaling and oncogenic transformation. This experimental approach resulted in the isolation of Cbl-b, a signal transduction molecule highly related to the mammalian c-cbl proto-oncogene product and to the C. elegans Sli-1 protein, a negative regulator of the EGF-receptor-like Let23 protein. The interaction between Vav and Cbl-b requires the entire SH3-SH2-SH3 carboxy-terminal domain of Vav and a long stretch of proline-rich sequences present in the central region of Cbl-b. Stimulation of quiescent rodent fibroblasts with either epidermal or platelet-derived growth factors induces an increased affinity of Vav for Cbl-b and results in the subsequent formation of a Vav-dependent trimeric complex with the ligand-stimulated tyrosine kinase receptors. During this process, Vav, but not Cbl-b, becomes highly phosphorylated on tyrosine residues. Overexpression of Cbl-b inhibits the signal transduction pathway of Vav that leads to the stimulation of c-Jun N-terminal kinase. By contrast, expression of truncated Cbl-b proteins and of missense mutants analogous to those found in inactive Sli-1 proteins have no detectable effect on Vav activity. These results indicate that Vav and Cbl-b act coordinately in the first steps of tyrosine protein kinase receptor-mediated signaling and suggest that members of the Sli-1/Cbl family are also negative regulators of signal transduction in mammalian cells.