Shulamit Katzav

Vav proteins, masters of the world of cytoskeleton organization

Vav proteins are evolutionarily conserved from nematodes to mammals and play a pivotal role in many aspects of cellular signaling, coupling cell surface receptors to various effectors functions. In mammals, there are three family members; Vav1 is specifically expressed in the hematopoietic system, whereas Vav2 and Vav3 are more ubiquitously expressed. Vav proteins contain multiple domains that enable their function in various fashions. The participation of the Vav proteins in several processes that require cytoskeletal reorganization, such as the formation of the immunological synapse (IS), phagocytosis, platelet aggregation, spreading, and transformation will be discussed in this review. We will also cover how the Vav proteins succeed in controlling these processes by their function as guanine nucleotide exchange factors (GEFs) for the Rho/Rac family of GTPases. The contribution of the Vav proteins in a GEF-independent manner to the organization of the cytoskeleton will also be deliberated. The scope of this review is to highlight the numerous roles of the Vav signal transducer proteins in actin organization.

Oncogenic transformation induces tumor angiogenesis: a role for PAR1 activation

The formation of new blood vessels is a critical determinant of tumor progression. We find that Par1 gene expression plays a central role in blood vessel recruitment in animal models. By in vivo injection of either Matrigel plugs containing Par1‐expressing cells or of rat prostatic carcinoma cells transfected with tetracycline‐inducible Par1 expression vectors, we show that Par1 significantly enhances both angiogenesis and tumor growth. Several vascular endothelial growth factor (VEGF) splice forms are induced in cells expressing Par1. Activation of PAR1 markedly augments the expression of VEGF mRNAs and of functional VEGFs as determined by in vitro assays for endothelial tube alignment and bovine aortic endothelial cell proliferation. Because neutralizing anti‐VEGF antibodies potently inhibited Par1‐induced endothelial cell proliferation, we conclude that Par1‐induced angiogenesis requires VEGF. Specific inhibitors of protein kinase C (PKC), Src, and phosphatidylinositol 3‐kinase (PI3K) inhibit Par1‐induced VEGF expression, suggesting the participation of these kinases in the process. We also show that oncogenic transformation by genes known to be part of PAR1 signaling machinery is sufficient to increase VEGF expression in NIH 3T3 cells. These data support the novel notion that initiation of cell signaling either by activating PAR1 or by the activated forms of oncogenes is sufficient to induce VEGF and hence angiogenesis. Yin, Y.‐J., Salah, Z., Maoz, M., Cohen Even Ram, S., Ochayon, S., Neufeld, G., Katzav, S., Bar‐Shavit, R. Oncogenic transformation induces tumor angiogenesis: a role for PAR1 activation. FASEB J. 17, 163–174 (2003)

Guanine nucleotide exchange factors for RhoGTPases: Good therapeutic targets for cancer therapy?

Rho guanosine triphosphatases (GTPases) are a family of small proteins which function as molecular switches in a variety of signaling pathways following stimulation of cell surface receptors. RhoGTPases regulate numerous cellular processes including cytoskeleton organization, gene transcription, cell proliferation, migration, growth and cell survival. Because of their central role in regulating processes that are dysregulated in cancer, it seems reasonable that defects in the RhoGTPase pathway may be involved in the development of cancer. RhoGTPase activity is regulated by a number of protein families: guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs) and guanine nucleotide-dissociation inhibitors (GDIs). This review discusses the participation of RhoGTPases and their regulators, especially GEFs in human cancers. In particular, we focus on the involvement of the RhoGTPase GEF, Vav1, a hematopoietic specific signal transducer which is involved in human neuroblastoma, pancreatic ductal carcinoma and lung cancer. Finally, we summarize recent advances in the design and application of a number of molecules that specifically target individual RhoGTPases or their regulators or effectors, and discuss their potential for cancer therapy.

Vav1 and Ly-GDI two regulators of Rho GTPases, function cooperatively as signal transducers in T cell antigen receptor-induced pathways.

The Rho family GTPases are pivotal for T cell signaling; however, the regulation of these proteins is not fully known. One well studied regulator of Rho GTPases is Vav1; a hematopoietic cell-specific guanine nucleotide exchange factor critical for signaling in T cells, including stimulation of the nuclear factor of activated T cells (NFAT). Surprisingly, Vav1 associates with Ly-GDI, a hematopoietic cell-specific guanine nucleotide dissociation inhibitor of Rac. Here, we studied the functional significance of the interaction between Vav1 and Ly-GDI in T cells. Upon organization of the immunological synapse, both Ly-GDI and Vav1 relocalize to T cell extensions in contact with the antigen-presenting cell. Ly-GDI is phosphorylated on tyrosine residues following T cell receptor stimulation, and it associates with the Src homology 2 region of an adapter protein, Shc. In addition, the interaction between Ly-GDI and Vav1 requires tyrosine phosphorylation. Overexpression of Ly-GDI alone is inhibitory to NFAT stimulation and calcium mobilization. However, when co-expressed with Vav1, Ly-GDI enhances Vav1 induction of NFAT activation, phospholipase Cγ phosphorylation, and calcium mobilization. Moreover, Ly-GDI does not alter the regulation of these phenomena when coexpressed with oncogenic Vav1. Since oncogenic Vav1 does not bind Ly-GDI, this suggests that the functional cooperativity of Ly-GDI and Vav1 is dependent upon their association. Thus, our data suggest that the interaction of Vav1 and Ly-GDI creates a fine tuning mechanism for the regulation of intracellular signaling pathways leading to NFAT stimulation.

The haematopoietic specific signal transducer Vav1 is expressed in a subset of human neuroblastomas

Vav1 is a signal transducer protein expressed exclusively in the haematopoietic system, where it plays a pivotal role in growth factor‐induced differentiation and proliferation. Vav1 couples tyrosine kinase signals with the activation of the Rho/Rac GTPases, leading to cell differentiation and/or proliferation. Vav1 was originally detected as an oncogene, but its involvement in human malignancies has not been reported thus far. We report here that Vav1 is expressed in a neuroblastoma cell line, SK‐N‐MC. Molecular analysis indicated that there are no gross rearrangements or mutations in the Vav1 gene in SK‐N‐MC cells. Vav1 protein from SK‐N‐MC cells was similar to wild‐type Vav1 in apparent molecular weight, phosphorylation state, and ability to associate with active EGFR. We also analysed the expression of Vav1 in 42 specimens of human neuroblastoma. Vav1 was expressed in the majority of these tumours. Our results suggest that Vav1 may play a role in the neoplastic process in a subset of neuroblastomas. Copyright

Signalling pathways induced by protease‐activated receptors and integrins in T cells

Recent characterization of the thrombin receptor indicates that it plays a role in T‐cell signalling pathways. However, little is known regarding the signalling events following stimulation of additional members of the protease‐activated receptor (PAR) family, i.e. PAR2 and PAR3. Most of the postligand cascades are largely unknown. Here, we illustrate that in Jurkat T‐leukaemic cells, activation of PAR1, PAR2 and PAR3 induce tyrosine phosphorylation of Vav1. This response was impaired in Jurkat T cells deficient in p56lck (JCaM1.6). Activation of PARs also led to an increase in tyrosine phosphorylation of ZAP‐70 and SLP‐76, two key proteins in T‐cell receptor (TCR) signalling. We also demonstrated that p56lck is meaningful for integrin signalling. Thus, JCaM1.6 cells exhibited a marked reduction in their adherence to fibronectin‐coated plates, as compared to the level of adherence of Jurkat T cells. While the phosphorylation of Vav1 in T cells is augmented following adhesion, no additional increase was noted following treatment of the adhered cells with PARs. Altogether, we have identified key components in the postligand‐signalling cascade of PARs and integrins. Furthermore, we have identified Lck as a critical and possibly upstream component of PAR‐induced Vav1 phosphorylation, as well as integrin activation, in Jurkat T cells.

Vav, a GDP/GTP nucleotide exchange factor, interacts with GDIs, proteins that inhibit GDP/GTP dissociation

Vav functions as a specific GDP/GTP nucleotide exchange factor which is regulated by tyrosine phosphorylation in the hematopoietic system. Loss of the amino‐terminus sequences of Vav was sufficient to control its transforming potential and its function in T cells. We report here the identification of the hematopoietic GDP dissociation inhibitor protein, Ly‐GDI, as a protein that interacts with the amino‐terminus of Vav. Further analysis confirmed that Vav and Ly‐GDI interact both in in vitro and in in vivo assays. This association is maximal only when the amino region of Vav is intact and requires an intact carboxy‐terminus of Ly‐GDI. The interaction between Vav and Ly‐GDI is not dependent on the tyrosine phosphorylation status of Vav. In addition, Rho‐GDI, the highly homologous protein to Ly‐GDI, associates with Vav as well. The contribution of the interaction between Vav and GDIs, proteins that are involved in the GDP/GTP exchange processes, to the biological function of Vav is further discussed.

Vav1: a hematopoietic signal transduction molecule involved in human malignancies.

Vav1 encodes a unique protein with several motifs known to play a role in tyrosine mediated signal transduction, including a DBL homology (DH) domain, a pleckstrin homology (PH) domain, a Src homology 2 (SH2) domain, and two Src homology 3 (SH3) domains. Physiological Vav1 expression is restricted to the hematopoietic system, where it functions primarily as a specific GDP/GTP nucleotide exchange factor (GEF), a function strictly regulated by tyrosine phosphorylation. In hematopoietic cells, Vav1 is phosphorylated following cell surface receptor activation, triggering re-organization of the cytoskeleton and regulation of other cellular functions including transcription, cytokine production, cell cycle progression, and Ca(2+) mobilization. Vav1 also functions as an adapter, facilitating interaction between other proteins. A truncated Vav1 was first isolated as an oncogene, and its wild-type form has recently been implicated in mammalian malignancies. These properties make Vav1 a promising target for new therapeutic approaches to organ transplantation and cancer therapy.

The haematopoietic specific signal transducer Vav1 is aberrantly expressed in lung cancer and plays a role in tumourigenesis.

Lung cancer is the leading cause of cancer death worldwide. The spectrum of aberrations affecting signalling pathways in lung cancer pathogenesis has not been fully elucidated. Physiological expression of Vav1 is restricted to the haematopoietic system, where its best‐known function is as a GDP/GTP nucleotide exchange factor for Rho/RacGTPases, an activity strictly controlled by tyrosine phosphorylation downstream of cell surface receptors. Here we find Vav1 expression in 42% of 78 lung cancer cell lines analysed. Moreover, immunohistochemical analysis of primary human lung cancer tissue samples revealed Vav1 expression in 26/59 malignant samples, including adenocarcinoma, squamous cell carcinoma and bronchioloalveolar carcinoma. Stronger Vav1 staining was associated with larger tumour size. siRNA‐mediated knockdown of Vav1 in lung cancer cells reduced proliferation in agar and tumour growth in nude mice, while control siRNA had no effect, suggesting that Vav1 plays a critical role in the tumorigenicity of lung cancer cells. Vav1 is tyrosine‐phosphorylated in lung cancer cells following activation by the growth factors EGF and TGFα, suggesting its participation in signalling events in these cells. Depletion of Vav1 reduced Rac‐GTP activation and decreased expression of TGFα, an autocrine growth factor. These data suggest that Vav1 plays a role in the neoplastic process in lung cancer, identifying it as a potential therapeutic target for lung cancer therapy. Copyright

Mutagenic analysis of Vav reveals that an intact SH3 domain is required for transformation.

The vav proto-oncogene encodes a protein with multiple modulae domains that enable it to function as a mediator, linking tyrosine signaling to downstream events in hematopoietic cells. Circumstantial evidence suggests that protein-protein interactions exerted by two of these domains, the Src homology 2 (SH2) and the Src homology 3 (SH3), play an important role in the regulation of Vav activity. To study the relevance of the SH3 domain for the function of vav as a transforming gene, we have created several mutations in the SH3 domain located at its carboxy region. Substitution of the non-conserved aspartic acid 797 (to asparagine, D797N) retained the transforming potential of the vav oncogene, whereas substitutions of five highly conserved amino-acids: alanine 789 (to asparagine, A789N), leucine 801 (to arginine, L801R), tryptophan 821 (to arginine, W821R), glycine 830 (to valine, G830V) and valine 837 (to glutamic acid, V837E) greatly reduced its transforming potential. The mutant proteins resemble Vav in many biochemical properties; however, while the transforming mutant protein (D797N) associates with several unidentified proteins in a manner similar to that of Vav, the non-transforming mutant Vav proteins react very poorly with these proteins. Among the known Vav-interacting proteins, hnRNP-K associates with all mutant proteins except A789N and V837E whereas binding of Zyxin to any of the mutant proteins is not affected. Taken together, our results clearly demonstrate that the SH3 domain has a positive effect on vav activity and is needed for vav transformation. The vavSH3C associating protein(s) that are crucial for its activity as a transforming gene have probably not yet been identified.