Victor Tybulewicz: Projects

Vav1

Vav1 is a guanine nucleotide exchange factor (GEF) for members of the Rho-family of small GTPases, including Rac1, Cdc42 and RhoA. This activity is activated by tyrosine phosphorylation. Using gene targeting we have shown that Vav1 plays a critical role in T cell development. Vav1-deficient mice show a partial block in thymic development at the pre-TCR checkpoint which monitors for successful rearrangement of the TCRβ genes, as well as stronger blocks in positive and negative selection of double positive thymocytes into the peripheral T cell pool (Tarakhovsky et al, 1995; Turner et al, 1997).

Analysis of TCR signalling in these mice showed that Vav1 was required for TCR-induced calcium flux, ERK and NF-κB activation (Costello et al, 1999). Furthermore we showed that the calcium defect was due to defective activation of phospholipase Cγ1 probably because of a failure to activate phophatidylinositide-3-kinase (PI3K) and the Tec-family kinases Itk and Tec (Reynolds et al, 2002).

Using knock-in gene targeting, we have generated mice expressing an enzymatically inactive Vav1. This showed that the GEF activity of Vav1 was important for some, but not all of its functions (Saveliev et al, 2009). In particular, Vav1's GEF activity is required to transduce signals leading to PI3K activation and actin polymerization, but not to calcium flux, ERK activation or T cell polarisation. The latter pathways are dependent on a GEF-independent function of Vav1, which is currently under investigation.

In collaboration with Katrin Rittinger, we have analysed the structure of the catalytically active part of Vav1. This showed that the PH and C1 domains of the protein play a critical role in supporting catalysis by the DH domain (Rapley et al, 2008). The structure suggested a possible allosteric regulation of Vav1.

Structure of Vav1 and Rac1

Structure of Vav1 and Rac1 Diagram showing structure of Vav1 and Rac1. Ribbon diagram of the DH, PH and C1 domains of Vav1 (grey) in complex with Rac1 (gold) (Rapley et al., 2008). Amino acids Leu 334 and Lys 335 (red) were mutated to generate an enzymatically inactive Vav1 (see Saveliev et al.). (Click to view larger image)

Selected Publications

Tarakhovsky, A., M. Turner, S. Schaal, P.J. Mee, L.P. Duddy, K. Rajewsky, and V.L.J. Tybulewicz. (1995) Defective antigen receptor-mediated proliferation of B and T cells in the absence of Vav. Nature 374, 467-470

Turner, M., P.J. Mee, A. Walters, M.E. Quinn, A.L. Mellor, R. Zamoyska, and V.L.J. Tybulewicz. (1997) A requirement for the Rho-family GTP exchange factor Vav in positive and negative selection of thymocytes. Immunity 7, 451-460

Reynolds, L.F., L.A. Smyth, T. Norton, N. Freshney, J. Downward, D. Kioussis, and V.L.J. Tybulewicz. (2002) Vav1 transduces T cell receptor signals to the activation of phospholipase C-γ1 via phosphoinositide 3-kinase-dependent and -independent pathways. Journal of Experimental Medicine 195, 1103-1114

Prisco, A; Vanes, L; Ruf, S; Trigueros, C and Tybulewicz, VL (2005) Lineage-specific requirement for the PH domain of Vav1 in the activation of CD4+ but not CD8+ T cells Immunity 23, 263-274

Rapley, J; Tybulewicz, VLJ and Rittinger, K (2008) Crucial structural role for the PH and C1 domains of the Vav1 exchange factor. EMBO Reports 9, 655-661

Saveliev, A; Vanes, L; Ksionda, O; Rapley, J; Smerdon, SJ; Rittinger, K and Tybulewicz, VLJ (2009) Function of the nucleotide exchange activity of Vav1 in T cell development and activation. Science Signaling 2, ra83

Ksionda, O., Saveliev, A., Rapley, J., Köchl, R., Faroudi, M., Smith-Garvin, J. E., Wülfing, C., Rittinger, K., Carter, T., Tybulewicz, V. L. J. (2012). Mechanism and function of Vav1 localization in TCR signaling. J Cell Sci, 125, 5302-5314.

Syk

Syk is a protein tyrosine kinase activated by BCR stimulation (Mocsai et al, 2010). Several years ago we made mice deficient in Syk and showed that the mutation causes a complete block in B cell development. This block is due to defects at two stages. Firstly there is a partial block at the pre-BCR signalling checkpoint, and then a complete block between the immature B cell and mature recirculating B cell compartment (Turner et al, 1995; Turner et al, 1997). More recently we have discovered that the partial block at the pre-BCR checkpoint is due to redundancy between Syk and a related tyrosine kinase, ZAP-70. Mice mutant in both Syk and ZAP-70 show a complete block at the pre-BCR checkpoint as well as a failure of heavy chain allelic exclusion (Schweighoffer et al, 2003).

More recently we have used a conditional allele of Syk to study signaling pathways controlling B cell survival. We have shown that BAFFR, a receptor for BAFF transduces signals via the BCR to the activation of Syk and that this pathway is critical for B cell survival (Schweighoffer et al, 2013). We are currently using genetic and proteomic techniques to understand how this crosstalk between receptors functions.

Schematic structure of Syk/ZAP-70 family protein tyrosine kinases

Schematic structure of Syk/ZAP-70 family protein tyrosine kinases. Schematic structure oThe tandem SH2 domains and the kinase domain are shown as orange and green cylinders respectively. Purple tubes represent the linker region connecting the SH2 and kinase domains. In comparison with the alternatively spliced isoform Syk-B, Syk contains an insert of 23 amino acids (dark purple) within the linker region. Tyrosine residues (Y) in Syk which have been shown to undergo phosphorylation are indicated – these may be important in regulating enzymatic activity or recruiting other signaling proteins (Turner et al., 2000).

Selected Publications

Turner, M., P.J. Mee, P.S. Costello, O. Williams, A.A. Price, L.P. Duddy, M.T. Furlong, R.L. Geahlen, and V.L.J. Tybulewicz (1995) Perinatal lethality and blocked B-cell development in mice lacking the tyrosine kinase Syk. Nature 378, 298-302

Turner, M., A. Gulbranson-Judge, M.E. Quinn, A.E. Walters, I.C.M. MacLennan, and V.L.J. Tybulewicz (1997) Syk tyrosine kinase is required for the positive selection of immature B cells into the recirculating B cell pool. Journal of Experimental Medicine 186, 2013-2021

Turner, M; Schweighoffer, E; Colucci, F; Di Santo, JP and Tybulewicz, VLJ (2000) Tyrosine kinase SYK: Essential functions for immunoreceptor signalling. Immunology Today 21, 148-154

Schweighoffer E, Vanes L, Mathiot A, Nakamura T, Tybulewicz VL (2003) Unexpected requirement for ZAP-70 in pre-B cell development and allelic exclusion. Immunity 18(4), 523-33

Mócsai, A., Ruland, J. and Tybulewicz, V. L. J. (2010). The Syk tyrosine kinase: a crucial player in diverse biological functions. Nat Rev Immunol, 10, 387-402.

Schweighoffer, E., Vanes, L., Nys, J., Cantrell, D., McCleary, S., Smithers, N., and Tybulewicz, V. L. J. (2013). The BAFF receptor transduces survival signals by co-opting the B cell antigen receptor signaling pathway. Immunity, 38, 475-488.

Rac1 and Rac2

Rac1 and Rac2 are small GTPases of the Rho-family, which have been implicated in the control of the actin cytoskeleton, signal transduction, cell proliferation, and apoptosis (Tybulewicz and Henderson, 2009). Since the knockout of Rac1 is an early embryonic lethal, we have generated a conditional allele of Rac1, flanked by loxP sites which allows deletion of the gene by the Cre recombinase. Using this we have been able to inactivate Rac1 in a tissue-specific manner, and have shown that deletion of Rac1 and Rac2 results in a severe block in late B cell development, at the transitional B cell stage in the spleen (Walmsley et al, 2003). We have gone on to show that this is due to an inability of the transitional cells to migrate into the white pulp of the spleen and proposed that this migration is a key developmental checkpoint during B cell positive selection (Henderson et al, 2010).

Deletion of Rac1 and Rac2 during early T cell development, leads to a strong developmental block in the thymus caused by defective pre-TCR signaling (Dumont et al, 2009). In contrast, deletion of both GTPases in mature T cells results in defective migration of T cells into and through lymph nodes (Faroudi et al, 2010). The GTPases are important for homing to the lymph node, adhesion to the high endothelial venules, transmigration across them, interstitial migration within the lymph node parenchyma and egress from the lymph node.

Rac GTPases control entry into splenic white pulp

Rac GTPases control entry into splenic white pulp. Rac2-deficient B cells (green) are excluded from the white pulp of the spleen, whose edges are identified by expression of MadCAM-1 (red). (Click to view larger image)

Selected Publications

Walmsley MJ, Ooi SK, Reynolds LF, Smith SH, Ruf S, Mathiot A, Vanes L, Williams DA, Cancro MP, Tybulewicz VL (2003) Critical roles for Rac1 and Rac2 GTPases in B cell development and signaling. Science 302(5644), 459-62

Dumont, C; Corsoni-Tadrzak, A; Ruf, S; de Boer, J; Williams, A; Turner, M; Kioussis, D and Tybulewicz, VL (2009) Rac GTPases play critical roles in early T cell development. Blood 113, 3990-3998

Tybulewicz, VLJ and Henderson, RB (2009) Rho family GTPases and their regulators in lymphocytes. Nature Reviews Immunology 9, 630-644

Henderson, RB; Grys, K; Vehlow, A; de Bettignies, C; Zachacz, A; Henley, T; Turner, M; Batista, F and Tybulewicz, VLJ (2010) A novel Rac-dependent checkpoint in B cell development controls entry into the splenic white pulp and cell survival. Journal of Experimental Medicine

Mouse models of Down syndrome

Trisomy of human chromosome 21 (Hsa21) occurs in ∼1 in 750 live births, and the resulting gene dosage imbalance gives rise to Down syndrome (DS), the most common known genetic form of mental retardation. DS is a constellation of different phenotypes: while all people with DS have hypotonia, mental retardation and neurodegeneration, most also present with a spectrum of other disorders such as heart defects, leukaemias, autoimmune disorders, etc.

In collaboration with Professor Elizabeth Fisher (Institute of Neurology, UCL) we are interested in identifying dosage sensitive genes on Hsa21 which, when present in three copies, cause the many different phenotypes seen in DS. We are addressing this using mouse models. We have created a novel mouse strain, termed Tc1, which carries a freely segregating copy of Hsa21. Tc1 mice show defects in memory, synaptic plasticity, locomotor function, heart development and in the craniofacial skeleton (O'Doherty et al, 2005; Morice et al, 2008; Galante et al 2009).

Human DS people have increased rates of mekaryoblastic leukaemia, but lower rates of solid tumours. We have shown that Tc1 mice have perturbed megakaryopoiesis, a pathology that may contribute to the increased rates of leukaemia in human DS (Alford et al, 2010). In contrast, studies of Tc1 mice have shown that the reduction in solid tumours may be due to defective angiogenesis (Reynolds et al, 2010).

Currently our main aim is to identify 'dosage-sensitive' genes, which, when present in three copies cause DS phenotypes. To do this we have constructed a series of novel mouse strains with duplications and deletions of regions mouse chromosomes orthologous to Hsa21. Analysis of these mouse strains is allowing us to pinpoint the dosage-sensitive genes and to understand pathological mechanisms causing specific phenotypes.

Generation of the transchromosomic Tc1 mice

Generation of the transchromosomic Tc1 mice. Using gene targeting we inserted a neomycin resistance gene into human chromosome 21 (Hsa21) in a human cell line. These cells carrying a tagged Hsa21 were arrested in metaphase and then centrifuged to isolate microcells carrying one or just a few chromosomes. The microcells were fused to murine embryonic stem (ES) cells, which were then selected with G418 for uptake of the neomycin resistance gene, and screened for lines carrying Hsa21. These ES lines were injected into mouse blastocysts to generate chimeric mice, which were bred to establish the Tc1 mouse strain carrying a freely segregating Hsa21. (Click to view larger image)

Fluorescence in situ hybridization of Tc1 cells

Fluorescence in situ hybridization of Tc1 cells. Fluorescence in situ hybridization (FISH) of a spread of metaphase chromosomes from a splenocyte taken from a Tc1 mouse. The blue colour indicates DAPI staining which shows all chromosomes, green is a probe specific for human chromosome 21 (Hsa21) and red is a probe specific for mouse chromosome X (MmuX).

Selected Publications

O'Doherty, A; Ruf, S; Mulligan, C; Hildreth, V; Errington, ML; Cooke, S; Sesay, A; Modino, S; Vanes, L; Hernandez, D ; Linehan, JM; Sharpe, PT; Brandner, S; Bliss, TVP; Henderson, DJ; Nizetic, D; Tybulewicz, VLJ and Fisher, EMC (2005) (2005) An aneuploid mouse strain carrying human chromosome 21 with Down syndrome phenotypes. Science 309, 2033-2037

Morice, E; Andreae, LC; Cooke, SF; Vanes, L; Fisher, EMC; Tybulewicz, VLJ and Bliss, TVP (2008) Preservation of long-term memory and synaptic plasticity despite short-term impairments in the Tc1 mouse model of Down syndrome. Learning & Memory 15, 492-500

Galante, M; Jani, H; Vanes, L; Daniel, H; Fisher, EMC; Tybulewicz, VLJ; Bliss, TVP and Morice, E (2009) Impairments in motor coordination without major changes in cerebellar plasticity in the Tc1 mouse model of Down syndrome. Human Molecular Genetics 18, 1449-1463

Alford, K; Slender, A; Vanes, L; Li, Z; Fisher, EMC; Nizetic, D; Orkin, SH; Roberts, I and Tybulewicz, VLJ (2010) Perturbed hematopoiesis in the Tc1 mouse model of Down syndrome. Blood 115, 2928-2937

Victor Tybulewicz

victor.t@crick.ac.uk
+44 (0)20 379 61612

  • Qualifications and history
  • 1984 PhD MRC Laboratory of Molecular Biology, Cambridge, UK
  • 1986-1991 Postdoctoral fellow, Whitehead Institute, MIT, Cambridge, MA, USA
  • 1991-2015 Group Leader, Medical Research Council National Institute for Medical Research, London, UK
  • 2015 Group Leader, the Francis Crick Institute, London, UK