Studies of the function and regulation of vasodilator-stimulated phosphoprotein

Sepsis-induced renal failure is associated with damage to the proximal epithelial tubule and is associated with disruption of the actin cytoskeleton and cell shedding. Nitric oxide is associated with the changes induced by pro-inflammatory cytokines in early sepsis. We hypothesised that it acts via phosphorylation of vasodilator-stimulated phosphoprotein (VASP) altering its ability to act as a link between cell:cell and cell:matrix junctions and the actin cytoskeleton. Transfection of iNOS into epithelial cells was associated with a loss of VASP from its normal location at the cell membrane and with disruption of the actin cytoskeleton. Immunoblotting revealed that iNOS transfection was associated with a rise in the cGMP-dependent protein kinase preferred phosphorylation site at Ser239, a region close to G-actin binding and F-actin polymerising domains. We also showed that Ser239 phosphorylation was only seen when Seri57 phosphorylation had already occurred, that it was a transient effect and that it was nitric oxide dependent. We hypothesised that this was due to a conformational change in VASP and that Ser239 phosphorylation was dependent on the level of Serl57-phosphorylated VASP available. This was demonstrated in RAW 264.7 macrophages in which very high levels of Seri57 are achievable. Given the permissive effect of Seri57 phosphorylation on Ser239 phosphorylation, we investigated the effects of cAMP on this as the Seri57 site has been demonstrated to be preferentially phosphorylated via cAMP-dependent protein kinase. The addition of cAMP alone was not associated with an increase in Ser239 phosphorylation. However, when given to iNOS transfected cells, prolongation of the presence of the dually phosphorylated form of VASP was seen. We therefore proposed that the interaction of both cyclic nucleotide-dependent protein kinase pathways is important in the control of VASP phosphorylation. VASP is present at highly dynamic areas of the cell membrane such as lamellopodia and is important in cell motility. We hypothesised that it would be important in the formation of new epithelial sheets following injury. To investigate this we used a dominant-negative form of VASP (DN-VASP) consisting of amino acids 277-383 of the full-length protein. Expression of DN-VASP in a preformed epithelial monolayer did not appear to be associated with breakdown of the sheet even though it did disrupt actin fibres. However, expression of DN-VASP in a newly forming sheet did appear to be involved with cell loss and a reduced ability to adhere to the sub-stratum. Therefore, VASP may be of greater importance in the formation of an epithelial sheet than in maintenance of its integrity. The ability of the actin cytoskeleton to reorganise in response to external stimuli is also of crucial importance in T cell activation. A T cell adaptor protein, ADAP contains an EVH 1 binding domain and is therefore capable of binding VASP. ADAP and VASP are amongst a group of proteins that are localised to the T cell: antigen presenting cell interface. ADAP knockouts show a decreased ability to cluster the integrin LFA-1 to the immunological synapse following stimulation. We investigated the effects of disrupting VASP function in T cells via the use of DN-VASP. Transfection of DN-VASP into T cells was associated with an inability to polarise actin in response to TCR ligation and a significant decrease in interleukin-2 production. However it was not associated with a decrease in the ability to bind to beads coated with the LFA-1 ligand, ICAM-1. We further investigated the effects of DN-VASP transfection on signal transduction pathways and demonstrated that it appears to disrupt MAP kinase activation though not through phosphorylation of early steps of the cascade. It did not appear to have as great an effect on NFAT and NF-kB pathways. We hypothesised that VASP is important in T cell activation via its effects on signal transduction and that, in vivo these effects may be modulated through phosphorylation of VASP. In summary, this work shows that VASP is affected by NO-induced phosphorylation and that appears to be more complex than first expected, involving cAMP-dependent pathways also. VASP appears to be important in the formation of new epithelial sheets but is of less importance in a pre-formed monolayer. VASP also appears crucial to T cell activation and DN-VASP appears to induce T cell anergy, specifically disrupting MAP kinase pathways