Thomas Trenkle

Vav3 Mediates Receptor Protein Tyrosine Kinase Signaling, Regulates GTPase Activity, Modulates Cell Morphology, and Induces Cell Transformation

ABSTRACT A recently reported new member of the Vav family proteins, Vav3 has been identified as a Ros receptor protein tyrosine kinase (RPTK) interacting protein by yeast two-hybrid screening. Northern analysis shows that Vav3 has a broad tissue expression profile that is distinct from those of Vav and Vav2. Two species of Vav3 transcripts, 3.4 and 5.4 kb, were detected with a differential expression pattern in various tissues. Transient expression of Vav in 293T and NIH 3T3 cells demonstrated that ligand stimulation of several RPTKs (epidermal growth factor receptor [EGFR], Ros, insulin receptor [IR], and insulin-like growth factor I receptor [IGFR]) led to tyrosine phosphorylation of Vav3 and its association with the receptors as well as their downstream signaling molecules, including Shc, Grb2, phospholipase C (PLC-γ), and phosphatidylinositol 3 kinase. In vitro binding assays using glutathione S-transferase-fusion polypeptides containing the GTPase-binding domains of Rok-α, Pak, or Ack revealed that overexpression of Vav3 in NIH 3T3 cells resulted in the activation of Rac-1 and Cdc42 whereas a deletion mutant lacking the N-terminal calponin homology and acidic region domains activated RhoA and Rac-1 but lost the ability to activate Cdc42. Vav3 induced marked membrane ruffles and microspikes in NIH 3T3 cells, while the N-terminal truncation mutants of Vav3 significantly enhanced membrane ruffle formation but had a reduced ability to induce microspikes. Activation of IR further enhanced the ability of Vav3 to induce membrane ruffles, but IGFR activation specifically promoted Vav3-mediated microspike formation. N-terminal truncation of Vav3 activated its transforming potential, as measured by focus-formation assays. We conclude that Vav3 mediates RPTK signaling and regulates GTPase activity, its native and mutant forms are able to modulate cell morphology, and it has the potential to induce cell transformation.

Reduced complexity probes for DNA arrays.

Publisher Summary There have been many advances in methods to manufacture and probe arrays of DNAs. Membranes with hundreds or thousands of polymerase chain reaction (PCR) products from cDNA clones are commercially available. Any cDNA clones on the array that are from the most abundant few thousand of mRNAs in a cell can easily be detected using a radiolabeled probe containing the full complexity of the mRNA population in the cell. The only limitation is background hybridization to all clones. This chapter explains a strategy based on cDNA fingerprints generated by RNA arbitrarily primed PCR (RAP-PCR). This method generates different subsets of the mRNA population, depending on the primers used. Some of the mRNAs that are difficult to detect using total cDNA probes are sufficiently represented in these reduced complexity probes to be easily detected on arrays of colonies. This method has the further advantage that it generates a labeled probe using hundreds-fold less RNA than is currently used by any other array probing methods. The chapter discusses the use of statistically primed PCR (SP-PCR) to generate probes.