1. 16kDa Prolactin Fragment Inhibits VEGF-induced Ras Activation Through Decreased p62 Dok Phosphorylation A 16-kDalton fragment of prolactin (16Prl) has been found to inhibit angiogenesis, the proliferation of tumor blood vessels, by specifically inhibiting Vascular Endothelial Growth Factor (VEGF)-induced activation of Ras. It has been found that phosphorylated p62 Dok inhibits RasGAP (guanosine triphosphatase-activating protein) preventing active RasGTP conversion to inactive RasGDP, therefore potentiating Ras. We hypothesize that 16Prl inhibits p62 Dok phosphorylation resulting in increased RasGap activity, and inactivation of Ras. Bovine Brain Endothelial cells will be treated with VEGF and 16Prl and the p62 Dok phosphorylation status, ratio of RasGTP/GDP, and Raf-1 phosphorylation status as a functional measure of Ras activity will be determined. We predict 16Prl will decrease p62 Dok phosphorylation resulting in a decreased RasGTP/GDP ratio and decreased Raf-1 phosphorylation. If this is a part of the inhibition mechanism of 16Prl, further research may lead to improved cancer treatments by this mechanism. William Blymire, Jr. York College of Pennsylvania, Department of Biological Sciences Introduction 1. D’Angelo, G., Martini, J., Iiri, T., Fantl, W.J., Martial, J. and Weiner, R.I. (1999). 16K human prolactin inhibits vascular endothelial growth factor-induced activation of Ras in capillary cells. Molecular Endocrinology 13:692-704. 2. Kashige, V., Carpino, N. and Kobayashi, R. (2000). Tyrosine phosphorylation of p62 dok by p210 bcr-abl inhibits RasGAP activity. Proceedings of the National Academy of Science USA 97:2093-2098. Predicted Results Research Design Acknowledgments Ron Kaltreider, PhD. Faculty Mentor Future Direction Project Summary Literature Cited If 16 kDa prolactin fragment blocks VEGF induced p62 Dok phosphorylation other molecules that blocks phosphorylation may be used to develop new cancer treatments. Figure 2. Predicted results of the western blot of tyrosine- phosphorylated proteins (A) p62 Dok and (B) Raf-1 when treated with VEGF, VEGF & 16 kDa prolactin, and 16 kDa prolactin. Objectives 1. To determine if 16 kDa prolactin fragment inhibits the VEGF-induced Ras activation by inhibiting the phosphorylation of p62 Dok 2. To confirm if 16 kDa prolactin fragment alters RasGAP activity in BBE cells by quantifying the ratio of RasGTP/GDP. 3. To confirm that Ras is inhibited by 16 kDa prolactin by measuring the phosphorylation status of Raf-1 in VEGF- induced BBE cells. Figure 3. Predicted Inhibitory effect of 16kDa prolactin on VEGF-induced Ras activation using quantitation of TLC by phosphorimaging of RasGTP and Ras-GDP. Numbers are predicted density ratios of RasGDP/GTP. Figure 1. Proposed Mechanism of 16 kDa prolactin inhibition of phosphorylation of p62 Dok and subsequent function of Ras-GAP inhibiting Ras. Dotted lines ending in a perpendicular line denote inhibition. Solid arrows denote signaling events (Adapted from 2). Review of Literature Angiogenesis is the proliferation of blood vessels from existing vessels normally occurs during wound healing but abnormally occurs to provide a blood supply for cancer tumors. The development of angiogenesis is thought to be a balance in which inhibitors and activators counter balance each other out until one overcomes the other. A build-up of activators leads to agiogenesis while a build-up of inhibitors prevents angiogenesis. In recent years, a great deal of research has been devoted to find ways to inhibit this event which would starve the tumor of needed resources for growth. One way in which to stop angiogenesis is to inhibit the cell signaling mechanisms that initiate angiogenesis. This can occur in several ways, including inhibiting the initiation of the signaling by interfering with receptors or by interfering with a step in the signaling process. One specific pathway that has been linked to angiogenesis initiation is the Ras pathway (Figure 1), which culminates in altered gene expression. This pathway is activated when a molecule interacts with a specific receptor, such as Vascular Endothelial Growth Factor (VEGF) or basic fibroblast growth factor (bFGF). Recent research has looked at the anti-angiogenic properties of molecules such as angiostatin, platelet factor 4, and a 16 kDa fragment of prolactin.  VEGF initiates the Ras pathway in Bovine Brain capillary Endothelial (BBE) cells (1).  RasGAP converts active RasGTP to inactive RasGDP effectively inhibiting Ras (1).  16 kDa prolactin fragment inhibits Ras by a increased RasGAP activity (1).  p62 Dok, when phosphorylated, deactivates RasGAP activity leading to a potentiation of Ras signaling(2). 1. 16 kDa prolactin fragment will decrease VEGF- induced phosphorylation of p62 Dok (Figure 2). 2. 16 kDa prolactin fragment will decrease RasGAP activity (increased RasGDP) (Figure 3). 3. 16 kDa prolactin fragment will decrease Ras activity (decreased Raf-1 phosphorylation) (Figure 2). Cell Culture -Culture in DMEM with 10% calf serum, L-glutamine, and 1% penicillin/streptomycin. -Treat with bFGF every other day (1). -Serum starve cells 48 hrs prior to treatment. Cell Treatment Cells treated for 5 minutes and then proteins isolated. -Untreated -VEGF (1nM) -VEGF (1nM) + 16 kDa Prolactin (1nM) -16 kDa Prolactin (1nM) Protein Isolation -Wash cells with cold Tris-buffered saline. -Lyse using a RIPA buffer. -Homogenize cells. -Centrifuge to isolate protein. -Quantitate total protein using standard Bradford assay Immunoprecipitation Use monoclonal antibodies precouples to A-sepharose indicated for 12 hours @ 4 o C. Purify complexes and elute using rabbit anti-rat IgG. Western Blot -Run Precipitants (20  g) on 8%PAGE stand- ardizing to total protein -Transfer to Imobilon-P membranes -Probe with anti-p62 Dok and anti-Raf antibodies to standardize total protein levels and detect. -Strip blots using additional antibodies. -Probe with anti-phosphotyrosine mouse monoclonal antiserum and detect. Anti-p62 Dok or Anti-Raf Antibodies Anti-Ras Antibody Detection and Quantification -Horseradish peroxidase-couples secondary antibodies enhanced with chemiluminescence reagent -Expose to Reflection NEF Films -Quantitate autoradiographs using NIH Image Thin Layer Chromatography -Resolve the eluted Ras-associated guanyl- nucleotides on polyethyleneimine cellulose plates with KH 2 PO 4 solvent (1). Detection and Quantification -Visualize labeled nucleotides using autoradiography. -Determine radioactivity in GTP and GDP using phosphorimaging. -Quantify using NIH Image. Expected Results