Transcript 16kDa Prolactin Fragment Inhibits VEGF

16kDa Prolactin Fragment Inhibits VEGF-induced Ras
Dok
Activation Through Decreased p62
Phosphorylation
Predicted Results
William Blymire, Jr.
York College of Pennsylvania, Department of Biological Sciences
Review of Literature
Project Summary
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 p62Dok inhibits RasGAP (guanosine
triphosphatase-activating protein) preventing active
RasGTP conversion to inactive RasGDP, therefore
potentiating Ras. We hypothesize that 16Prl inhibits
p62Dok phosphorylation resulting in increased
RasGap activity, and inactivation of Ras. Bovine
Brain Endothelial cells will be treated with VEGF and
16Prl and the p62Dok 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 p62Dok
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.
Research Design
 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).
p62Dok, when phosphorylated, deactivates
RasGAP activity leading to a potentiation of Ras
signaling(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
Introduction
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.
Figure 2. Predicted results of the western blot of tyrosinephosphorylated proteins (A) p62Dok and (B) Raf-1 when
treated with VEGF, VEGF & 16 kDa prolactin, and 16 kDa
prolactin.
Figure 1. Proposed Mechanism of 16 kDa
prolactin inhibition of phosphorylation of p62Dok
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).
Expected Results
Anti-p62Dok
or Anti-Raf
Antibodies
Immunoprecipitation
Use monoclonal antibodies precouples
to A-sepharose indicated for 12 hours
@ 4oC. Purify complexes and elute
using rabbit anti-rat IgG.
Western Blot
Objectives
1. To determine if 16 kDa prolactin
fragment inhibits the VEGF-induced Ras
activation by inhibiting the
phosphorylation of p62Dok
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 VEGFinduced 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.
-Run Precipitants (20 g) on 8%PAGE standardizing to total protein
-Transfer to Imobilon-P membranes
-Probe with anti-p62Dok 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.
Detection and Quantification
-Horseradish peroxidase-couples secondary
antibodies enhanced with chemiluminescence
reagent
-Expose to Reflection NEF Films
-Quantitate autoradiographs using NIH Image
Anti-Ras
Antibody
Thin Layer Chromatography
-Resolve the eluted Ras-associated guanylnucleotides on polyethyleneimine
cellulose plates with KH2PO4 solvent (1).
Detection and Quantification
-Visualize labeled nucleotides using
autoradiography.
-Determine radioactivity in GTP and GDP
using phosphorimaging.
-Quantify using NIH Image.
Acknowledgments
Ron Kaltreider, PhD. Faculty Mentor
1. 16 kDa prolactin fragment will decrease VEGFinduced phosphorylation of p62Dok (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).
Future Direction
If 16 kDa prolactin fragment blocks VEGF induced
p62Dok phosphorylation other molecules that blocks
phosphorylation may be used to develop new
cancer treatments.
Literature Cited
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 p62dok by p210bcr-abl inhibits RasGAP activity.
Proceedings of the National Academy of Science USA 97:2093-2098.