Kinomic profiling approach identifies Trk as a novel radiation modulator

Abstract

Background Ionizing radiation treatment is used in over half of all cancer patients, thus determining the mechanisms of response or resistance is critical for the development of novel treatment approaches. Materials and methods In this report, we utilize a high-content peptide array platform that performs multiplex kinase assays with real-time kinetic readout to investigate the mechanism of radiation response in vascular endothelial cells. We applied this technology to irradiated human umbilical vein endothelial cells (HUVEC). Results We identified 49 specific tyrosine phosphopeptides that were differentially affected by irradiation over a time course of one hour. In one example, the Tropomyosin receptor kinase (Trk) family members, TrkA and TrkB, showed transient activation between 2–15 minutes following irradiation. When we targeted TrkA and TrkB using small molecule inhibitors, HUVEC were protected from radiation damage. Conversely, stimulation of TrkA using gambogic amide promoted radiation enhancement. Conclusions Thus, we show that our approach not only can identify rapid changes in kinase activity but also identify novel targets such as TrkA. TrkA inhibition resulted in radioprotection that correlated with enhanced repair of radiation-induced damage while TrkA stimulation by gambogic amide produced radiation sensitization.

Traumatic Brain Injury Induces Alterations in Cortical Glutamate Uptake without a Reduction in Glutamate Transporter-1 Protein Expression

“…The signal intensities for each peptide were analyzed using BioNavigator 5.2 Software (PamGene). 27 Rodent brain samples evaluated here using the array are comparable to previous data acquired by us in human samples. 28 A control array was run without the addition of ATP to identify non-specific binding of labeled antibody to the array substrate (data not shown).…”

“…The software captures FITC labeled anti-phospho antibodies binding to each phosphorylated peptide substrate every 6 seconds for 90 minutes. 27 Primary analyses were performed from integrated exposure times for each spot (10ms, 20ms, 50ms, 100ms, and 200ms) using steady state as quality control. Integrated spot intensities with the 99 th percentile were used to calculate a minimal positive shift and data were log 2 transformed.…”

Altered serine/threonine kinase activity in schizophrenia

“…The degree of phosphorylation per well is measured in real time using Evolve (PamGene) kinetic image capture software. The software captures FITC labeled anti-phospho antibodies binding to each phosphorylated peptide substrate every 6 seconds for 90 minutes (Jarboe et al, 2012). Integrated spot intensities (slope of exposure brightness over multiple exposure times multiplied by 100) within the 99th percentile were then used to calculate a minimal positive shift using steady state as quality control and data were log 2 transformed.…”

“…+1.5 and -1.5 represent equal magnitudes of change in opposite directions). The signal intensities for each peptide were analyzed using BioNavigator 5.2 Software (PamGene)(Jarboe et al, 2012). …”

Kinomic exploration of temozolomide and radiation resistance in Glioblastoma multiforme xenolines

“…Kinomic profiling of xenoline lysates (lysed in M-Per lysis buffer, Pierce) containing 1:100 Halt’s protease and phosphatase inhibitors (Pierce cats. 78420, 78415) was conducted in the UAB Kinome Core (www.kinomecore.com) using the PamStation®12 platform, manufactured by PamGene (‘s-Hertogenbosch, Netherlands) similar to our previous work [34]. Briefly after protein quantification (BCA protein determination, Pierce Scientific), total protein lysates were loaded onto the appropriate PamChip® [PTK (tyrosine kinome) or STK (serine/threonine kinome)] in kinase buffer.…”