Abstract LB-414: Study of the kinase activity using NAPPA protein microarray expressed with Human IVTT system

Abstract

Abstract Protein kinases are highly involved in every type of cancer. They play important roles in all cancer hallmarks, ranging from cell proliferation to metastasis, apoptosis to immortality, to name just a few. The complete understanding of this key class of proteins is important for cancer diagnosis and treatment. In order to study protein kinases we are employing protein microarrays, a solid platform that allows the study of thousand of proteins simultaneously. Our platform of choice is NAPPA (Nucleic Acid Programmable Protein Microarray), which permits the use of fresh expressed proteins for functional assays. On NAPPA, the cDNA coding for the gene of interest, which is fused to a tag, is co-printed on the array with an antibody against the tag. Printed slides are expressed using an in vitro transcription and translation system (IVTT) and the newly synthesized proteins are captured by the anti-tag antibody. Following expression, the slides can be used for a variety of applications including biomarker screening and protein-protein interaction studies. In the past, NAPPA slides have been expressed using Rabbit Reticulocyte (Promega) as the IVTT system. However the main concerns with this system included high background, poor expression of genes from other species including humans, and poor lot-to-lot reproducibility. The recent launch of the first Human IVTT system purified from HeLa cells (ThermoFisher Scientific) has the potential to overcome those shortcomings and expand the number of applications that can be carried out with NAPPA arrays. In this study, we evaluate the use of Human IVTT system in NAPPA arrays to study protein kinases. An initial analysis of protein expression using western blot revealed higher expression of proteins in Human IVTT system compared to rabbit reticulocyte lysate. Further benchmarking between the two expression systems was carried out using protein display on NAPPA slides. Proteins expressed in Human IVTT system were at least 10-fold higher compared to rabbit reticulocyte lysate system. Using Human IVTT system we identified autophosphorylation in many tyrosine kinases, including Abl1 and Src. This autophosphorylation could be selectively repressed by prior addition of specific kinase inhibitors. Further we have demonstrated that dephosphorylated proteins can be phosphosphorylated by addition of human cell extracts and this reaction can be measured using anti-phospho tyrosine antibodies. These results suggest that NAPPA arrays can be used to measure kinase activity in a high throughput format not only in the kinases expressed in vitro but also in the cell extracts that are a source for kinases. Taken together our results suggest that Human IVTT system can be used in the NAPPA platform and this system is suitable to study protein kinases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-414. doi:1538-7445.AM2012-LB-414