Abstract 1270: Identification and functional analysis of Pim-1 kinase substrates in prostate cancer

Abstract

Abstract Protein kinases constitute one of the largest and most pleiotropic families of proteins. Mutations leading to activation of protein kinases and loss of function of phosphatases can have deleterious consequences that lead to disease. Pim-1, a serine/threonine kinase, is one of many kinases that may play a role in the etiology of malignant diseases including lymphoma and prostate cancer. Recent studies indicate Pim-1 is overexpressed in prostate cancer and is a highly plausible therapeutic target. Paradoxically, a decrease in Pim-1 expression in later stages of prostate cancer correlates with poor prognosis. Pim-1 has been known to be an oncogene for more than two decades, however, our understanding of how it functions is incomplete. A key underlying factor in this lack of knowledge is that, to date, fewer than fifteen Pim-1 substrates have been identified. The identification and validation of novel Pim-1 substrates could elucidate biomarkers of Pim-1 activity and provide insight into the molecular basis of Pim-1 function. A recent, innovative kinase-substrate profiling method termed the Reverse In-gel Kinase Assay (RIKA) reverses the role of kinase and substrate in a traditional in-gel kinase assay. The assay involves the partial immobilization of an active kinase in a denaturing polyacrylamide gel to determine which substrates from cell lysates can be phosphorylated by the kinase within the gel. The potential substrates are excised from the gel and identified using mass spectrometry. Using a Pim-1 RIKA approach, 40S Ribosomal Protein 7 (RPS7) was identified as a potential novel Pim-1 substrate. RPS7 was expressed in E.coli to conduct preliminary in vitro studies. These in vitro studies demonstrated that Pim-1 robustly phosphorylates RPS7. Further studies were performed to identify sites of phosphorylation on RPS7, and alanine-substitution mutants were generated that completely abolished phosphorylation of RPS7 by Pim-1. These data indicated that RPS7 may be a novel Pim-1 substrate. Studies to validate RPS7 as a direct target of Pim-1 in vivo, and to determine the functional consequences of Pim-1 phosphorylation of RPS7 are in progress. 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 1270. doi:1538-7445.AM2012-1270