Abstract 2450: Identifying novel substrates of PLK2 using a chemical genetics approach
Authors: Ramkumar Poornima , Levin Rebecca S. , Sansó Miriam , Jatiani Shashidhar , Dar Arvin C. , Fisher Robert P. , Shokat Kevan M. , Reddy E Premkumar 
Published: August 2015

Abstract
Polo like kinase 2 (PLK2/SNK) is a Ser/Thr kinase with roles identified in G1-S phase transition and in centriole duplication prior to entry into mitosis. PLK2 null embryonic fibroblasts have a slower proliferative rate and delayed entry into S-phase than their wild-type counterparts validating its role in cell cycle. Furthermore, wild-type p53, in response to DNA damage, induces PLK2 expression and checkpoint arrest. While these studies suggest that PLK2 is a tumor suppressor, studies have also shown that PLK2 binds to and phosphorylates mutant p53, but not wild-type p53, thereby promoting an oncogenic, auto-regulatory feedback loop, suggesting a potential role for PLK2 in proliferation. These observations suggest that PLK2 can function as an oncogene as well as a tumor suppressor. It is likely that cellular context and the nature of substrates are key to our understanding of the role that PLK2 plays in tumorigenesis. Although these reports highlight the importance of PLK2 in a variety of cellular processes, there is limited knowledge on the various proteins that are regulated by PLK2. We have optimized a chemical genetics system to identify novel substrates of PLK2. To that end, we mutated the conserved gatekeeper residue in the kinase domain of PLK2 (Leu159) to a smaller amino acid (Gly) to enlarge the ATP binding pocket. This engineered kinase, L159G-PLK2 (Plk2-as) is now able to utilize ATP analogs that carry a N6 modified bulky substituent of nucleoside residue such as N6-Phenylethyl ATP to phosphorylate substrates. We took advantage of this system to tag and isolate PLK2 substrates in vitro. Wild type and analog sensitive-PLK2 kinase domains were purified from bacteria and conditions were optimized for specific thio-phosphorylation of cell lysates by PLK2-as kinase. Labeling of cell lysates and subsequent mass spectrometry identified Signal transducer and activator of transcription 1 (STAT1) as a novel substrate of PLK2. In vitro PLK2 kinase assays in the presence or absence of specific PLK2 inhibitor, ON1231320, validated STAT1 to be a novel substrate of PLK2. Since mass spectrometry identified Ser708 as the potential phosphorylation site, we designed WT and mutant S708A peptides that span 16 amino acids of the phosphorylation site and used them as substrates for PLK1 and PLK2 in vitro kinase assays. While PLK2 readily phosphorylated the WT peptide, phosphorylation of the S708A mutant peptide was significantly reduced suggesting that PLK2 phosphorylates STAT1 at Ser708. This was further confirmed by our observation that ON1231320, a PLK2- specific inhibitor, inhibited PLK2-mediated phosphorylation of WT peptide. In addition, neither the WT nor the S708A peptide was phosphorylated by PLK1, indicating that Ser708 is a PLK2-specific phosphorylation site. Together, these studies validate that STAT1 is a bona fide target of PLK2.
Citation Format: Poornima Ramkumar, Rebecca S. Levin, Miriam Sansó, Shashidhar Jatiani, Arvin C. Dar, Robert P. Fisher, Kevan M. Shokat, E Premkumar Reddy. Identifying novel substrates of PLK2 using a chemical genetics approach. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2450. doi:10.1158/1538-7445.AM2015-2450