title: Identification of transglutaminase 2 kinase substrates using a novel on-chip activity assay.
abstract: Transglutaminase 2 (TG2) is an enzyme that plays a critical role in a wide variety of cellular processes through its multifunctional activities. TG2 kinase has emerged as an important regulator of apoptosis, as well as of chromatin structure and function. However, systematic investigation of TG2 kinase substrates is limited due to a lack of a suitable TG2 kinase activity assays. Thus, we developed a novel on-chip TG2 kinase activity assay for quantitative determination of TG2 kinase activity and for screening TG2 kinase substrate proteins in a high-throughput manner. Quantitative TG2 kinase activity was determined by selective detection of substrate protein phosphorylation on the surface of well-type amine arrays. The limit of detection (LOD) of this assay was 4.34μg/ml. We successfully applied this new activity assay to the kinetic analysis of 27 TG2-related proteins for TG2 kinase activity in a high-throughput manner and determined Michaelis-Menten constants (Km) of these proteins. We used the Km values and cellular locations of the TG2-related proteins to construct a substrate affinity map for TG2 kinase. Therefore, this on-chip TG2 kinase activity assay has a strong potential for the systematic investigation of substrate proteins and will be helpful for studying new physiological functions.
authors.affiliation: Department of Molecular and Cellular Biochemistry, Kangwon National University School of Medicine, Chuncheon, Kangwon-Do 200-701, Republic of Korea.
authors.forename: Se-Hui
authors.initials: SH
authors.lastname: Jung
authors.affiliation: Department of Molecular and Cellular Biochemistry, Kangwon National University School of Medicine, Chuncheon, Kangwon-Do 200-701, Republic of Korea.
authors.forename: Deok-Hoon
authors.initials: DH
authors.lastname: Kong
authors.affiliation: Department of Molecular and Cellular Biochemistry, Kangwon National University School of Medicine, Chuncheon, Kangwon-Do 200-701, Republic of Korea.
authors.forename: Hye-Yoon
authors.initials: HY
authors.lastname: Jeon
authors.affiliation: Department of Molecular and Cellular Biochemistry, Kangwon National University School of Medicine, Chuncheon, Kangwon-Do 200-701, Republic of Korea.
authors.forename: Su-Hyun
authors.initials: SH
authors.lastname: Ji
authors.affiliation: Department of Medical Environmental Biology and Tropical Medicine, Kangwon National University School of Medicine, Chuncheon, Kangwon-Do 200-701, Republic of Korea.
authors.forename: Eun-Taek
authors.initials: ET
authors.lastname: Han
authors.affiliation: Department of Physiology, Kangwon National University School of Medicine, Chuncheon, Kangwon-Do 200-701, Republic of Korea.
authors.forename: Won Sun
authors.initials: WS
authors.lastname: Park
authors.affiliation: Department of Internal Medicine, Kangwon National University School of Medicine, Chuncheon, Kangwon-Do 200-701, Republic of Korea.
authors.forename: Seok-Ho
authors.initials: SH
authors.lastname: Hong
authors.affiliation: Department of Molecular and Cellular Biochemistry, Kangwon National University School of Medicine, Chuncheon, Kangwon-Do 200-701, Republic of Korea; Department of Anesthesiology, Kangwon National University School of Medicine, Chuncheon, Kangwon-Do 200-701, Republic of Korea.
authors.forename: Min-Soo
authors.initials: MS
authors.lastname: Kim
authors.affiliation: Department of Molecular and Cellular Biochemistry, Kangwon National University School of Medicine, Chuncheon, Kangwon-Do 200-701, Republic of Korea.
authors.forename: Young-Myeong
authors.initials: YM
authors.lastname: Kim
authors.affiliation: Department of Molecular and Cellular Biochemistry, Kangwon National University School of Medicine, Chuncheon, Kangwon-Do 200-701, Republic of Korea. Electronic address: ksha@kangwon.ac.kr.
authors.forename: Kwon-Soo
authors.initials: KS
authors.lastname: Ha
mesh_terms: D017209:Apoptosis; D015374:Biosensing Techniques / Q000295:instrumentation*; D057075:Enzyme Assays / Q000295:instrumentation; D004867:Equipment Design; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D010766:Phosphorylation; D040081:Protein Array Analysis / Q000295:instrumentation*; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011494:Protein Kinases / Q000378:metabolism*; D013379:Substrate Specificity; D011503:Transglutaminases / Q000378:metabolism*
chemical_list: D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D011494:Protein Kinases; D019204:GTP-Binding Proteins
keywords: Michaelis-Menten constants (K(m)); On-chip TG2 kinase activity assay; Substrate affinity map; Transglutaminase 2