title: Threonine 209 phosphorylation on RUNX3 by Pak1 is a molecular switch for its dualistic functions.
abstract: P21 Activated Kinase 1 (Pak1), an oncogenic serine/threonine kinase, is known to have a significant role in the regulation of cytoskeleton and cellular morphology. Runx3 was initially known for its role in tumor suppressor function, but recent studies have reported the oncogenic role of Runx3 in various cancers. However, the mechanism that controls the paradoxical functions of Runx3 still remains unclear. In this study, we show that Runx3 is a physiologically interacting substrate of Pak1. We identified the site of phosphorylation in Runx3 as Threonine 209 by mass spectrometry analysis and site-directed mutagenesis, and further confirmed the same with a site-specific antibody. Results from our functional studies showed that Threonine 209 phosphorylation in Runx3 alters its subcellular localization by protein mislocalization from the nucleus to the cytoplasm and subsequently converses its biological functions. This was further supported by in vivo tumor xenograft studies in nude mouse models which clearly demonstrated that PANC-28 cells transfected with the Runx3-T209E clone showed high tumorigenic potential as compared with other clones. Our results from clinical samples also suggest that Threonine 209 phosphorylation by Pak1 could be a potential therapeutic target and of great clinical relevance with implications for Runx3 inactivation in cancer cells where Runx3 is known to be oncogenic. The findings presented in this study provide evidence of Runx3-Threonine 209 phosphorylation as a molecular switch in dictating the tissue-specific dualistic functions of Runx3 for the first time.
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Madras (IITM), Chennai, India.
authors.forename: A
authors.initials: A
authors.lastname: Kumar
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Madras (IITM), Chennai, India.
authors.forename: M
authors.initials: M
authors.lastname: Singhal
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Madras (IITM), Chennai, India.
authors.forename: C
authors.initials: C
authors.lastname: Chopra
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Madras (IITM), Chennai, India.
authors.forename: S
authors.initials: S
authors.lastname: Srinivasan
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Madras (IITM), Chennai, India.
authors.forename: R P
authors.initials: RP
authors.lastname: Surabhi
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Madras (IITM), Chennai, India.
authors.forename: R
authors.initials: R
authors.lastname: Kanumuri
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Madras (IITM), Chennai, India.
authors.forename: S
authors.initials: S
authors.lastname: Tentu
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Madras (IITM), Chennai, India.
authors.forename: S
authors.initials: S
authors.lastname: Jagadeeshan
authors.affiliation: Department of Pathology, Sri Ramachandra University, Chennai, India.
authors.forename: S
authors.initials: S
authors.lastname: Sundaram
authors.affiliation: Department of Pathology, Sri Ramachandra University, Chennai, India.
authors.forename: K
authors.initials: K
authors.lastname: Ramanathan
authors.affiliation: Department of Community Medicine, Sri Ramachandra University, Chennai, India.
authors.forename: R
authors.initials: R
authors.lastname: Shankar Pitani
authors.affiliation: Madras Diabetic Research Foundation (MDRF), Chennai, India.
authors.forename: B
authors.initials: B
authors.lastname: Muthuswamy
authors.affiliation: Madras Diabetic Research Foundation (MDRF), Chennai, India.
authors.forename: S
authors.initials: S
authors.lastname: Abhijit
authors.affiliation: Rajiv Gandhi Centre for Biotechnology (RGCB), Thiruvananthapuram, India.
authors.forename: A S
authors.initials: AS
authors.lastname: Nair
authors.affiliation: Department of Human Genetics, Sri Ramachandra University, Chennai, India.
authors.forename: G
authors.initials: G
authors.lastname: Venkatraman
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Madras (IITM), Chennai, India.
authors.forename: S K
authors.initials: SK
authors.lastname: Rayala
mesh_terms: D000818:Animals; D014408:Biomarkers, Tumor / Q000235:genetics*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000235:genetics; D050677:Core Binding Factor Alpha 3 Subunit / Q000235:genetics*; D003593:Cytoplasm; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009369:Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D010766:Phosphorylation; D013912:Threonine / Q000378:metabolism; D023041:Xenograft Model Antitumor Assays
chemical_list: D014408:Biomarkers, Tumor; D050677:Core Binding Factor Alpha 3 Subunit; D047908:Intracellular Signaling Peptides and Proteins; C430457:PAK1IP1 protein, human; C493729:Runx3 protein, human; D013912:Threonine