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Threonine 209 phosphorylation on RUNX3 by Pak1 is a molecular switch for its dualistic functions

Oncogene volume 35, pages 4857–4865 (2016)Cite this article

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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.

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Acknowledgements

We thank Prof Yoshiaki Ito, Cancer Science Institute of Singapore, Singapore, for providing us the Runx3 constructs. We profusely thank Prof Susanne M Gollin, University of Pittsburgh, for providing us with SCC-131 cells. We thank Dr Marsha Fraizer (University of Texas, M. D. Anderson Cancer Center, USA); for providing MDA Panc 28 cells. We thank Dr Rajeshwari, Bioklone, Chennai, for generating polyclonal Runx3-Threonine 209 phospho antibody. We thank DAE-BRNS (Grant # 35/14/41/2014-BRNS-RTAC), DBT (BT/PR14340/NNT/28/860/2015) Government of India for financial support to SKR and the Department of Biotechnology, Indian Institute of Technology Madras (IITM) for infrastructural facilities. We thank Mr Balabhaskara Rao and Ms Lakshmi Arivazhagan for the initial help with protein expression studies and Runx3 clones. We thank Professor SP Thyagarajan, Dean Research and Management of SRU for Support and encouragement.

Author contributions

AKC, MS, CC, SSS, RS, ST, RK and SJ – performed experiments; SS and KR – stained and scored the IHC slides; SA and DB – helped with immunofluorescence studies; RSP – performed statistical analysis; ASN – helped with clinical samples; GV, MS and SKR – designed the study and experiments and wrote the paper.

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Author notes

  1. A Kumar, M Singhal and C Chopra: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biotechnology, Indian Institute of Technology Madras (IITM), Chennai, India

    A Kumar, M Singhal, C Chopra, S Srinivasan, R P Surabhi, R Kanumuri, S Tentu, S Jagadeeshan & S K Rayala

  2. Department of Pathology, Sri Ramachandra University, Chennai, India

    S Sundaram & K Ramanathan

  3. Department of Community Medicine, Sri Ramachandra University, Chennai, India

    R Shankar Pitani

  4. Madras Diabetic Research Foundation (MDRF), Chennai, India

    B Muthuswamy & S Abhijit

  5. Rajiv Gandhi Centre for Biotechnology (RGCB), Thiruvananthapuram, India

    A S Nair

  6. Department of Human Genetics, Sri Ramachandra University, Chennai, India

    G Venkatraman

Authors
  1. A Kumar
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  2. M Singhal
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  3. C Chopra
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  7. S Tentu
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  8. S Jagadeeshan
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  9. S Sundaram
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  10. K Ramanathan
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  11. R Shankar Pitani
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  12. B Muthuswamy
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  13. S Abhijit
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  14. A S Nair
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  15. G Venkatraman
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  16. S K Rayala
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Corresponding authors

Correspondence to G Venkatraman or S K Rayala.

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The authors declare no conflict of interest.

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Supplementary Information accompanies this paper on the Oncogene website

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Kumar, A., Singhal, M., Chopra, C. et al. Threonine 209 phosphorylation on RUNX3 by Pak1 is a molecular switch for its dualistic functions. Oncogene 35, 4857–4865 (2016). https://doi.org/10.1038/onc.2016.18

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