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Mutations at the dimer interface and surface residues of Nm23-H1 metastasis suppressor affect its expression and function

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Abstract

The Nm23 metastasis suppressor family is involved in a variety of physiological and pathological processes including cell proliferation, differentiation, tumorigenesis, and metastasis. Given that Nm23 proteins may function as hexamers composed of different members of the family, especially Nm23-H1 and H2 isoforms, it is pertinent to assess the importance of interface and surface residues in defining the functional characteristics of Nm23 proteins. Using molecular modeling to identify clusters of residues that may affect dimer formation and isoform specificity, mutants of Nm23-H1 were constructed and assayed for their ability to modulate cell migration. Mutations of dimer interface residues Gly22 and Lys39 affected the expression level of Nm23-H1, without altering the transcript level. The reduced protein expression was not due to increased protein degradation or altered subcellular distribution. Substitution of the surface residues of Nm23-H1 with Nm23-H2-specific Ser131 and/or Lys124/135 affected the electrophoretic mobility of the protein. Moreover, in cell migration assays, several mutants with altered surface residues exhibited impaired ability to suppress the mobility of MDA-MB-231 cells. Collectively, the study suggests that disrupting the dimer interface may affect the expression of Nm23-H1, while the residues at α-helix and β-sheet on the surface of Nm23-H1 may contribute to its metastasis suppressive function.

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Acknowledgements

We thank Jacey Liu, Jason Tsang, and Wenjie Zhu for technical assistance.

Funding

This work was supported in part by grants from the University Grants Committee of Hong Kong (AoE/M-604/16), Hong Kong Research Grants Council Theme-based Research Scheme (T13-605/18-W), Innovation and Technology Commission of Hong Kong (ITCPD/17-9), and the Hong Kong Jockey Club.

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  1. Yuanjun Li

    Present address: Eye Center of Xiangya Hospital, Hunan Key Laboratory of Opthalmology, Xiangya Hospital, Central South University, Changsha, China

Authors and Affiliations

  1. Division of Life Science and the Biotechnology Research Institute, Hong Kong University of Science and Technology, Hong Kong, China

    Yuanjun Li, Wen Liu, Vasu Saini & Yung H. Wong

  2. State Key Laboratory of Molecular Neuroscience and the Molecular Neuroscience Center, Hong Kong University of Science and Technology, Hong Kong, China

    Yung H. Wong

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Contributions

Methodology, validation, analysis—YL, initial draft preparation—YL and YHW, data interpretation—YL, WL and YHW, writing-review editing—WL, VS, and YHW, molecular modeling—WL, cloning and fluorescence analysis—VS, supervision—YHW, funding acquisition—YHW, conception of the study and final approval of the manuscript YHW. All authors have read and agreed to the published version of the manuscript.

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Correspondence to Yung H. Wong.

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The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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11010_2020_3836_MOESM1_ESM.pdf

Supplementary Fig. S1 Interaction of Flag-Gβγ and Nm23 surface mutants. HEK293 cells were co-transfected with 2 μg each of Flag-Gβ, HA-Gγ and Nm23-H1/2 or vector for co-immunoprecipitation (Co-IP) assays. Cells were lysed 48 h after transfection, and immunoprecipitated (IP) with anti-Flag agarose affinity gel as indicated and finally subjected to Western blot analysis. Both Nm23-H1 and H2 could form protein complexes with Gβγ in the Co-IP assay, and this ability was also observed in the KHD and HD mutants, but not in the RPN mutant. (PDF 62 kb)

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Li, Y., Liu, W., Saini, V. et al. Mutations at the dimer interface and surface residues of Nm23-H1 metastasis suppressor affect its expression and function. Mol Cell Biochem 474, 95–112 (2020). https://doi.org/10.1007/s11010-020-03836-1

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