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Abstract: FR-OR45

FKBP12 Interacts with 14-3-3 and Synaptopodin to Maintain Actin Cytoskeleton and Processes in Podocytes

Session Information

Category: Pathology and Lab Medicine

  • 1600 Pathology and Lab Medicine

Authors

  • Yasuda, Hidenori, Department of Cell Biology, Kidney Research Center, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan
  • Fukusumi, Yoshiyasu, Department of Cell Biology, Kidney Research Center, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan
  • Zhang, Ying, Department of Cell Biology, Kidney Research Center, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan
  • Kawachi, Hiroshi, Department of Cell Biology, Kidney Research Center, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan
Background

FKBP12 is identified as a binding protein of Tacrolimus (Tac). We reported that FKBP12 is highly expressed in podocytes in kidney, and FKBP12 in podocytes is localized along the actin cytoskeleton. We also reported that FKBP12 is decreased in injured podocytes, and Tac ameliorates podocyte injury by restoring FKBP12 at the actin cytoskeleton (ASN 2019). However, the interaction of FKBP12 with actin-associated proteins and the molecular function of FKBP12 in podocyte are not elucidated yet.

Methods

The localization of FKBP12 with actin-associated proteins was analyzed by dual label immunostaining in glomeruli and human cultured podocytes. The subcellular distribution of FKBP12 was analyzed by western blot in the cultured podocytes. The interaction of FKBP12 with F-actin was analyzed by actin-binding assay with the cell lysate. The interaction of FKBP12 with the actin-associated proteins was analyzed by immunoprecipitation (IP) assay with the lysate of cultured podocytes and HEK293 transfected cells. The effect of FKBP12 siRNA and Tac treatment was analyzed in cultured podocytes.

Results

FKBP12 staining was co-localized with the actin-associated proteins 14-3-3β and synaptopodin (Synp) in glomeruli. The subcellular distribution of FKBP12 was similar to that of 14-3-3 in cultured podocytes. FKBP12 was co-localized and associated with F-actin in the podocytes. FKBP12 interacted with 14-3-3β in cultured podocytes. The IP assay with the HEK expression system also showed FKBP12 interacted with endogenous 14-3-3β. FKBP12 interacted with Synp in the HEK cells co-transfected with FKBP12 and Synp. The interaction of FKBP12 with Synp was not altered by the treatment of 14-3-3β siRNA. Tac enhanced the interaction of FKBP12 with Synp. The expression of 14-3-3β was decreased (63.0% to normal, P<0.01), the structure of F-actin is deranged (staining score, 2.0 vs. 2.9 of normal, P<0.05), and the process formation was impaired (40.4% to normal, P<0.005) in the podocytes treated with FKBP12 siRNA. Tac treatment to normal cells increased the expression of FKBP12 at F-actin in processes and enhanced process formation.

Conclusion

FKBP12 interacts with 14-3-3 and Synp to maintain the actin cytoskeleton and processes in podocyte. The enhanced interactions of FKBP12 with Synp and 14-3-3 by Tac treatment restores FKBP12 at actin cytoskeleton in podocyte.

Funding

  • Government Support – Non-U.S.