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Abstract: SA-PO333

c-Src and Caveolin-1 in Na-K-ATPase Signaling Complex: A Cys-Cys Crosslinking Analysis

Session Information

Category: Hypertension and CVD

  • 1403 Hypertension and CVD: Mechanisms

Authors

  • Bai, Fang, Marshall University JCE School of Medicine, Huntington, West Virginia, United States
  • Nie, Ying, Marshall University, Huntington, West Virginia, United States
  • Chaudhry, Muhammad A., Marshall University School of Medicine, Huntington, West Virginia, United States
  • Pratt, Rebecca, Marshall University School of Medicine, Huntington, West Virginia, United States
  • Liu, Jiang, Marshall University JCE School of Medicine, Huntington, West Virginia, United States
Background

Activation of Na/K-ATPase signaling cascade regulates sodium handling in proximal tubule, systemic oxidative stress, and uremic cardiomyopathy. This study is to investigate the formation of the signaling complex, especially interactions amongst the α1 subunit, c-Src, and caveolin-1 (cav-1) under native condition in live cells.

Methods

Crosslinking studies were performed in resting live LLC-PK1 cells with Cys-Cys crosslinkers BMH (non-cleavable) and DTME (cleavable). Blue Native-PAGE was used to identify the molecular weight and protein components of the complexes under native condition. Capillary immunoblotting spectra analysis was used to determine the interactions amongst α1, c-Src, and cav-1, by comparisons between LLC-PK1 and cav-1-knockdown C2-9 cells (generated from LLC-PK1), as well as between triple Src kinase (c-Src, Yes, Fyn)-null mouse fibroblasts SYF cells and c-Src-rescued SYF cells, SYF+c-Src cells.

Results

(1) In Blue Native-PAGE, control samples showed a predominant complex around 480 kD (480-band), and crosslinked samples showed an additional complex around 720 kDa (720-band). Mass spectrometry analysis showed that, both the Na/K-ATPase α1 and β1 subunits were present in both 720-band and 480-band, in both control and BMH-crosslinked samples. Cav-1 was only present in the 720-band but not the 480-band. Moreover, in control samples, c-Src was detected in the 480-band, comparing with that c-Src was detected in the 720-band in BMH-crosslinked samples. (2) In spectra analysis of control and crosslinked samples, there were interactions between the α1 subunit and c-Src, and between the α1 subunit and cav-1. While depletion of c-Src or cav-1 clearly reduced the involvement of the α1 subunit in the crosslinked protein complexes, depletion of cav-1 did not affect the interaction of c-Src with other proteins including the α1 subunit. (3) Furthermore, there are multiple bands showing immuno-reactivity with the α1 subunit, c-Src, and cav-1, indicating the existence of different sizes of protein complexes that might contain different protein components with different functions.

Conclusion

In live cells, present study indicated that there are direct interactions between the α1 subunit and c-Src as well as between the α1 subunit and cav-1, but not likely between c-Src and cav-1. The α1 subunit functions as a bridge to link c-Src and cav-1.

Funding

  • NIDDK Support