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The Na/K-ATPase ?1 and c-Src form signaling complex under native condition: A crosslinking approach.

ABSTRACT: The protein-protein interactions amongst the Na/K-ATPase ?1 subunit, c-Src, and caveolin-1 (cav-1) are essential for the Na/K-ATPase signaling functions. However, there are arguments concerning the interaction model. The present study aims to clarify the interactions amongst the endogenous native proteins in live cells under native resting condition. Under native condition, Blue Native-PAGE and Blue Native-PAGE/SDS-PAGE 2D analyses demonstrated co-existence of the ?1 subunit and c-Src in same protein complex, as well as a direct interaction between the ?1 subunit and c-Src. By comparison of cleavable and non-cleavable cysteine-cysteine crosslinked samples, capillary immunoblotting analysis demonstrated that depletion of Src kinase family members (c-Src, Yes, and Fyn) or cav-1 clearly reduced the interactions of the ?1 subunit with proteins, but depletion of cav-1 did not affect the interaction of c-Src with the ?1 subunit. The data indicated that there are direct interactions between the ?1 subunit and c-Src as well as between the ?1 subunit and cav-1, but argued about the interaction between c-Src and cav-1 under the condition. Furthermore, the data also indicated the existence of different protein complexes containing the ?1 subunit and c-Src, which might have different signaling functions.

SUBMITTER: Nie Y

PROVIDER: S-EPMC7138855 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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The Na/K-ATPase α1 and c-Src form signaling complex under native condition: A crosslinking approach.

Nie Ying Y Bai Fang F Chaudhry Muhammad A MA Pratt Rebecca R Shapiro Joseph I JI Liu Jiang J

Scientific reports 20200407 1

The protein-protein interactions amongst the Na/K-ATPase α1 subunit, c-Src, and caveolin-1 (cav-1) are essential for the Na/K-ATPase signaling functions. However, there are arguments concerning the interaction model. The present study aims to clarify the interactions amongst the endogenous native proteins in live cells under native resting condition. Under native condition, Blue Native-PAGE and Blue Native-PAGE/SDS-PAGE 2D analyses demonstrated co-existence of the α1 subunit and c-Src in same pr ...[more]

PMID: 32265464

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Project description:BackgroundIt has not been possible to study the pumping and signaling functions of Na/K-ATPase independently in live cells.ResultsBoth cell-free and cell-based assays indicate that the A420P mutation abolishes the Src regulatory function of Na/K-ATPase.ConclusionA420P mutant has normal pumping but not signaling function.SignificanceIdentification of Src regulation-null mutants is crucial for addressing physiological role of Na/K-ATPase. The α1 Na/K-ATPase possesses both pumping and signaling functions. However, it has not been possible to study these functions independently in live cells. We have identified a 20-amino acid peptide (Ser-415 to Gln-434) (NaKtide) from the nucleotide binding domain of α1 Na/K-ATPase that binds and inhibits Src in vitro. The N terminus of NaKtide adapts a helical structure. In vitro kinase assays showed that replacement of residues that contain a bulky side chain in the helical structure of NaKtide by alanine abolished the inhibitory effect of the peptide on Src. Similarly, disruption of helical structure by proline replacement, either single or in combination, reduced the inhibitory potency of NaKtide on Src. To identify mutant α1 that retains normal pumping function but is defective in Src regulation, we transfected Na/K-ATPase α1 knockdown PY-17 cells with expression vectors of wild type or mutant α1 carrying Ala to Pro mutations in the region of NaKtide helical structure and generated several stable cell lines. We found that expression of either A416P or A420P or A425P mutant fully restored the α1 content and consequently the pumping capacity of cells. However, in contrast to A416P, either A420P or A425P mutant was incapable of interacting and regulating cellular Src. Consequently, expression of these two mutants caused significant inhibition of ouabain-activated signal transduction and cell growth. Thus we have identified α1 mutant that has normal pumping function but is defective in signal transduction.

Dataset Information

The Na/K-ATPase ?1 and c-Src form signaling complex under native condition: A crosslinking approach.

Publications

The Na/K-ATPase α1 and c-Src form signaling complex under native condition: A crosslinking approach.

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