Src-independent ERK signaling through the rat α3 isoform of Na/K-ATPase

Abstract

The Na/K-ATPase α1 polypeptide supports both ion-pumping and signaling functions. The Na/K-ATPase α3 polypeptide differs from α1 in both its primary structure and its tissue distribution. The expression of α3 seems particularly important in neurons, and recent clinical evidence supports a unique role of this isoform in normal brain function. The nature of this specific role of α3 has remained elusive, because the ubiquitous presence of α1 has hindered efforts to characterize α3-specific functions in mammalian cell systems. Using Na/K-ATPase α1 knockdown pig kidney cells (PY-17), we generated the first stable mammalian cell line expressing a ouabain-resistant form of rat Na/K-ATPase α3 in the absence of endogenous pig α1 detectable by Western blotting. In these cells, Na/K-ATPase α3 formed a functional ion-pumping enzyme and rescued the expression of Na/K-ATPase β1 and caveolin-1 to levels comparable with those observed in PY-17 cells rescued with a rat Na/K-ATPase α1 (AAC-19). The α3-containing enzymes had lower Na affinity and lower ouabain-sensitive transport activity than their α1-containing counterparts under basal conditions, but showed a greater capacity to be activated when intracellular Na was increased. In contrast to Na/K-ATPase α1, α3 could not regulate Src. Upon exposure to ouabain, Src activation did not occur, yet ERK was activated through Src-independent pathways involving PI3K and PKC. Hence, α3 expression confers signaling and pumping properties that are clearly distinct from that of cells expressing Na/K-ATPase α1.

Retinoschisin is linked to retinal Na/K-ATPase signaling and localization

“…This Src pool is most likely not subjected to regulation by the retinoschisin–Na/K-ATPase complex. Another explanation for the weak effect of retinoschisin on Src is provided by a recent study showing that the α3-subunit isoform, which is the predominant α-isoform in photoreceptor cells (85% α3 vs. 15% α1; Schneider and Kraig, 1990 ), is incapable of inducing Src activation ( Madan et al. , 2017 ).…”

“…, 2007 ). Of interest, α3–Na/K-ATPases were reported to induce ERK signaling in a Src-independent but so-far-undetermined way ( Madan et al. , 2017 ).…”

Ouabain enhances cell-cell adhesion mediated by β1-subunits of the Na+,K+-ATPase in CHO fibroblasts

“…On the other hand, the inhibition of ERK signaling cascade had a minor effect in the ouabain-dependent cellular aggregation. This might be partially explained by the fact that ERK1/2 is not the only signaling effector activated by cSrc [75,76]. Moreover, our initial screening revealed various signaling proteins that were not studied here and that are probably involved such as TrkB, EphB4 and PDGFR.…”

Protein Interaction and Na/K-ATPase-Mediated Signal Transduction

“…Interestingly, these differences are also conserved in both α2 and α3 sequences ( Figure 3 ). In view of the importance of NaKtide sequence in α1 NKA-mediated Src interaction, we generated α2 and α3-expressing mammalian cells using a knock-down and rescue protocol, and demonstrated that both α2 and α3 NKA isoforms lack Src-interacting capacity [ 119 , 120 ]. As such, they do not carry Src-dependent signal transduction upon ouabain binding.…”