NaKtide, a Na/K-ATPase-derived Peptide Src Inhibitor, Antagonizes Ouabain-activated Signal Transduction in Cultured Cells

We have previously shown that the Na/K-ATPase binds and inhibits Src. Here, we report the molecular mechanism of Na/K-ATPase-mediated Src regulation and the generation of a novel peptide Src inhibitor that targets the Na/K-ATPase/Src receptor complex and antagonizes ouabain-induced protein kinase cascades. First, the Na/K-ATPase inhibits Src kinase through the N terminus of the nucleotide-binding domain of the α1 subunit. Second, detailed mapping leads to the identification of a 20-amino acid peptide (NaKtide) that inhibits Src (IC₅₀ = 70 nm) in an ATP concentration-independent manner. Moreover, NaKtide does not directly affect the ERK and protein kinase C family of kinases. It inhibits Lyn with a much lower potency (IC₅₀ = 2.5 μm). Third, highly positively charged leader peptide conjugates including HIV-Tat-NaKtide (pNaKtide) readily enter cultured cells. Finally, the following functional studies of pNaKtide demonstrate that this conjugate can specifically target the Na/K-ATPase-interacting pool of Src and act as a potent ouabain antagonist in cultured cells: 1) pNaKtide, unlike PP2, resides in the membranes. Consistently, it affects the basal Src activity much less than that of PP2. 2) pNaKtide is effective in disrupting the formation of the Na/K-ATPase/Src receptor complex in a dose-dependent manner. Consequently, it blocks ouabain-induced activation of Src, ERK, and hypertrophic growth in cardiac myocytes. 3) Unlike PP2, pNaKtide does not affect IGF-induced ERK activation in cardiac myocytes. Taken together, we suggest that pNaKtide may be used as a novel antagonist of ouabain for probing the physiological and pathological significance of the newly appreciated signaling function of Na/K-ATPase and cardiotonic steroids.The Na/K-ATPase is expressed in most eukaryotic cells and is essential for maintaining the transmembrane ion gradient by pumping Na⁺ out of and K⁺ into cells (1). Structurally, the enzyme consists of two non-covalently linked α and β subunits. Similar to other P-ATPases, the Na/K-ATPase α subunit has 10 transmembrane domains with both the N and C termini located in the cytoplasm (2, 3). Based on the published crystal structures of Na/K-ATPase (4), the α subunit consists of several well-characterized domains. The actuator (A)² domain consists of the N terminus and the second cytosolic domain (CD2) connected to transmembrane helices M2 and M3, and the highly conserved discontinuous phosphorylation (P) domain is close to the plasma membrane, while the nucleotide-binding (N) domain is relatively isolated (2). There is a significant amount of movement of both the A and N domains during the ion-pumping cycle as in the SR Ca²⁺-ATPase (4–6). It appears that the A domain rotates, while the N domain closes during the transport cycle. Interestingly, these domains have also been implicated in interacting with many protein partners, including inositol 1,4,5-trisphosphate receptors, phosphoinositide 3-kinase, phospholipase C-γ (PLC-γ), ankyrin, and cofilin (7–12).Src, a member of the Src family non-receptor kinases, plays an important role in the signal transduction pathways of many extracellular stimuli such as cytokines, growth factors, and stress responses (13) and has been considered as a promising target for therapeutic intervention in certain cancers (14) and bone diseases (15). Several endogenous inhibitors of Src have been documented previously, including the C-terminal Src kinase, CSK-homologous kinase, Wiscott-Aldrich syndrome protein, RACK1, and caveolin (16–19).Previously, we and others (20) have demonstrated that binding of cardiotonic steroids (CTS) such as ouabain to the Na/K-ATPase stimulates multiple protein kinase cascades. Moreover, the knock-out of Src prevents these cascades from being activated (10, 21, 22). More recently, we have observed that the Na/K-ATPase interacts directly with Src via at least two binding motifs. One of these interactions is between the CD2 of the α1 subunit and the Src SH2, and the other involves the third cytosolic domain (CD3) of the α1 subunit and the Src kinase domain. We propose that the formation of the Na/K-ATPase/Src complex serves as a receptor for ouabain to stimulate the aforementioned protein kinase cascades. Specifically, the CD3-Src kinase interaction maintains Src in an inactive form whereas the binding of ouabain to the Na/K-ATPase disrupts this interaction, resulting in the assembly and activation of different pathways including ERK cascades, PLC/PKC pathway and mitochondrial production of reactive oxygen species (23). Thus, the Na/K-ATPase functions as an endogenous negative Src regulator. This proposition is consistent with the fact that the basal Src activity is inversely correlated to the amount of Na/K-ATPase α1 subunit in both cultured cells (24) and in α1 heterozygous mouse tissues (25). Therefore, to better understand how the molecular interactions between the Na/K-ATPase and Src regulate Src activity, we have further mapped the Src-binding domains in the CD3 of α1. These studies led to the identification of a peptide Src inhibitor (pNaKtide) and the demonstration that pNaKtide can act as a novel ouabain antagonist capable of inhibiting ouabain-induced activation of protein kinase cascades and hypertrophic growth in cardiac myocytes.