Binding of Src to Na⁺/K⁺-ATPase Forms a Functional Signaling Complex

Abstract

We have shown that ouabain activates Src, resulting in subsequent tyrosine phosphorylation of multiple effectors. Here, we tested if the Na ؉ /K ؉ -ATPase and Src can form a functional signaling complex. In LLC-PK1 cells the Na ؉ /K ؉ -ATPase and Src colocalized in the plasma membrane. Fluorescence resonance energy transfer analysis indicated that both proteins were in close proximity, suggesting a direct interaction. GST pulldown assay showed a direct, ouabain-regulated, and multifocal interaction between the ␣1 subunit of Na ؉ /K ؉ -ATPase and Src. Although the interaction between the Src kinase domain and the third cytosolic domain (CD3) of ␣1 is regulated by ouabain, the Src SH3SH2 domain binds to the second cytosolic domain constitutively. Functionally, binding of Src to either the Na ؉ /K ؉ -ATPase or GST-CD3 inhibited Src activity. Addition of ouabain, but not vanadate, to the purified Na ؉ /K ؉ -ATPase/Src complex freed the kinase domain and restored the Src activity. Consistently, exposure of intact cells to ouabain apparently increased the distance between the Na ؉ /K ؉ -ATPase and Src. Concomitantly, it also stimulated tyrosine phosphorylation of the proteins that are associated with the Na ؉ /K ؉ -ATPase. These new findings illustrate a novel molecular mechanism of signal transduction involving the interaction of a P-type ATPase and a nonreceptor tyrosine kinase.

Translationally controlled tumor protein induces human breast epithelial cell transformation through the activation of Src

“…Binding of TCTP to the a1 subunit of Na,K-ATPase induces the release and activation of Src Recent studies suggest that phosphatidylinositol 3-kinase (PI3K) binds to the N-terminal proline-rich motif of Na,K-ATPase a subunit and that partial inhibition of Na,K-ATPase by ouabain induces Src binding to the Na,K-ATPase a subunit (Yudowski et al, 2000;Tian et al, 2006). We found that TCTP bound to the Na, K-ATPase a subunit partially inhibits its pumping activity (Jung et al, 2004).…”

“…Recently, it has been suggested that Src interacts with at least two cytoplasmic domains of Na,K-ATPase a subunit, and that this binding keeps Src in its normally inactive form, whereas binding of ouabain to the ectodomain of a subunit changes Src into its active form through a conformational change in the enzyme (Tian et al, 2006). In the current studies, we showed that Src, apparently tethered to the Na,K-ATPase a subunit, is released and activated by TCTP binding to the a subunit (Figure 1) in human breast epithelial cells.…”

“…It has been previously demonstrated that the ouabaininduced Src activation produces a signaling complex and subsequently transactivates EGFR (Tian et al, 2006). As we found that TCTP binding to Na,K-ATPase a1 induces the release and activation of Src, which is primarily tethered to Na,K-ATPase a1, we tested whether TCTP-induced active Src binds to EGFR and activates the receptor.…”

“…Partial inhibition of Na,K-ATPase by ouabain promotes Src kinase binding to Na,K-ATPase, with the formation of a signaling complex and transactivation of anti-epidermal growth factor receptor (EGFR), thereby activating different downstream signaling pathways (Haas et al, 2002;Tian et al, 2006). We previously reported that translationally controlled tumor protein (TCTP) binding to the cytoplasmic domain of Na, K-ATPase a subunit results in the activation of EGFR and its downstream signaling pathways related to cell survival and motility (Jung et al, 2004;Kim et al, 2009).…”

Expression of Mutant α1 Na/K-ATPase Defective in Conformational Transition Attenuates Src-mediated Signal Transduction*

“…The latter interaction keeps Src in an inactive state. Binding of cardiotonic steroids such as ouabain to the Na/K-ATPase disrupts the latter interaction, resulting in an activation of the pump-associated Src (6). Besides Src, the ␣1 Na/K-ATPase has many interacting partners including phosphoinositide 3-kinase, inositol triphosphate receptor, adducin, ankyrin, and caveolin-1 and is actively involved in multiple cellular processes such as intracellular Ca 2ϩ regulation and caveolae formation (3,(7)(8)(9)(10)(11)(12).…”