title: 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.
authors.affiliation: Department of Pharmacology, Medical University of Ohio, Toledo, OH 43614, USA.
authors.forename: Jiang
authors.initials: J
authors.lastname: Tian
authors.forename: Ting
authors.initials: T
authors.lastname: Cai
authors.forename: Zhaokan
authors.initials: Z
authors.lastname: Yuan
authors.forename: Haojie
authors.initials: H
authors.lastname: Wang
authors.forename: Lijun
authors.initials: L
authors.lastname: Liu
authors.forename: Michael
authors.initials: M
authors.lastname: Haas
authors.forename: Elena
authors.initials: E
authors.lastname: Maksimova
authors.forename: Xin-Yun
authors.initials: XY
authors.lastname: Huang
authors.forename: Zi-Jian
authors.initials: ZJ
authors.lastname: Xie
mesh_terms: D000818:Animals; D002460:Cell Line; D002645:Chickens; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D051379:Mice; D046912:Multiprotein Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010042:Ouabain / Q000494:pharmacology; D019000:Phosphotyrosine / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D015398:Signal Transduction; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013552:Swine; D019061:src-Family Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D046912:Multiprotein Complexes; D019000:Phosphotyrosine; D010042:Ouabain; D019061:src-Family Kinases; D000254:Sodium-Potassium-Exchanging ATPase