Binding of Src to Na+/K+-ATPase forms a functional signaling complex.
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.
Tian
Jiang
J
Department of Pharmacology, Medical University of Ohio, Toledo, OH 43614, USA.
Cai
Ting
T
Yuan
Zhaokan
Z
Wang
Haojie
H
Liu
Lijun
L
Haas
Michael
M
Maksimova
Elena
E
Huang
Xin-Yun
XY
Xie
Zi-Jian
ZJ
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*
D046912:Multiprotein Complexes; D019000:Phosphotyrosine; D010042:Ouabain; D019061:src-Family Kinases; D000254:Sodium-Potassium-Exchanging ATPase