Binding of Src to Na+/K+-ATPase forms a functional signaling complex
Authors: Zhaokan Yuan
Michael Haas
Ting Cai
Haojie Wang
Xin-Yun Huang
Jiang Tian
Lijun Liu
Zi-Jian Xie
Elena Maksimova
Journal: Molecular Biology of the Cell (2006)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: ouabain; Src; Na+/K+-ATPase; signaling complex; colocalized
Affiliations: Departments of Pharmacology and Medicine, Medical University of Ohio, Toledo, OH 43614, Department of Physiology, Cornell University, Weill Medical College, New York, NY 10021

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. 

Retracted: No

PubPeer comments:
 There seems to be image duplication of the bands in Figure 3B: ![file] In Figure 7A, there seems to be image manipulation. White boxes have been placed between bands to cover up background and or other bands: ![file] an adjusted image helps see the boxes more clearly: ![file] Another area of concern is Figure 4D: ![file] , 
 ![file] There seems to be image duplication of the bands in Figure 7A , 
 Perhaps some background will help put this paper in context. For those not familiar with the Na,K-ATPase field, the role of this enzyme in regulating cell growth and the effects of cardiac glycosides on cell growth through signaling pathways has been a source of some disagreement. These effects on growth, which are independent of inhibition of the activity of the pump, were described by two groups. M. Peng , L. Huang, Z. Xie, W. H. Huang, and A. Askari (1996), Partial inhibition of Na+ K+-ATPase by ouabain induces Ca++-dependent expressions of early-response genes in cardiac myocytes. J. Biol. Chem., 271,10372-10378. O. Aizman , P. Uhlen, M. Lal, H. Brismar and A. Aperia. (2001) Ouabain, a steroid hormone that signals with slow calcium oscillations. Proc. Natl. Acad. Sci. USA., 98, 13420-13424. It is clear that the pump does mediate effects on cell growth. What is not so clear is the nature of the molecular mechanism and the signaling pathway(s) that mediate this effect. Xie and colleagues proposed that the effects were mediated through interaction of the pump with Src. The dispute involving Src, and other unresolved issues in the Na,K-ATPase field, are reviewed critically in a recent article by the late Amir Askari, who was on the opposing side in the debate. A. Askari (2019) The sodium pump and digitalis drugs: Dogmas and fallacies Pharmacol Res Perspect. e00505. One point that is clear is that a number of groups were unable to reproduce the results supporting interaction of the pump with Src, as reported by Xie s laboratory in Tian et al (2006) Mol. Biol. Cell, 17, 317u2013326. R. J Clifford, J. H. Kaplan (2013) Human breast tumor cells are more resistant to cardiac glycoside toxicity than non-tumorigenic breast cells. PLoSONE. 2013, 8:e84306. M. E. Gable, S, L. Abdallah, S. M. Najjar, L. Liu, A. Askari (2014) Digitalis-induced cell signaling by the sodim pump. Biochem. Biophys. Res. Commun.,446,1151-1154. K. M, Weigand, H. G. Swarts, N. U. Fedosova, F. G. Russel, J. B. Koenderink JB. Na, K-ATPase activity modulates activation: a role for ATP ADP ratio. Biochim. Biophys. Acta., 1818, 1269-1273. E. Yosef, A. Katz, Y. Peleg, T. Mehlman, S. J. Karlish (2016) Do Src kinase and caveolin interact directly with Na, K-ATPase? J. Biol. Chem., 291, 11736-11750. , 
 We thank you for the comments on our paper and figures. 1. Fig. 3B, the middle three bands do look similar. It was a Coommassie blue staining. The original figure is shown below: ![file] 2. Fig. 7A, we think that if you adjust the image from a PDF file with contrast, it will show box-like area all over the image, even the characters. However, if you only adjust the image part, it will not show that kind of artificial effect. Please see the blots below: ![file] , 
 #4 First, Joseph I Shapiro is not an author on this paper. His connection to this paper and research group is unclear, as is his role in supplying responses and supposed original gel images. What is going on here? Second, in Figure 3B, it is abundantly obvious that the bands in lanes 3,4,5 are identical. The provided  original figure is no different from the published panel, other than the blue tone. It provides no further clarification or exculpatory information. The supplied explanations are not compelling at all. These issues require an objective investigation. I hope that someone has brought them to the attention of the journal and an institutional research integrity officer. , 
 Thank you very much for responding to our comments. 1. In your original Fig. 3B, the pattern of white dots on those middle 3 bands still look unexpectedly similar. 2. In Fig. 7A, even in the image you post, I can still see a grey box of smooth background that differs unexpectedly from the adjoining background of the visible bands. Do you have the original image for this blot? , 
 In addition to the three bands in figure 3B looking remarkably similar as noted by commentator 1, I was also struck by the uniformity of the background; it is essentially featureless. Error level analysis of the image suggests that the background in the immediate vicinity of the bands differs from the background in the rest of the image. ![file] , 
 There are three significant issues with this paper: 1. Figure 7A appears to have boxes placed over parts of two images (comment #1). 2. Another problem with figure 7A is the apparent duplication of bands (comment #2). 3. There is band duplication within the image in figure 3B (comments #1 and #7); there is an usual featureless background with bands that appear to have been pasted in place. In addition, Figure 2A is unusual in that the background, as with figure 3B, is featureless and the bands appear washed out. Analysis of this image, which is shown in what follows, suggests that the background around the bands is different than the background elsewhere on the gel. The relatively low quality of the image available from the article makes evaluating it more difficult and analysis would be more definitive with a better image. Nonetheless, the features surrounding the bands in of this picture are consistent with the bands having been cut from somewhere else and pasted in place on a different background. So far the only response to any of the comments has been from Joseph I. Shapiro, whose connection to this work is still a bit unclear. It might help if the first author, Jiang Tian, could comment. ![file] ![file] ,