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A Model for Domain-Specific Regulation of Src kinase by alpha-1 subunit of Na/K-ATPase

Banerjee, Moumita
http://rave.ohiolink.edu/etdc/view?acc_num=mco1385040354

Abstract Details

2013, Doctor of Philosophy (PhD), University of Toledo, College of Medicine.
Our previous results indicate that alpha-1 (α1) subunit of Na/K-ATPase interacts with Src kinase via two separate domains. While the nucleotide binding domain of α1 interacts with the Src kinase domain, the second cytosolic domain (CD2) binds the Src SH2 domain. The following studies were conducted to assess the functionality of this putative CD2/SH2 interaction in cell culture. LLC-PK1 cells were transfected with either YFP or YFP-CD2. We found that expressed CD2 can bind to Src kinase in cells and prevent Src from being targeted to different signaling complexes. CD2 was found to act as a Src SH2 domain ligand in cells, increasing its basal activity but impairing its signaling function. As a consequence, Src mediated signaling pathways like those activated by ouabain and fibronectin, were inhibited. Furthermore, CD2 expression resulted in inhibition of cell spreading and proliferation, both of which require the involvement of Src. Next, we found that CD2 is phosphorylated at tyrosine (Y260) located in its C terminal region. Since Src SH2 domain has a preference for binding to phosphorylated tyrosines, we hypothesized that this phosphorylation may facilitate its binding to Src. Indeed, mutation of this Y to alanine (A) reduced the growth inhibitory properties of CD2. To further characterize the importance of this Y, we generated Y260A mutant rat α1 Na/K-ATPase and expressed the mutant in a α1 knockdown cell line. This mutation abolished the ouabain mediated Src-ERK signaling function of Na/K-ATPase without hindering its ion transporting function. Co-immunoprecipitation studies revealed that the Y260A mutant is compromised in its ability to bind Src kinase as compared with its normal counterpart. Finally we have developed two Src inhibitory peptides from CD2, spanning the Y260 region- one of which is phosphorylated at Y260 (pNaSH2) and the other is unphosphorylated (NaSH2). In vitro assays indicated that both the peptides were successful in preventing CD2-Src interaction, although pNaSH2 was more effective. However cell studies clearly suggested that pNaSH2 was more potent as it inhibited ouabain mediated signal transduction as well as cell spreading, far better than NaSH2. Taken together these studies indicate a very important role for CD2 in Src kinase interaction with the Na/K-ATPase for the formation of the receptor complex. Also there are indications that the Y260 and its phosphorylation might influence the dynamics of Src regulation in cells.
ZiJian Xie, PhD (Advisor)
Kevin Pan, PhD (Committee Member)
Sandrine Pierre, PhD (Committee Member)
Jiang Tian, PhD (Committee Member)
Andrew Beavis, PhD (Committee Member)
116 p.
Biomedical Research; Cellular Biology; Pharmacology
sodium potassium atpase; src kinase; ouabain; cell signaling

Recommended Citations

Citations

  • Banerjee, M. (2013). A Model for Domain-Specific Regulation of Src kinase by alpha-1 subunit of Na/K-ATPase [Doctoral dissertation, University of Toledo]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=mco1385040354

    APA Style (7th edition)

  • Banerjee, Moumita. A Model for Domain-Specific Regulation of Src kinase by alpha-1 subunit of Na/K-ATPase. 2013. University of Toledo, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=mco1385040354.

    MLA Style (8th edition)

  • Banerjee, Moumita. "A Model for Domain-Specific Regulation of Src kinase by alpha-1 subunit of Na/K-ATPase." Doctoral dissertation, University of Toledo, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=mco1385040354

    Chicago Manual of Style (17th edition)

mco1385040354
276
© 2013, all rights reserved.
This open access ETD is published by University of Toledo Health Science Campus and OhioLINK.