Na/K-ATPase Signaling: from Bench to Bedside
Abstract (Summary)The Na/K-ATPase interacts with Src to form a functional receptor complex. Bind of ouabain to this receptor complex stimulates Src and subsequently activates multiple protein kinase cascades. To understand the molecular mechanism of Na/K-ATPase/Src interaction, we have further mapped the contacting sites between these two proteins. These studies have produced the following new findings. First, GST pulldown assays identified the N-termini of the a1 N-domain (ND1) and the Src kinase domain (KD1) as the contacting sites that are responsible for Src inhibition. Second, while ND1 works as the Na/K-ATPase and inhibits Src, the KD1 is capable of blocking Na/K-ATPase- or ND1-induced Src inhibition. In addition, expression of YFP-ND1 targets the Na/K-ATPase/Src receptor complex, suggesting that ND1 may function as a ouabain antagonist. Third, further mapping analyses lead to the identification and development of 20 amino acid NaKtide as a potent Src inhibitor. Moreover, coupling of NaKtide with a cell-penetrating peptide GRKKRRQRRRPPQ makes NaKtide cell permeable and allows targeting of this conjugated peptide (pNaKtide) to the plasma membrane. Functionally, pNaKtide is a potent Src inhibitor with an IC50 of 5 nM. Significantly, it has no effect on PKC family of kinases and inhibits Lyn kinase, a closely related Src family kinase, with a much higher IC50. When applied to cell cultures, pNaKtide disrupts the formation of Na/K-ATPase/Src receptor complex and blocks ouabain-induced activation of ERKs in both LLC-PK1 cells and cardiac myocytes. In comparison to PP2, a widely used Src inhibitor, the effects of pNaKtide appear to be more specific to the Na/K-ATPase-interacting Src. While PP2 inhibits IGF-induced Src activation, pNaKtide shows no effect. Taken together, pNaKtide represents a novel ouabain antagonist and can be utilized for probing the physiological function of receptor Na/K-ATPase/Src complex in isolated organs or in vivo. Because Src and Src family kinases are highly expressed in many cancer cells, we have determined whether downregulation of Na/K-ATPase contributes to the elevated Src activity and thus proliferation of these cells. It appears that Na/K-ATPase expression is low in some prostate (DU145) and breast (MCF-7) cancer cells that exhibit high Src activity. Moreover, expression of YFP-ND1 is sufficient to inhibit Src activity in these cells. Significantly, expression of this mini-gene reduces cell growth in DU145 cells. It also inhibits the cell attachment. These findings suggest that the Na/K-ATPase may be a tumor suppressor by regulating the Src activity. Consistently, addition of pNaKtide to cancer cells that express less Na/K-ATPase has proven to be effective in inhibiting cell growth and attachment. Furthermore, in vivo studies have demonstrated the effectiveness of pNaKtide in blocking the formation and growth of xenografted prostate tumors in NOD/SCID mice. Taken together, these findings suggest pNaKtide as a potential therapeutics against tumors that the Na/K-ATPase expression is decreased.
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:na k atpase ouabain src peptide inhibitor
Date of Publication:01/01/2008