NaKtide, a Na/K-ATPase-derived peptide Src inhibitor, antagonizes ouabain-activated signal transduction in cultured cells.
We have previously shown that the Na/K-ATPase binds and inhibits Src. Here, we report the molecular mechanism of Na/K-ATPase-mediated Src regulation and the generation of a novel peptide Src inhibitor that targets the Na/K-ATPase/Src receptor complex and antagonizes ouabain-induced protein kinase cascades. First, the Na/K-ATPase inhibits Src kinase through the N terminus of the nucleotide-binding domain of the alpha1 subunit. Second, detailed mapping leads to the identification of a 20-amino acid peptide (NaKtide) that inhibits Src (IC50 = 70 nm) in an ATP concentration-independent manner. Moreover, NaKtide does not directly affect the ERK and protein kinase C family of kinases. It inhibits Lyn with a much lower potency (IC50 = 2.5 microm). Third, highly positively charged leader peptide conjugates including HIV-Tat-NaKtide (pNaKtide) readily enter cultured cells. Finally, the following functional studies of pNaKtide demonstrate that this conjugate can specifically target the Na/K-ATPase-interacting pool of Src and act as a potent ouabain antagonist in cultured cells: 1) pNaKtide, unlike PP2, resides in the membranes. Consistently, it affects the basal Src activity much less than that of PP2. 2) pNaKtide is effective in disrupting the formation of the Na/K-ATPase/Src receptor complex in a dose-dependent manner. Consequently, it blocks ouabain-induced activation of Src, ERK, and hypertrophic growth in cardiac myocytes. 3) Unlike PP2, pNaKtide does not affect IGF-induced ERK activation in cardiac myocytes. Taken together, we suggest that pNaKtide may be used as a novel antagonist of ouabain for probing the physiological and pathological significance of the newly appreciated signaling function of Na/K-ATPase and cardiotonic steroids.
Li
Zhichuan
Z
Departments of Physiology and Pharmacology, University of Toledo, Toledo, Ohio 43614, USA.
Cai
Ting
T
Tian
Jiang
J
Xie
Joe X
JX
Zhao
Xiaochen
X
Liu
Lijun
L
Shapiro
Joseph I
JI
Xie
Zijian
Z
D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D002463:Cell Membrane Permeability / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D006801:Humans; D006984:Hypertrophy; D051379:Mice; D008969:Molecular Sequence Data; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000473:pathology; D010042:Ouabain / Q000494:pharmacology*; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology*; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology*; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects*; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000494:pharmacology; D034421:Sus scrofa; D019061:src-Family Kinases / Q000037:antagonists & inhibitors*
C544203:NaKtide; D010446:Peptide Fragments; D010455:Peptides; D021122:Protein Subunits; D010042:Ouabain; D019061:src-Family Kinases; D000254:Sodium-Potassium-Exchanging ATPase