Joe Xie

The Hippo pathway plays a key role in organ size control by regulating cell proliferation and apoptosis in Drosophila. Although recent genetic studies have shown that the Hippo pathway is regulated by the NF2 and Fat tumor suppressors, the physiological regulations of this pathway are unknown. Here we show that in mammalian cells, the transcription coactivator YAP (Yes-associated protein), is inhibited by cell density via the Hippo pathway. Phosphorylation by the Lats tumor suppressor kinase leads to cytoplasmic translocation and inactivation of the YAP oncoprotein. Furthermore, attenuation of this phosphorylation of YAP or Yorkie (Yki), the Drosophila homolog of YAP, potentiates their growth-promoting function in vivo. Moreover, YAP overexpression regulates gene expression in a manner opposite to cell density, and is able to overcome cell contact inhibition. Inhibition of YAP function restores contact inhibition in a human cancer cell line bearing deletion of Salvador (Sav), a Hippo pathway component. Interestingly, we observed that YAP protein is elevated and nuclear localized in some human liver and prostate cancers. Our observations demonstrate that YAP plays a key role in the Hippo pathway to control cell proliferation in response to cell contact.

Aims: Pathologic evidence supports unique sex-specific mechanisms as precursors for acute cardiovascular (CV) events. Current evidence on long-term CV risk among women when compared with men based on measures of coronary artery calcium (CAC) remains incomplete. Methods and results: A total of 63 215 asymptomatic women and men were enrolled in the multicentre, CAC Consortium with median follow-up of 12.6 years. Pooled cohort equation (PCE) risk scores and risk factor data were collected with the Agatston score and other CAC measures (number of lesions and vessels, lesion size, volume, and plaque density). Cox proportional hazard models were employed to estimate CV mortality (n = 919). Sex interactions were calculated. Women and men had average PCE risk scores of 5.8% and 9.1% (P < 0.001). Within CAC subgroups, women had fewer calcified lesions (P < 0.0001) and vessels (P = 0.017), greater lesion size (P < 0.0001), and higher plaque density (P = 0.013) when compared with men. For women and men without CAC, long-term CV mortality was similar (P = 0.67), whereas detectable CAC was associated with 1.3-higher hazard for CV death among women when compared with men (P < 0001). Cardiovascular mortality was higher among women with more extensive, numerous, or larger CAC lesions. The relative hazard for cardiovascular disease (CVD) mortality for women and men was 8.2 vs. 5.1 for multivessel CAC, 8.6 vs. 5.9 for ≥5 CAC lesions, and 8.5 vs. 4.4 for a lesion size ≥15 mm3, respectively. Additional explorations revealed that women with larger sized and more numerous CAC lesions had 2.2-fold higher CVD mortality (P < 0.0001) as compared to men. Moreover, CAC density was not predictive of CV mortality in women (P = 0.51) but was for men (P < 0.001), when controlling for CAC volume and cardiac risk factors. Conclusion: Our overall findings support that measures beyond the Agatston score provide important clues to sex differences in atherosclerotic plaque and may further refine risk detection and focus preventive strategies of care.

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 α1 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 μm). 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. 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 α1 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 μm). 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. The Na/K-ATPase is expressed in most eukaryotic cells and is essential for maintaining the transmembrane ion gradient by pumping Na+ out of and K+ into cells (1Skou J.C. Biochim. Biophys. Acta. 1957; 23: 394-401Crossref PubMed Scopus (1286) Google Scholar). Structurally, the enzyme consists of two non-covalently linked α and β subunits. Similar to other P-ATPases, the Na/K-ATPase α subunit has 10 transmembrane domains with both the N and C termini located in the cytoplasm (2Kaplan J.H. Annu. Rev. Biochem. 2002; 71: 511-535Crossref PubMed Scopus (885) Google Scholar, 3Jorgensen P.L. Hakansson K.O. Karlish S.J. Annu. Rev. Physiol. 2003; 65: 817-849Crossref PubMed Scopus (439) Google Scholar). Based on the published crystal structures of Na/K-ATPase (4Morth J.P. Pedersen B.P. Toustrup-Jensen M.S. Sorensen T.L. Petersen J. Andersen J.P. Vilsen B. Nissen P. Nature. 2007; 450: 1043-1049Crossref PubMed Scopus (702) Google Scholar), the α subunit consists of several well-characterized domains. The actuator (A) 2The abbreviations used are: A domainactuator domainCD2second cytosolic domainCD3third cytosolic domainCTScardiotonic steroidsECFPenhanced cyan fluorescent proteinEYFPenhanced yellow fluorescent proteinERKextracellular signal-regulated protein kinaseFRETfluorescence resonance energy transferGSTglutathione S-transferaseIGF-1insulin-like growth factor 1N domainnucleotide-binding domainP domainphosphorylation domainPKCprotein kinase CPLCphospholipase CPP24-amino-5-[4-chlorophenyl]-7-[t-butyl]pyrazolo[3,4-d]pyrimidineROIregion of interestPBSphosphate-buffered salineFITCfluorescein isothiocyanteSRsarcoplasmic reticulum. 2The abbreviations used are: A domainactuator domainCD2second cytosolic domainCD3third cytosolic domainCTScardiotonic steroidsECFPenhanced cyan fluorescent proteinEYFPenhanced yellow fluorescent proteinERKextracellular signal-regulated protein kinaseFRETfluorescence resonance energy transferGSTglutathione S-transferaseIGF-1insulin-like growth factor 1N domainnucleotide-binding domainP domainphosphorylation domainPKCprotein kinase CPLCphospholipase CPP24-amino-5-[4-chlorophenyl]-7-[t-butyl]pyrazolo[3,4-d]pyrimidineROIregion of interestPBSphosphate-buffered salineFITCfluorescein isothiocyanteSRsarcoplasmic reticulum. domain consists of the N terminus and the second cytosolic domain (CD2) connected to transmembrane helices M2 and M3, and the highly conserved discontinuous phosphorylation (P) domain is close to the plasma membrane, while the nucleotide-binding (N) domain is relatively isolated (2Kaplan J.H. Annu. Rev. Biochem. 2002; 71: 511-535Crossref PubMed Scopus (885) Google Scholar). There is a significant amount of movement of both the A and N domains during the ion-pumping cycle as in the SR Ca2+-ATPase (4Morth J.P. Pedersen B.P. Toustrup-Jensen M.S. Sorensen T.L. Petersen J. Andersen J.P. Vilsen B. Nissen P. Nature. 2007; 450: 1043-1049Crossref PubMed Scopus (702) Google Scholar, 5Toyoshima C. Nakasako M. Nomura H. Ogawa H. Nature. 2000; 405: 647-655Crossref PubMed Scopus (1609) Google Scholar, 6Toyoshima C. Nomura H. Nature. 2002; 418: 605-611Crossref PubMed Scopus (807) Google Scholar). It appears that the A domain rotates, while the N domain closes during the transport cycle. Interestingly, these domains have also been implicated in interacting with many protein partners, including inositol 1,4,5-trisphosphate receptors, phosphoinositide 3-kinase, phospholipase C-γ (PLC-γ), ankyrin, and cofilin (7Jordan C. Püschel B. Koob R. Drenckhahn D. J. Biol. Chem. 1995; 270: 29971-29975Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 8Barwe S.P. Anilkumar G. Moon S.Y. Zheng Y. Whitelegge J.P. Rajasekaran S.A. Rajasekaran A.K. Mol. Biol. Cell. 2005; 16: 1082-1094Crossref PubMed Scopus (134) Google Scholar, 9Lee K. Jung J. Kim M. Guidotti G. Biochem. J. 2001; 353: 377-385Crossref PubMed Scopus (47) Google Scholar, 10Yuan Z. Cai T. Tian J. Ivanov A.V. Giovannucci D.R. Xie Z. Mol. Biol. Cell. 2005; 16: 4034-4045Crossref PubMed Scopus (185) Google Scholar, 11Zhang S. Malmersjö S. Li J. Ando H. Aizman O. Uhlén P. Mikoshiba K. Aperia A. J. Biol. Chem. 2006; 281: 21954-21962Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 12Yudowski G.A. Efendiev R. Pedemonte C.H. Katz A.I. Berggren P.O. Bertorello A.M. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 6556-6561Crossref PubMed Scopus (162) Google Scholar). actuator domain second cytosolic domain third cytosolic domain cardiotonic steroids enhanced cyan fluorescent protein enhanced yellow fluorescent protein extracellular signal-regulated protein kinase fluorescence resonance energy transfer glutathione S-transferase insulin-like growth factor 1 nucleotide-binding domain phosphorylation domain protein kinase C phospholipase C 4-amino-5-[4-chlorophenyl]-7-[t-butyl]pyrazolo[3,4-d]pyrimidine region of interest phosphate-buffered saline fluorescein isothiocyante sarcoplasmic reticulum. actuator domain second cytosolic domain third cytosolic domain cardiotonic steroids enhanced cyan fluorescent protein enhanced yellow fluorescent protein extracellular signal-regulated protein kinase fluorescence resonance energy transfer glutathione S-transferase insulin-like growth factor 1 nucleotide-binding domain phosphorylation domain protein kinase C phospholipase C 4-amino-5-[4-chlorophenyl]-7-[t-butyl]pyrazolo[3,4-d]pyrimidine region of interest phosphate-buffered saline fluorescein isothiocyante sarcoplasmic reticulum. Src, a member of the Src family non-receptor kinases, plays an important role in the signal transduction pathways of many extracellular stimuli such as cytokines, growth factors, and stress responses (13Thomas S.M. Brugge J.S. Annu. Rev. Cell Dev. Biol. 1997; 13: 513-609Crossref PubMed Scopus (2161) Google Scholar) and has been considered as a promising target for therapeutic intervention in certain cancers (14Yeatman T.J. Nat. Rev. Cancer. 2004; 4: 470-480Crossref PubMed Scopus (949) Google Scholar) and bone diseases (15Boyce B.F. Xing L. Yao Z. Yamashita T. Shakespeare W.C. Wang Y. Metcalf 3rd, C.A. Sundaramoorthi R. Dalgarno D.C. Iuliucci J.D. Sawyer T.K. Clin. Cancer Res. 2006; 12: 6291s-6295sCrossref PubMed Scopus (36) Google Scholar). Several endogenous inhibitors of Src have been documented previously, including the C-terminal Src kinase, CSK-homologous kinase, Wiscott-Aldrich syndrome protein, RACK1, and caveolin (16Chong Y.P. Ia K.K. Mulhern T.D. Cheng H.C. Biochim. Biophys. Acta. 2005; 1754: 210-220Crossref PubMed Scopus (88) Google Scholar, 17Miller L.D. Lee K.C. Mochly-Rosen D. Cartwright C.A. Oncogene. 2004; 23: 5682-5686Crossref PubMed Scopus (28) Google Scholar, 18Schulte R.J. Sefton B.M. Biochemistry. 2003; 42: 9424-9430Crossref PubMed Scopus (19) Google Scholar, 19Li S. Couet J. Lisanti M.P. J. Biol. Chem. 1996; 271: 29182-29190Abstract Full Text Full Text PDF PubMed Scopus (674) Google Scholar). Previously, we and others (20Xie Z. Cai T. Mol. Interv. 2003; 3: 157-168Crossref PubMed Scopus (350) Google Scholar) have demonstrated that binding of cardiotonic steroids (CTS) such as ouabain to the Na/K-ATPase stimulates multiple protein kinase cascades. Moreover, the knock-out of Src prevents these cascades from being activated (10Yuan Z. Cai T. Tian J. Ivanov A.V. Giovannucci D.R. Xie Z. Mol. Biol. Cell. 2005; 16: 4034-4045Crossref PubMed Scopus (185) Google Scholar, 21Tian J. Gong X. Xie Z. Am. J. Physiol. Heart Circ. Physiol. 2001; 281: H1899-H1907Crossref PubMed Google Scholar, 22Aydemir-Koksoy A. Abramowitz J. Allen J.C. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). we have that the Na/K-ATPase directly with Src two binding of these is the of the α1 subunit and the Src and the other the third cytosolic domain of the α1 subunit and the Src kinase We that the formation of the Na/K-ATPase/Src complex as a receptor for ouabain to the protein kinase cascades. the kinase Src in an the binding of ouabain to the Na/K-ATPase this in the and activation of pathways including ERK and of J. Cai T. Z. Wang H. L. M. Xie Mol. Biol. Cell. 2006; PubMed Scopus Google Scholar). the Na/K-ATPase as an endogenous Src is with the that the basal Src activity is to the amount of Na/K-ATPase α1 subunit in both cultured cells M. Cai T. Tian J. Xie J. Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus Google Scholar) and in α1 Y. Cai T. Wang H. Li Z. Xie Z. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). to the molecular the Na/K-ATPase and Src Src we have the domains in the of studies to the identification of a peptide Src inhibitor (pNaKtide) and the that pNaKtide can act as a novel ouabain antagonist of ouabain-induced activation of protein kinase cascades and hypertrophic growth in cardiac myocytes. PP2, a Src kinase and a from The following from and and The from The from the the of from and from Src, and from The from The and A from and the with the and by The of as previously J. Cai T. Z. Wang H. L. M. Xie Mol. Biol. Cell. 2006; PubMed Scopus Google Scholar). acid acid acid acid acid and acid on the of Na/K-ATPase α1 subunit by the from the J. S. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) and into A for by the into and by of the from the α1 by and and Src cells from cells from cells as M. Cai T. Tian J. Xie J. Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus Google Scholar). cultured in and and cells for and Src cells cultured in the for and used for the to the of cardiac as previously with M. L. Xie Z. A. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). from of by with and by for a of in in and in for on to and by the and Na/K-ATPase from the as previously M. L. Xie Z. A. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar), and the with and used in this expressed in and glutathione from the glutathione with and in the to the in the of the as previously J. Cai T. Z. Wang H. L. M. Xie Mol. Biol. Cell. 2006; PubMed Scopus Google Scholar). of on glutathione and with of in of phosphate-buffered saline in the of for The with the The on and by with the cells with and in 1 1 1 1 10 10 and Cell by for the with a by and to an and The signal the enhanced and a as previously M. Wang H. Tian J. Xie Z. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). the activation of and Src, with and the and with for the Na/K-ATPase affect kinase the Src Lyn with amount of the Na/K-ATPase in for The for and by of the Src and Lyn by to activation J. S. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). of by phosphorylation for of as in the of for with the of of of 1 of peptide and 10 The to on a for charged peptide the while peptide the The of the an of the into as previously J.S. C.H. Mochly-Rosen D. Circ. Res. 1996; PubMed Scopus Google Scholar). cells and in by two of with the 10 and the for 10 in an cells with and on for a for with to the cells for used for by as previously J. Cai T. Z. Wang H. L. M. Xie Mol. Biol. Cell. 2006; PubMed Scopus Google Scholar). and α1 into cells. cells on to for 1 with cells with for The used for with the The cells that expressed both and α1 to the A region of interest 1) the by of and the of by and the of and The by the of in the region it to the region 2) in the cells cultured on and with the cells for with with for and with The cells with a α1 in for 1 with cells to for 1 and a of the cells and cardiac to 1 with and by fluorescence a cardiac to ouabain with with pNaKtide for cells and and with a as the the and significance We have shown that the of the α1 subunit with and inhibits Src J. Cai T. Z. Wang H. L. M. Xie Mol. Biol. Cell. 2006; PubMed Scopus Google Scholar). in the Na/K-ATPase consists of both and N domains. The of Na/K-ATPase that the N domain is the domain is relatively close to the (4Morth J.P. Pedersen B.P. Toustrup-Jensen M.S. Sorensen T.L. Petersen J. Andersen J.P. Vilsen B. Nissen P. Nature. 2007; 450: 1043-1049Crossref PubMed Scopus (702) Google Scholar). Src is a cytosolic protein, it is that the N domain with the Src kinase this we and it binds to used as a as a that the N domain of α1 subunit with Src and in the N domain acid the binding in the N we and as in and the the The that Src with while a of Src in and of the that the N terminus of is highly and does not to ATP binding M. G. P. S.M. J.P. Nat. Biol. 2003; PubMed Scopus Google Scholar, T. S. K. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). Moreover, the domain in SR Ca2+-ATPase is to T. K. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). we the N terminus of with Src. in we two and and binding to Src in we that the not the with Src. Moreover, the binding of and in the we that both amount of not inhibits Src as the we Src with of in the and Src by used in the as a and also in both and as effective as the in Src Moreover, the of on Src dose-dependent nm) be used to Src in we Src in cells with We that of and Src activity with that of cells The studies demonstrate the of as a Src inhibitor in cultured cells. can target the plasma Na/K-ATPase/Src we the following of First, as in we that expressed as a we a pool of with Na/K-ATPase α1 in the plasma Second, to this pool of has the to with Src, we cells with and and J. Cai T. Z. Wang H. L. M. Xie Mol. Biol. Cell. 2006; PubMed Scopus Google Scholar). The an energy transfer from to in a significant = that and the plasma in close a these two Finally, by an we that we that is most of interacting with the Src. the suggest that binds and inhibits Src, we that the domain to of Src Based on the crystal of the Na/K-ATPase (4Morth J.P. Pedersen B.P. Toustrup-Jensen M.S. Sorensen T.L. Petersen J. Andersen J.P. Vilsen B. Nissen P. Nature. 2007; 450: 1043-1049Crossref PubMed Scopus (702) Google Scholar), 1 and and may an in 1 peptide a of Src while peptide a the other both 1 and the we that peptide potent in Src with an of 70 to that of peptide 1 peptide is from the we it peptide 1 has on Src, it used as a we it NaKtide is relatively to Src, we dose-dependent on Src family kinase shown in NaKtide a dose-dependent of the 2.5 than that for Src NaKtide affects kinases, we a family kinase α and β with NaKtide and and the kinase shown in unlike family kinase NaKtide as as to 10 that NaKtide does not act as an ATP as a Src inhibitor PP2, we on Src in the of of We that in ATP from to not affect Src not studies have demonstrated that the of to a of positively charged can into cultured cells as as S.P. 2005; 4: PubMed Scopus Google Scholar, M. 2006; PubMed Scopus Google Scholar). we HIV-Tat-NaKtide (pNaKtide) also and used as a we also kinase that pNaKtide a highly potent Src inhibitor while the Interestingly, of the potency of NaKtide in this in the from 70 to also not the of pNaKtide and we with in of cells that pNaKtide to of and pNaKtide be in Moreover, unlike a of pNaKtide in the plasma in the other the to we that most of in Unlike pNaKtide, of the in the plasma in to affect the of the NaKtide the We have demonstrated that the Na/K-ATPase and Src in the plasma to a functional of ouabain to this receptor complex the of in a and activation of the Src J. Cai T. Z. Wang H. L. M. Xie Mol. Biol. Cell. 2006; PubMed Scopus Google Scholar). pNaKtide, binding to Src from the cytosolic function as an effective ouabain antagonist by the formation of Na/K-ATPase/Src complex by ouabain-induced activation of Src. this we to the of pNaKtide on the formation of Na/K-ATPase/Src receptor complex in the plasma cells with and and to of We on pNaKtide this conjugate in the plasma the receptor Na/K-ATPase/Src in both and to the plasma Moreover, a significant in cells as we previously J. Cai T. Z. Wang H. L. M. Xie Mol. Biol. Cell. 2006; PubMed Scopus Google Scholar). of pNaKtide a dose-dependent in as as the of cells that that pNaKtide is effective as in interacting with the plasma pool of Src, the formation of a Na/K-ATPase/Src It is important to that the of pNaKtide as a Src inhibitor is in the the for the formation of Na/K-ATPase/Src complex is 1 in potency may be of and to the plasma Src. 1 pNaKtide used in the following of to and as an ouabain First, to pNaKtide as a Src we and the cells to 1 pNaKtide for Cell to of Src and lower in We that of Src with the Na/K-ATPase in cells J. Cai T. Z. Wang H. L. M. Xie Mol. Biol. Cell. 2006; PubMed Scopus Google Scholar). cells from cells M. Cai T. Tian J. Xie J. Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus Google Scholar). of the Na/K-ATPase in these cells the pool of Na/K-ATPase and the basal Src and activity M. Cai T. Tian J. Xie J. Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus Google Scholar). in and pNaKtide of basal Src activity in cells. this not significant = the other it a significant of basal is not is a of Src. Interestingly, the of pNaKtide on Src and in cells much than that in cells we suggest that pNaKtide may target the Na/K-ATPase-interacting pool of of pNaKtide, and on in used in for Src activity with as for with 1 pNaKtide for 1 with 1 for cells with and to 1 NaKtide for 1 Src in and of = to cardiac with 1 pNaKtide for 1 with 1 for used in for Src activity with as for with 1 pNaKtide for 1 with 1 for cells with and to 1 NaKtide for 1 Src in and of = to in a that the of pNaKtide on is to of Src, we the in Src and cells. shown in pNaKtide on in cells Src family and pNaKtide activity in cells the plasma pNaKtide a inhibitor of the Src we cells with NaKtide in the of shown in of a of basal Src activity by Interestingly, the of on basal Src pNaKtide, it the basal Src activity in cells. Second, we the of pNaKtide on ouabain-induced activation of ERK in cells. we that 1 pNaKtide ouabain-induced activation in cells be that this is not a we the in of cardiac myocytes. shown in ouabain-induced activation of Src and also by Third, we pNaKtide and PP2, a Src shown in both pNaKtide and have a on Src on basal Src activity than that of pNaKtide in both and cardiac Moreover, cardiac by PP2, not pNaKtide, a significant of ERK activation Finally, we the of pNaKtide on ouabain-induced of cardiac myocytes. The of cardiac to pNaKtide for 1 and with ouabain for pNaKtide ouabain-induced in a dose-dependent ouabain-induced by pNaKtide the of this we have the of the Na/K-ATPase α1 subunit that and inhibits Src. Based on these we have a novel peptide Src inhibitor that can target the Na/K-ATPase/Src receptor complex and function as an effective ouabain antagonist in cultured cells. and other important in the following The of Na/K-ATPase α1 subunit consists of both N and domains. We that binds the Src kinase domain and inhibits Src kinase activity in J. Cai T. Z. Wang H. L. M. Xie Mol. Biol. Cell. 2006; PubMed Scopus Google Scholar). Based on the newly published crystal of the N domain is the domain is relatively close to the (4Morth J.P. Pedersen B.P. Toustrup-Jensen M.S. Sorensen T.L. Petersen J. Andersen J.P. Vilsen B. Nissen P. Nature. 2007; 450: 1043-1049Crossref PubMed Scopus (702) Google Scholar). Consistently, we that the N domain binds and inhibits Src. Interestingly, it is that the less N terminus of SR Ca2+-ATPase N domain with T. K. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). we have demonstrated that the the acid of the α1 N inhibits Src. mapping that the domain in on Src kinase and on Src. Based on the crystal as as (4Morth J.P. Pedersen B.P. Toustrup-Jensen M.S. Sorensen T.L. Petersen J. Andersen J.P. Vilsen B. Nissen P. Nature. 2007; 450: 1043-1049Crossref PubMed Scopus (702) Google Scholar, M. G. P. S.M. J.P. Nat. Biol. 2003; PubMed Scopus Google Scholar), both a that the and the Src kinase may be for the Src A that several endogenous and Src. and Wiscott-Aldrich syndrome inhibits Src with the domain M. Cartwright C.A. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), does by binding to the Src kinase domain R.J. Sefton B.M. Biochemistry. 2003; 42: 9424-9430Crossref PubMed Scopus (19) Google Scholar). detailed is for inhibitors of other kinases, most Src inhibitors ATP T.K. R. in Cancer. Scholar). the we that in ATP not Src Moreover, it is that NaKtide as a Src inhibitor L. Cancer Res. 1997; Google Scholar) the peptide does not NaKtide a novel of Src a Src both and NaKtide potent and The is close to to most of Src Finally, the of NaKtide on other we that NaKtide appears to be relatively to Src It on family of on on the of Src that it is not an ERK inhibitor can affect ERK signaling by Src. is with the that of Src that as a protein and basal Src as effective as and Moreover, a of in the plasma and that this plasma the the Na/K-ATPase and Src Taken together, these suggest that and NaKtide may be used as an effective Src inhibitor a relatively ouabain antagonist on is by the following First, we that NaKtide by into cells of basal Src activity as by of Second, we demonstrated that a positively charged leader peptide such as to NaKtide it readily Moreover, that a of pNaKtide in the plasma and much less on basal Src activity in both and cardiac not Interestingly, pNaKtide to cells the pool of Na/K-ATPase is in to that in pNaKtide of Src suggest that the plasma pNaKtide may have a relatively on the Na/K-ATPase-interacting pool of Src. Third, we that pNaKtide effective in the formation of Na/K-ATPase/Src receptor complex ouabain-induced activation of in cells by 1 pNaKtide Moreover, pNaKtide effective in ouabain-induced activation of and in cardiac Finally, the of pNaKtide the Na/K-ATPase/Src complex demonstrated by that PP2, not pNaKtide, a significant of IGF-induced activation to several important to be First, we have not acid in NaKtide that and Src and not in the kinase domain NaKtide binds to and the binding inhibits Src. of these the molecular mechanism of Na/K-ATPase-mediated Src Second, we that in pNaKtide, it also on basal Src it is it be of interest to can ouabain-induced Moreover, may have a on Src is to a role in these Finally, we have not the of pNaKtide as an ouabain antagonist in isolated We for the