The endosomal trafficking regulator LITAF controls the cardiac Nav1.5 channel via the ubiquitin ligase NEDD4-2
2020; Elsevier BV; Volume: 295; Issue: 52 Linguagem: Inglês
10.1074/jbc.ra120.015216
ISSN1083-351X
AutoresNilüfer N. Turan, Karni S. Moshal, Karim Roder, Brett Baggett, Anatoli Y. Kabakov, Saroj Dhakal, Ryota Teramoto, David Y. Chiang, Mingwang Zhong, An Xie, Yichun Lu, Samuel C. Dudley, Calum A. MacRae, Alain Karma, Gideon Koren,
Tópico(s)Ion channel regulation and function
ResumoThe QT interval is a recording of cardiac electrical activity. Previous genome-wide association studies identified genetic variants that modify the QT interval upstream of LITAF (lipopolysaccharide-induced tumor necrosis factor-α factor), a protein encoding a regulator of endosomal trafficking. However, it was not clear how LITAF might impact cardiac excitation. We investigated the effect of LITAF on the voltage-gated sodium channel Nav1.5, which is critical for cardiac depolarization. We show that overexpressed LITAF resulted in a significant increase in the density of Nav1.5-generated voltage-gated sodium current INa and Nav1.5 surface protein levels in rabbit cardiomyocytes and in HEK cells stably expressing Nav1.5. Proximity ligation assays showed co-localization of endogenous LITAF and Nav1.5 in cardiomyocytes, whereas co-immunoprecipitations confirmed they are in the same complex when overexpressed in HEK cells. In vitro data suggest that LITAF interacts with the ubiquitin ligase NEDD4-2, a regulator of Nav1.5. LITAF overexpression down-regulated NEDD4-2 in cardiomyocytes and HEK cells. In HEK cells, LITAF increased ubiquitination and proteasomal degradation of co-expressed NEDD4-2 and significantly blunted the negative effect of NEDD4-2 on INa. We conclude that LITAF controls cardiac excitability by promoting degradation of NEDD4-2, which is essential for removal of surface Nav1.5. LITAF-knockout zebrafish showed increased variation in and a nonsignificant 15% prolongation of action potential duration. Computer simulations using a rabbit-cardiomyocyte model demonstrated that changes in Ca2+ and Na+ homeostasis are responsible for the surprisingly modest action potential duration shortening. These computational data thus corroborate findings from several genome-wide association studies that associated LITAF with QT interval variation. The QT interval is a recording of cardiac electrical activity. Previous genome-wide association studies identified genetic variants that modify the QT interval upstream of LITAF (lipopolysaccharide-induced tumor necrosis factor-α factor), a protein encoding a regulator of endosomal trafficking. However, it was not clear how LITAF might impact cardiac excitation. We investigated the effect of LITAF on the voltage-gated sodium channel Nav1.5, which is critical for cardiac depolarization. We show that overexpressed LITAF resulted in a significant increase in the density of Nav1.5-generated voltage-gated sodium current INa and Nav1.5 surface protein levels in rabbit cardiomyocytes and in HEK cells stably expressing Nav1.5. Proximity ligation assays showed co-localization of endogenous LITAF and Nav1.5 in cardiomyocytes, whereas co-immunoprecipitations confirmed they are in the same complex when overexpressed in HEK cells. In vitro data suggest that LITAF interacts with the ubiquitin ligase NEDD4-2, a regulator of Nav1.5. LITAF overexpression down-regulated NEDD4-2 in cardiomyocytes and HEK cells. In HEK cells, LITAF increased ubiquitination and proteasomal degradation of co-expressed NEDD4-2 and significantly blunted the negative effect of NEDD4-2 on INa. We conclude that LITAF controls cardiac excitability by promoting degradation of NEDD4-2, which is essential for removal of surface Nav1.5. LITAF-knockout zebrafish showed increased variation in and a nonsignificant 15% prolongation of action potential duration. Computer simulations using a rabbit-cardiomyocyte model demonstrated that changes in Ca2+ and Na+ homeostasis are responsible for the surprisingly modest action potential duration shortening. These computational data thus corroborate findings from several genome-wide association studies that associated LITAF with QT interval variation. The voltage-gated sodium channel Nav1.5 is responsible for the initial upstroke of cardiac action potential (1Rook M.B. Evers M.M. Vos M.A. Bierhuizen M.F. Biology of cardiac sodium channel Nav1.5 expression.Cardiovasc. Res. 2012; 93 (21937582): 12-2310.1093/cvr/cvr252Crossref PubMed Scopus (125) Google Scholar, 2Abriel H. Kass R.S. Regulation of the voltage-gated cardiac sodium channel Nav1.5 by interacting proteins.Trends Cardiovasc. Med. 2005; 15 (15795161): 35-4010.1016/j.tcm.2005.01.001Crossref PubMed Scopus (127) Google Scholar). Post-translational modifications such as phosphorylation or ubiquitination are essential for correct expression and function of Nav1.5 (1Rook M.B. Evers M.M. Vos M.A. Bierhuizen M.F. Biology of cardiac sodium channel Nav1.5 expression.Cardiovasc. Res. 2012; 93 (21937582): 12-2310.1093/cvr/cvr252Crossref PubMed Scopus (125) Google Scholar). The activity and density of Nav1.5 channels at the membrane depend on forward trafficking, stability, and domain-targeting mediated by anchoring proteins and retrograde trafficking (3Balse E. Boycott H.E. Ion channel trafficking: control of ion channel density as a target for arrhythmias?.Front. Physiol. 2017; 8 (29089904): 80810.3389/fphys.2017.00808Crossref PubMed Scopus (19) Google Scholar). Retrograde trafficking of cardiac Nav1.5 depends on the E3 ubiquitin ligase NEDD4-2 (neural precursor cell-expressed developmentally down-regulated 4 type 2), which accelerates the degradation of Nav1.5 by ubiquitination (4Abriel H. Kamynina E. Horisberger J.D. Staub O. Regulation of the cardiac voltage-gated Na+ channel (H1) by the ubiquitin-protein ligase Nedd4.FEBS Lett. 2000; 466 (10682864): 377-38010.1016/S0014-5793(00)01098-XCrossref PubMed Scopus (105) Google Scholar, 5van Bemmelen M.X. Rougier J.S. Gavillet B. Apothéloz F. Daidié D. Tateyama M. Rivolta I. Thomas M.A. Kass R.S. Staub O. 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SIMPLE interacts with NEDD4 and TSG101: evidence for a role in lysosomal sorting and implications for Charcot–Marie–Tooth disease.J. Neurosci. Res. 2005; 82 (16118794): 43-5010.1002/jnr.20628Crossref PubMed Scopus (51) Google Scholar, 22Eaton H.E. Desrochers G. Drory S.B. Metcalf J. Angers A. Brunetti C.R. SIMPLE/LITAF expression induces the translocation of the ubiquitin ligase itch towards the lysosomal compartments.PLoS One. 2011; 6 (21326863)e1687310.1371/journal.pone.0016873Crossref PubMed Scopus (22) Google Scholar). The N terminus of LITAF contains two PXY motifs, which are essential for interacting with members of the NEDD4 family of HECT (homologous to the E6-AP C terminus) domain ubiquitin ligases via their WW domains (17Shirk A.J. Anderson S.K. Hashemi S.H. Chance P.F. Bennett C.L. SIMPLE interacts with NEDD4 and TSG101: evidence for a role in lysosomal sorting and implications for Charcot–Marie–Tooth disease.J. Neurosci. 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Charcot–Marie–Tooth disease-linked protein SIMPLE functions with the ESCRT machinery in endosomal trafficking.J. Cell Biol. 2012; 199 (23166352): 799-81610.1083/jcb.201204137Crossref PubMed Scopus (45) Google Scholar, 17Shirk A.J. Anderson S.K. Hashemi S.H. Chance P.F. Bennett C.L. SIMPLE interacts with NEDD4 and TSG101: evidence for a role in lysosomal sorting and implications for Charcot–Marie–Tooth disease.J. Neurosci. Res. 2005; 82 (16118794): 43-5010.1002/jnr.20628Crossref PubMed Scopus (51) Google Scholar, 18Zhu H. Guariglia S. Yu R.Y. Li W. Brancho D. Peinado H. Lyden D. Salzer J. Bennett C. Chow C.W. Mutation of SIMPLE in Charcot–Marie–Tooth 1C alters production of exosomes.Mol. Biol. Cell. 2013; 24 (23576546): 1619-163710.1091/mbc.E12-07-0544Crossref PubMed Scopus (48) Google Scholar). Mutations clustered around the hydrophobic region required for membrane localization in LITAF cause Charcot–Marie–Tooth disease, an inherited peripheral neuropathy. They also result in mislocalization and impaired endosome-to-lysosome trafficking of membrane proteins (16Lee S.M. Chin L.S. Li L. Charcot–Marie–Tooth disease-linked protein SIMPLE functions with the ESCRT machinery in endosomal trafficking.J. Cell Biol. 2012; 199 (23166352): 799-81610.1083/jcb.201204137Crossref PubMed Scopus (45) Google Scholar, 24Lacerda A.F. Hartjes E. Brunetti C.R. LITAF mutations associated with Charcot–Marie–Tooth disease 1C show mislocalization from the late endosome/lysosome to the mitochondria.PLoS One. 2014; 9 (25058650)e10345410.1371/journal.pone.0103454Crossref PubMed Scopus (15) Google Scholar). Importantly, the genetic variant rs8049607 located within an intergenic enhancer region (25Tan W.L.W. Anene-Nzelu C.G. Wong E. Lee C.J.M. Tan H.S. Tang S.J. Perrin A. Wu K.X. Zheng W. Ashburn R.J. Pan B. Lee M.Y. Autio M.I. Morley M.P. Tam W.L. et al.Epigenomes of human hearts reveal new genetic variants relevant for cardiac disease and phenotype.Circ. Res. 2020; 127 (32529949): 761-77710.1161/CIRCRESAHA.120.317254Crossref PubMed Scopus (5) Google Scholar) is associated with a very modest QT interval prolongation of 1.2 ms (11Newton-Cheh C. Eijgelsheim M. Rice K.M. de Bakker P.I. Yin X. Estrada K. Bis J.C. Marciante K. Rivadeneira F. Noseworthy P.A. Sotoodehnia N. Smith N.L. Rotter J.I. Kors J.A. Witteman J.C. et al.Common variants at ten loci influence QT interval duration in the QTGEN Study.Nat. Genet. 2009; 41 (19305408): 399-40610.1038/ng.364Crossref PubMed Scopus (324) Google Scholar, 12Arking D.E. Pulit S.L. Crotti L. van der Harst P. Munroe P.B. Koopmann T.T. Sotoodehnia N. Rossin E.J. Morley M. Wang X. Johnson A.D. Lundby A. Gudbjartsson D.F. Noseworthy P.A. Eijgelsheim M. et al.Genetic association study of QT interval highlights role for calcium signaling pathways in myocardial repolarization.Nat. Genet. 2014; 46 (24952745): 826-83610.1038/ng.3014Crossref PubMed Scopus (161) Google Scholar, 13Pfeufer A. Sanna S. Arking D.E. Müller M. Gateva V. Fuchsberger C. Ehret G.B. Orrú M. Pattaro C. Köttgen A. Perz S. Usala G. Barbalic M. Li M. Pütz B. et al.Common variants at ten loci modulate the QT interval duration in the QTSCD Study.Nat. Genet. 2009; 41 (19305409): 407-41410.1038/ng.362Crossref PubMed Scopus (300) Google Scholar). This variant (rs8049607) is associated with reduced LITAF mRNA transcript levels in the left ventricle (26GTEx ConsortiumThe Genotype-Tissue Expression (GTEx) project.Nat. Genet. 2013; 45 (23715323): 580-58510.1038/ng.2653Crossref PubMed Scopus (3155) Google Scholar, 27Moshal K.S. Roder K. Kabakov A.Y. Werdich A.A. Chiang D.Y.-E. Turan N.N. Xie A. Kim T.Y. Cooper L.L. Lu Y. Zhong M. Li W. Terentyev D. Choi B.-R. Karma A. et al.LITAF (lipopolysaccharide-induced tumor necrosis factor) regulates cardiac L-type calcium channels by modulating NEDD (neural precursor cell expressed developmentally downregulated protein) 4-1 ubiquitin ligase.Circ. Genom. Precis. Med. 2019; 12 (31462068): 407-42010.1161/CIRCGEN.119.002641Crossref PubMed Scopus (3) Google Scholar). Thus, a reduction in LITAF prolongs the QT interval. Based on the genome-wide association studies' findings (11Newton-Cheh C. Eijgelsheim M. Rice K.M. de Bakker P.I. Yin X. Estrada K. Bis J.C. Marciante K. Rivadeneira F. Noseworthy P.A. Sotoodehnia N. Smith N.L. Rotter J.I. Kors J.A. Witteman J.C. et al.Common variants at ten loci influence QT interval duration in the QTGEN Study.Nat. Genet. 2009; 41 (19305408): 399-40610.1038/ng.364Crossref PubMed Scopus (324) Google Scholar, 12Arking D.E. Pulit S.L. Crotti L. van der Harst P. Munroe P.B. Koopmann T.T. Sotoodehnia N. Rossin E.J. Morley M. Wang X. Johnson A.D. Lundby A. Gudbjartsson D.F. Noseworthy P.A. Eijgelsheim M. et al.Genetic association study of QT interval highlights role for calcium signaling pathways in myocardial repolarization.Nat. Genet. 2014; 46 (24952745): 826-83610.1038/ng.3014Crossref PubMed Scopus (161) Google Scholar, 13Pfeufer A. Sanna S. Arking D.E. Müller M. Gateva V. Fuchsberger C. Ehret G.B. Orrú M. Pattaro C. Köttgen A. Perz S. Usala G. Barbalic M. Li M. Pütz B. et al.Common variants at ten loci modulate the QT interval duration in the QTSCD Study.Nat. Genet. 2009; 41 (19305409): 407-41410.1038/ng.362Crossref PubMed Scopus (300) Google Scholar) and LITAF's functional role in endosome-to-lysosome trafficking, we hypothesized that LITAF is a candidate for regulation of cardiac excitation, likely acting as an effector of ion channel complex trafficking or degradation. Indeed, we have recently shown that LITAF acts as an adaptor protein promoting NEDD4-1–mediated ubiquitination and subsequent degradation of L-type calcium channels (LTCCs), and gain of function of LITAF is associated with shortening of action potential duration (APD) (27Moshal K.S. Roder K. Kabakov A.Y. Werdich A.A. Chiang D.Y.-E. Turan N.N. Xie A. Kim T.Y. Cooper L.L. Lu Y. Zhong M. Li W. Terentyev D. Choi B.-R. Karma A. et al.LITAF (lipopolysaccharide-induced tumor necrosis factor) regulates cardiac L-type calcium channels by modulating NEDD (neural precursor cell expressed developmentally downregulated protein) 4-1 ubiquitin ligase.Circ. Genom. Precis. Med. 2019; 12 (31462068): 407-42010.1161/CIRCGEN.119.002641Crossref PubMed Scopus (3) Google Scholar). In this study, we present data that support an additional role for LITAF in modulating membrane density and function of cardiac Nav1.5 via the ubiquitin ligase NEDD4-2. Uniquely, gain of function of LITAF increases the expression of sodium channels in the membrane. To investigate any possible effect of LITAF on the Nav1.5 channel and its generated voltage-gated sodium current INa, we used cultured 3-week-old rabbit cardiomyocytes (3wRbCM). We developed and used this model to study various ion channels underlying action potential duration (27Moshal K.S. Roder K. Kabakov A.Y. Werdich A.A. Chiang D.Y.-E. Turan N.N. Xie A. Kim T.Y. Cooper L.L. Lu Y. Zhong M. Li W. Terentyev D. Choi B.-R. Karma A. et al.LITAF (lipopolysaccharide-induced tumor necrosis factor) regulates cardiac L-type calcium channels by modulating NEDD (neural precursor cell expressed developmentally downregulated protein) 4-1 ubiquitin ligase.Circ. Genom. Precis. Med. 2019; 12 (31462068): 407-42010.1161/CIRCGEN.119.002641Crossref PubMed Scopus (3) Google Scholar, 28Kabakov A.Y. Moshal K. Lu Y. Roder K. Nilufer T. Li W. Murphy K. Terentyev D. Koren G. Week-old rabbit cardiomyocytes (3wRbCM): a novel cellular model for studying cardiac excitation.Biophys. J. 2019; 116: 230a10.1016/j.bpj.2018.11.1267Abstract Full Text Full Text PDF Google Scholar). For example, 3-week-old rabbit cardiomyocytes cultured for 48 h display a stable INa current (Fig. 1A). The cells were transduced with adenovirus encoding GFP and hemagglutinin (HA)–tagged LITAF. Overexpression of LITAF caused a significant increase (27.4%) in peak INa density (from −19.3 ± 2.2 pA/pF to −24.6 ± 2.21 pA/pF; p = 0.0073; Fig. 1B), yet there were no changes in voltage-dependent activation and inactivation kinetics (Fig. 1C). Western blotting results show that total Nav1.5 protein levels were significantly up-regulated (76.7%) in LITAF-overexpressing 3wRbCM (p < 0.05; Fig. 1D). Next, we switched to HEK cells as they are frequently used to study Nav1.5 in vitro (5van Bemmelen M.X. Rougier J.S. Gavillet B. Apothéloz F. Daidié D. Tateyama M. Rivolta I. Thomas M.A. Kass R.S. Staub O. Abriel H. Cardiac voltage-gated sodium channel Nav1.5 is regulated by Nedd4-2 mediated ubiquitination.Circ. Res. 2004; 95 (15217910): 284-29110.1161/01.RES.0000136816.05109.89Crossref PubMed Scopus (164) Google Scholar, 29Liu M. Shi G. Yang K.C. Gu L. Kanthasamy A.G. Anantharam V. Dudley S.C. Role of protein kinase C in metabolic regulation of the cardiac Na.Heart Rhythm. 2017; 14: 440-44710.1016/j.hrthm.2016.12.026Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 30Ponce-Balbuena D. Guerrero-Serna G. Valdivia C.R. Caballero R. Diez-Guerra F.J. Jiménez-Vázquez E.N. Ramírez R.J. Monteiro da Rocha A. Herron T.J. Campbell K.F. Willis B.C. Alvarado F.J. Zarzoso M. Kaur K. Pérez-Hernández M. et al.Cardiac Kir2.1 and Na.Circ. Res. 2018; 122 (29514831): 1501-151610.1161/CIRCRESAHA.117.311872Crossref PubMed Scopus (26) Google Scholar). We used HEK cells that stably co-express Nav1.5 and GFP, to confirm our data obtained with 3wRbCM. HEK cells were co-transfected with expression plasmids for LITAF and red fluorescent protein (DsRed) or GFP and DsRed (control). Patch clamp results show that LITAF increased (20.1%) Nav1.5 current density from −80.6 ± 12.9 pA/pF to −96.8 ± 13.5 pA/pF; p < 0.05; Fig. 2B). Importantly, a significant concomitant increase in total Nav1.5 channel expression was noted (Fig. 2C). Surface biotinylation experiments were carried out to confirm that membrane expression of Nav1.5 was also significantly elevated upon LITAF overexpression (Fig. 2D), which is consistent with higher INa peak density in the presence of exogenous LITAF (Fig. 2C). Because our data suggested a functional interaction between LITAF and Nav1.5, we performed co-immunoprecipitation experiments on HEK cells stably expressing V5-tagged Nav1.5. The cells were co-transfected with plasmid encoding HA-tagged LITAF or empty control vector. Cell extracts were incubated with V5 antiserum, immunoprecipitated, separated by SDS-PAGE, transferred to membrane, and probed with anti-Nav1.5 antibody. Western blotting analyses suggest that LITAF is found in a protein complex with Nav1.5 (Fig. 3A). Additionally, we performed in situ PLA to look for any co-localization between these two molecules in 3wRbCM (Fig. 3B). The specificity of the assay was shown by the lack of staining using mouse anti-LITAF or rabbit anti-Nav1.5 as negative controls. The appearance of puncta with the combination of mouse anti-LITAF and rabbit anti-Nav1.5 supports proximity between LITAF and Nav1.5 in cardiomyocytes. NEDD4-2–dependent ubiquitination is a prerequisite for the degradation of surface Nav1.5 (4Abriel H. Kamynina E. Horisberger J.D. Staub O. Regulation of the cardiac voltage-gated Na+ channel (H1) by the ubiquitin-protein ligase Nedd4.FEBS Lett. 2000; 466 (10682864): 377-38010.1016/S0014-5793(00)01098-XCrossref PubMed Scopus (105) Google Scholar, 5van Bemmelen M.X. Rougier J.S. Gavillet B. Apothéloz F. Daidié D. Tateyama M. Rivolta I. Thomas M.A. Kass R.S. Staub O. Abriel H. Cardiac voltage-gated sodium channel Nav1.5 is regulated by Nedd4-2 mediated ubiquitination.Circ. Res. 2004; 95 (15217910): 284-29110.1161/01.RES.0000136816.05109.89Crossref PubMed Scopus (164) Google Scholar). Because we have previously established physical and functional interactions between LITAF and the ubiquitin ligase NEDD4-1 (27Moshal K.S. Roder K. Kabakov A.Y. Werdich A.A. Chiang D.Y.-E. Turan N.N. Xie A. Kim T.Y. Cooper L.L. Lu Y. Zhong M. Li W. Terentyev D. Choi B.-R. Karma A. et al.LITAF (lipopolysaccharide-induced tumor necrosis factor) regulates cardiac L-type calcium channels by modulating NEDD (neural precursor cell expressed developmentally downregulated protein) 4-1 ubiquitin ligase.Circ. Genom. Precis. Med. 2019; 12 (31462068): 407-42010.1161/CIRCGEN.119.002641Crossref PubMed Scopus (3) Google Scholar), we entertained the possibility LITAF may also regulate NEDD4-2 with respect to Nav1.5. Therefore, we first looked for any physical interaction between NEDD4-2 and LITAF, which could be mediated by four WW domains of NEDD4-2 and two PXY motifs of LITAF (17Shirk A.J. Anderson S.K. Hashemi S.H. Chance P.F. Bennett C.L. SIMPLE interacts with NEDD4 and TSG101: evidence for a role in lysosomal sorting and implications for Charcot–Marie–Tooth disease.J. Neurosci. Res. 2005; 82 (16118794): 43-5010.1002/jnr.20628Crossref PubMed Scopus (51) Google Scholar, 31Boase N.A. Kumar S. NEDD4: The founding member of a family of ubiquitin-protein ligases.Gene. 2015; 557 (25527121): 113-12210.1016/j.gene.2014.12.020Crossref PubMed Scopus (71) Google Scholar). Total lysates of stable HEK cells transiently expressing HA-tagged LITAF and FLAG-tagged NEDD4-2 or FLAG-tagged NEDD4-2 were immunoprecipitated with FLAG antibody. The resulting immunoprecipitates were subjected to Western blotting. Fig. 4A shows co-precipitated LITAF indicating that LITAF and NEDD4-2 are found in the same protein complex. We also noticed that LITAF significantly reduced levels of co-expressed NEDD4-2 by 39% (Fig. 4A). Next, we wanted to assess the possible role of LITAF and NEDD4-2 in the homeostasis of Nav1.5. To this end, we transiently transfected HEK cells stably expressing Nav1.5 and GFP. Not surprisingly, co-expressed NEDD4-2 significantly decreased peak INa density (e.g. by 68%, viz. from −71.1 ± 12.7 pA/pF to −22.5 ± 3.8 pA/pF; −10 mV; p < 0.01; Fig. 4B), which is in agreement with a previous study by van Bemmelen et al. (5van Bemmelen M.X. Rougier J.S. Gavillet B. Apothéloz F. Daidié D. Tateyama M. Rivolta I. Thomas M.A. Kass R.S. Staub O. Abriel H. Cardiac voltage-gated sodium channel Nav1.5 is regulated by Nedd4-2 mediated ubiquitination.Circ. Res. 2004; 95 (15217910): 284-29110.1161/01.RES.0000136816.05109.89Crossref PubMed Scopus (164) Google Scholar). Co-expressed LITAF, however, partially reversed the negative effect of NEDD4-2 on INa density (from −22.5 ± 3.8 pA/pF to −37.6 ± 13.0 pA/pF; −10mV; p < 0.01; Fig. 4B). Thus, this 52% recovery of INa in the presence of co-expressed LITAF is in line with the aforementioned 39% LITAF-dependent drop in NEDD4-2 levels. In summary, these functional data corroborate a role for LITAF modulating membrane expression of Nav1.5 by regulating NEDD4-2 ubiquitination-mediated degradation. Because the data presented in Fig. 4 suggest a functional interaction between LITAF and NEDD4-2 regulating Nav1.5 expression on the membrane, we wanted to investigate the possible role of LITAF in the regulation of NEDD4-2. To this end, we measured endogenous NEDD4-2 levels in 3-week-old and neonatal rabbit cardiomyocytes (NRbCM) overexpressing LITAF. We noted that LITAF overexpression reduced total levels of NEDD4-2 by ∼30% (3wRbCM) and 50% (NRbCM), respectively (Fig. 5, A, B, and E). Similarly, LITAF overexpression down-regulated endogenous NEDD4-2 levels by ∼60% in HEK cells (Fig. 5, C and E). Lastly, co-expression of LITAF and FLAG-tagged NEDD4-2 in HEK cells resulted in an ∼90% down-regulation of NEDD4-2–FLAG (Fig. 5, D and E). We reasoned that LITAF overexpression likely caused ubiquitin-mediated degradation of NEDD4-2 in the various cell types tested. To test this hypothesis, we co-transfected expression plasmids for HA-tagged ubiquitin, FLAG-tagged NEDD4-2, FLAG-tagged LITAF, or control plasmid into HEK cells
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