Abstract
T-cadherin (T-Cad), a unique member of the cadherin family of proteins, plays an important role in cell adhesion and cell signaling. Recently, we demonstrated that T-Cad is transcriptionally repressed by DNA methyltransferase 3b during nerve growth factor (NGF)-induced neuronal differentiation of PC12 cells. Here, we show that T-Cad expression is also regulated at the post-translational level by the proteasomal pathway in these cells, which is facilitated upon NGF treatment. Pulse-chase experiments demonstrated that NGF treatment significantly reduced the half-life of T-Cad. Degradation of T-Cad was blocked upon treatment of PC12 cells with the proteasomal inhibitor ZLLL or lactacystin. Ectopic expression of Cdh1 (CDC20 homolog 1), one of the substrate recognition components of anaphase promoting complex (E3 ligase), stimulated T-Cad degradation. Deletion of CD1, one of the five extracellular cadherin domains (CD), promoted degradation of T-Cad, especially in the presence of NGF. On the contrary, deletion of CD2 stabilized this protein maximally. Ubiquitination of different deletion mutants indicates that T-Cad harbors multiple ubiquitination signals. Furthermore, genistein, a protein-tyrosine kinase inhibitor, impeded T-Cad degradation in PC12 cells, implicating requirement of tyrosine phosphorylation in this process. Mutation at tyrosine 327 (Y327F) markedly increased the half-life of T-Cad, suggesting that phosphorylation of this tyrosine residue located within CD2 is critical for this process. These results show that T-cadherin is subject to dual regulation during NGF-induced differentiation of PC12 cells, namely transcriptional repression mediated by Dnmt3b and post-translational degradation through the proteasomal pathway. These data, together with the inhibitory role of T-Cad in neurite outgrowth of PC12 cells upon NGF treatment, underscore the significance of its stringent regulation during this differentiation process.
Highlights
We show that T-Cad is regulated at the post-translational level by degradation through the ubiquitin-proteasome system, which requires tyrosine phosphorylation and APC/ Ccdh1 (E3) ligase. These results suggest that T-Cad is regulated at both RNA and protein levels, which is probably essential for NGF-induced neurite outgrowth in PC12 cells
The levels of the polyubiquitinated form of different proteins (⌬Cadherin Extracellular Domain 1 (CD1) Ͼ Nsp⌬CD1 Ͼ and Ͼ ⌬CD3–5 Ͼ WT Ͼ ⌬CD 2–5, Fig. 6E, right panel) correlated inversely with their stability (⌬CD1 Ͻ Nsp⌬CD1 Ͻ and Ͻ ⌬CD3–5 Ͻ WT Ͻ ⌬CD2–5, Fig. 6C). These results show that multiple lysines in different cadherin domains of T-Cad can be ubiquitinated in vivo
In an earlier study we demonstrated that NGF-induced differentiation of PC12 cells causes up-regulation of DNA methyltransferase 3b and down-regulation of DNA methyltransferase 1 and 3a [18]
Summary
Inhibitor ZLLL o7r Ala,catancydst7inD. .ETchtoepioc reixgpinreasslidonatoaf aCnddh1oritgoirnoaflalxyosnugbromwtitht,easdevfiidgeunrtefrsomweTr-Ceand-omtediated inhibition (CDC20 homolog a1v),aoinlaebolfethfeosruebsvtaraltuearteicoongn.iTtiohnecaoumtphoo- rsowf neerueritaeboluetgtroowlothcaduterinsgoNmGeF-dinadtuacefdoPrC12 cell differennTe-Cntasdodfeagnraadpahtaisoenr.peDropemleetoaitotinnegoxfcpCoemDrp1i,mleoxnee(nEot3fstlhicgeoafsniev)de, uestxcitmtreaucdlealtaleutdthtneieauttiiromonnes(4bo) oaftnhtdharesedaoursciutgiboisnntraianltuwnmeouarrniktde, weaxshteaincsshoiolunbolef spinal motor recombinant lar cadherin domtahinesy(CsDta),tperosmupotpedordtegtrhaedactioonn colfuTs-ioCnads, ofptrhoteeinpa(5p).er, and offered to prepare especially in the npreewsenfcieguofrNesGFb.aOsnetdheocnontthraarty,ddaelteati.oHn oofweveTr-,CtahdeisJdoifuferrnenatliadlleycelxipnreesdsetdhiantdiofffefreenr,t neuronal popuCenDt2dsetlaebtiiloiznedmtuhtiasanptrdsoetiencidnisicmiaotaenxsimwthaialtlthy.TUw-bChiaqiducihhtiantrahbteoiornsauomftudhlitofifperrlse- dislpaaattigoternresneadn.udTrhiinsegremaguuolttaohtreodarxisnonsatgtaermtoewptothhra(al6lty).aaIntndiyssdpiastciraelltyelryesdtirsitcrtiebdubiquitination siegrnraolsr. sFuinrthtehremocroen, gsetnriustcetinio, anporoftfeiignu-tryeros- inutthede ipnatpheerredgoionnodetvaoildteorf tghroewstchieconntei,fiwchich suggests a sine kinase inhibitorc, iomnpcelduesdiTo-nCsadodfetghraedawtioonrkin, PaCn1d2 ctehlles,y stpaotnedntibalyrotlheeofrTe-pCraoddinutchiebrileigtuylaotifonthoef specific neurite outipmropcleicsast.iMngutraetqiounireamt teynrtosoinf ety3er2xo7spi(neYer3i2pm7hFoe)snpmthaaorlrkydeldaaltytioainnacirnnedatshetidhs e cgwrohoenwrctehlau.rTysl-ihCoyanddsrloeocvaefrlbtisohnaelrseopceaelpepvteoartrel.idgainndcasrrdeiparceassnidtsveaxscpurelasrsitoisnsu(7e)s. These results show that T-cadherin is subject to dual regulation factors can modulate T-Cad expression [9]. During NGF-induced differentiation of PC12 cells, namely tran- T-Cad gene is silenced by promoter hypermethylation in a scriptional repression mediated by Dnmt3b and post-translational variety of cancers [10, 11], and is associated with poor prognosis degradation through the proteasomal pathway. PC12 cells upon NGF treatment, underscore the significance of its Dnmt3b represses T-Cad expression that requires histone stringent regulation during this differentiation process
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