Akt Regulates TPP1Oligomerization And Telomere Protection

Telomeres are specialized structures at the ends of eukaryotic chromosomesthat are important for maintaining genome stability and integrity. Telomeredysfunction has been linked to aging and cancer development. In mammalian cells,extensive studies have been carried out to illustrate how core telomeric proteinsassemble on telomeres to recruit the telomerase and additional factors for telomeremaintenance and protection. However, how changes in growth signaling pathwaysimpact telomeres and telomere-binding proteins remains largely unexplored. Thephosphatidylinositol3-kinase (PI3-K)/Akt (also known as PKB) pathway, one ofthe best characterized growth signaling cascades, regulates a variety of cellularfunction including cell proliferation, survival, metabolism, and DNA repair, anddysregulation of PI3-K/Akt signaling has been linked to aging and diseases such ascancer and diabetes. In this study, we provide evidence that the Akt signalingpathway plays an important role in telomere protection. Akt inhibition either bychemical inhibitors or small interfering RNAs induced telomere dysfunction.Furthermore, we found that TPP1could oligomerize through its OB fold, a processthat was dependent on the Akt kinase. Telomere damage and reduced TPP1 dimerization as a result of Akt inhibition was also accompanied by diminishedrecruitment of TPP1and POT1to the telomeres. Our findings highlight apreviously unknown link between Akt signaling and telomere protection.Main research results are as follows:(1) Akt activity is important for telomere end protection. When humanHTC75cells were grown under starvation conditions (0.01%serum), we noticedincreased53BP1foci at telomeres that accompanied growth inhibition in thesecells. These observations suggest that telomeres may be sensitive to changes innutrients, and that cell growth signaling may play a positive role in telomeremaintenance. We treated cells with triciribine, an Akt specific inhibitor, andanalyzed telomere dysfunction induced foci (TIF) in these cells. Akt activityrecovered following removal of triciribine which coincided with the decrease inTIF formation in these cells, indicating that Akt activity indeed is important fortelomere end protection.(2) Akt activity is important for TPP1and POT1recruitment to the telomeres.We did not observe any drastic changes after triciribine treatment in the levels ofcore telomeric proteins that we examined. We performed chromatinimmunoprecipitation (ChIP) assays to examine association of core telomereproteins with telomeres in cells treated with triciribine. Of the six core telomericproteins, TPP1and POT1were the only ones that consistently exhibiteddiminished telomere binding with drug treatment.(3) The Akt1isoform is the predominant player in telomere protection.Depletion of Akt1by all three siRNAs elevated TIF formation. In comparison,knocking down Akt2and Akt3isoforms had no apparent effect on TIF formation inthese cells, suggesting that Akt1is the key Akt isoform that mediates telomere endprotection. Expression of RNAi-resistant wild-type Akt1, but not kinase-deadAkt1(Akt1K179M), could rescue the TIF phenotype of the Akt1knockdown cells,further supporting the notion that Akt1is critical for telomere protection andunderscoring the importance of its kinase activity in this process.(4) The TPP1OB fold can mediate TPP1oligomerization. Both Bi-molecular Fluorescence Complementation (BiFC/PCA) assay and Co-IP can prove that TPP1can form oligomer in vivo. We carried out in vitro GST pull-down assays usingbacterially expressed GST or MBP-tagged TPP1OB fold proteins and found thatTPP1could form dimer through OB fold. Expression of wild-type TPP1inAkt1-depleted cells led to a reduction of TIFs to levels comparable to control cells;supporting the idea that Akt1can function through TPP1to regulate telomeres. Inaddition, deletion of the OB fold abolished the rescue ability of TPP1, againconfirming the importance of OB fold and suggesting that OB-fold dependentdimerization of TPP1might be important for telomere protection.(5) TPP1oligomerization depends on Akt1activity. Both treating cells withtriciribine, an Akt specific inhibitor, and knocking down Akt1using siRNAs couldabolish TPP1oligomerization. In control knockdown cells, ectopic expression ofAkt1enhanced TPP1dimer formation as well. These data support the notion thatAkt1may participate in telomere protection through regulating TPP1oligomerization. Importantly, an active Akt1kinase is required in this process,because the kinase-dead mutant Akt1K179M failed to rescue TPP1-TPP1interaction in the BiFC assay. Collectly theses results indicate that Akt1activity isimportant in regulating TPP1oligomerization.