IRIS Institutional Research Information System - AIR Archivio Istituzionale della Ricerca

IRIS è il sistema di gestione integrata dei dati della ricerca (persone, progetti, pubblicazioni, attività) adottato dall'Università degli Studi di Milano.

AIR nasce in UNIMI nel 2006 con lo scopo di raccogliere, documentare e conservare le informazioni sulla produzione scientifica dell'Università degli Studi di Milano e costituisce di fatto l'Anagrafe della ricerca dell'Ateneo.

Introduction: Notch and AKT/PI3K signaling are two key oncogenic pathways closely associated in T-cell acute lymphoblastic leukemia (T-ALL). These pathways collaborate in controlling proliferation, survival and migration of T-ALL cells and are deregulated in 60% (Notch pathway) and 48%(AKT/PI3K) of T-ALL cases. Recent evidences indicate the reciprocal regulation between the constitutively active Notch and AKT/PI3K pathways in T-ALL cells. Here we identified a novel way by which AKT regulates Notch activity and investigated the underlying mechanism. Methods: Molt4 T-ALL cell line and HEK293T cell line were grown in RPMI-1640 and DMEM respectively, supplemented with 10% heat-inactivated FBS. 1 g RNA isolated from cells was retro-transcribed in 20 l by M-MuLV reverse transcriptase using random hexamer primers. RT-PCR analysis was performed using primers for Notch1, HES1, preTCR, GAPDH. Apoptotic cells were identified by Annexin-V and propidium iodide staining. Protein expression was detected by Western blot analysis of whole cell lysates. Immunoprecipitation (IP) of ubiquitin-conjugated proteins was performed using the UbiQapture-Q Kit (Biomol, Exeter, UK), as described by the manufacturer. Co-immunoprecipitation (Co-IP) analysis was performed using Protein G Agarose beads, eluted immunoprecipitates were analyzed by Western blot. Immunofluorescent staining was done on HEK293T cells that were incubated with anti-Flag or anti-c-Cbl primary antibodies and then with the appropriate AlexaFluor-conjugated secondary antibodies. Images were acquired with a Leica TCS SP2 confocal microscope. A co-localization area was determined based on a 2D cytofluorogram and density analysis was performed by Multicolor Analysis Leica Confocal software. Results: The influence of AKT signaling on Notch1 levels was investigated by an inhibitory approach using the PI3K inhibitor LY294002. LY294002-mediated withdrawal of AKT reduced Notch1 protein levels and activity, without affecting Notch1 transcript. We showed that Notch1 protein loss was due to lysosomal degradation of the Notch1 membrane-bound form. IP and Co-IP analyses revealed that AKT withdrawal resulted in an increased tyrosine phosphorylation of Notch1 followed by binding to an E3 ubiquitin ligase, c-Cbl that mono-ubiquitinated Notch1 resulting in lysosomal degradation. Conclusions: For the first time we identified and elucidated a mechanism used by the AKT pathway to control Notch1 levels through the regulation of its lysosomal degradation. Thus, in a deregulated environment such as T-ALL cells, hyperactive AKT signaling may contribute to Notch1 increase reducing its lysosomal degradation. These findings contribute to a better understanding of the cooperative roles of Notch and AKT pathways in cancer.

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