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BCR-ABL suppresses autophagy via BECLIN-1
    presses autophagy. Moreover, addition of interleukin-3 (IL-3), which rescues cells from nilotinib-induced cell death but does not rescue BCR-ABL inhibition, could not block autophagy (Online Supplementary Figure S3D-F).
Based on our mouse model data, we hypothesized that BECLIN-1 may be an essential player in autophagy sup- pression by BCR-ABL. BECLIN-1 has a crucial role in autophagosome formation as being part of the UVRAG- VPS15-ATG14-VPS34-RUBICON-BECLIN-1 complex. Interestingly, we could show that the formation of the UVRAG-VPS15-ATG14-VPS34-RUBICON-BECLIN-1 complex was altered in a BCR-ABL positive human cell line (K562) after nilotinib treatment (Online Supplementary Figure S3G): recruitment of positive regulators of autophagosome formation (VPS15, VPS34, UVRAG and ATG14) to BECLIN-1 was increased upon BCR-ABL inhi- bition, whereas the recruitment of the negative regulator RUBICON to BECLIN-1 was impaired after nilotinib treatment. These results indicate that BCR-ABL kinase activity modulates the BECLIN-1 complex composition and thereby leads to autophagy suppression.
BCR-ABL interacts with BECLIN-1
Next, we aimed to investigate how BCR-ABL kinase activity modulates BECLIN-1 complex composition. We found that BCR-ABL strongly binds to BECLIN-1, inde- pendent of ABL kinase activity.
Furthermore, BECLIN-1 was tyrosine phosphorylated in a complex with kinase active BCR-ABL indicating that BCR-ABL may directly phosphorylate BECLIN-1 (Figure 2A). Immunoprecipitation of endogenous BCR-ABL in K562 cells confirmed the BCR-ABL/BECLIN-1 interaction (Figure 2B-C) and GST-pulldown-assays using purified BECLIN-1 suggest that BCR-ABL and BECLIN-1 may bind directly to each other (Figure 2D).
We could also detect BCR-ABL/BECLIN-1 co-localiza- tion using immunofluorescence staining (Online Supplementary Figure S4A). To investigate which region of BCR-ABL binds to BECLIN-1, we performed binding assays by overexpressing either BCR or ABL together with Beclin-1 in 293T cells. Immunoprecipitations revealed that BCR interacts with BECLIN-1 but not ABL (Figure 2E).
BCR-ABL directly phosphorylates BECLIN-1 at specific tyrosine residues Y233 and Y352
We next investigated, whether BECLIN-1 is exclusively phosphorylated by BCR-ABL. Interestingly, all other test- ed oncogenic kinases (FLT3-ITD, NPM-ALK and PDGFRA-D842V) failed to induce BECLIN-1 phosphory- lation, implying that BECLIN-1 is a specific substrate of BCR-ABL and not a general target of oncogenic tyrosine kinase signaling (Figure 2F). Moreover, we were able to confirm BECLIN-1 tyrosine phosphorylation in all tested samples of primary CML patient material, whereas BECLIN-1 phosphorylation was absent in healthy control samples (Online Supplementary Figure S4B).
To test whether BECLIN-1 is a direct target of BCR-ABL we performed an in vitro kinase assay, and identified spe- cific phosphorylation in two distinct regions of BECLIN-1: One spanning amino acid (aa) region 141 - 277 and anoth- er aa region 338 - 450 (Figure 3A). Furthermore, we gener- ated a series of tyrosine residue mutants to determine spe- cific BECLIN-1 tyrosine residues phosphorylated by BCR- ABL. Strong phosphorylation by BCR-ABL could be detected on BECLIN-1 tyrosine residues Y233 and Y352,
whereas Y162 and Y338 show minor phosphorylation (Figure 3B). Western blot analyses of single and double phosphorylation-deficient mutants of those distinct BECLIN-1 tyrosine residues validated our results (Figure 3C) and demonstrated that BCR-ABL phosphorylates BECLIN-1 specifically at tyrosine residues Y233 and Y352. Interestingly, tyrosine Y352 (AA352-355 YCSG) is part of a STAT5 Src Homology 2 (SH2) domain binding motif (Y[VLTFIC]xx).37
Phospho-mimic mutant Beclin-1 Y233E/Y352E suppresses autophagy through BECLIN-1 complex alterations whereas the phospho-deficient Beclin-1 Y233F/Y352F mutant induces autophagy
To evaluate whether phosphorylation of BECLIN-1 reg- ulates autophagy, we generated a BECLIN-1 phosphoryla- tion-mimic (Y233E/Y352E) and a phosphorylation-defi- cient mutant (Y233F/Y352F). In an LC3 puncta assay in K562 cells, we found that the phosphorylation-mimic BECLIN-1 mutant suppresses autophagy, whereas the phosphorylation-deficient BECLIN-1 mutant induces increased autophagy (Figure 4A-B). By immunoblotting, we could confirm that the phosphorylation-mimic mutant Y233E/Y352E decreases autophagy, whereas expression of the phosphorylation-deficient mutant Y233F/Y352F induces autophagy (Figure 4C). Our findings therefore suggest that phosphorylation of BECLIN-1 by BCR-ABL suppresses autophagy induction.
Next we sought to know, whether the impaired autophagy induction of the phospho-mimic mutant Y233E/Y352E may be due to an altered recruitment of complex components to BECLIN-1. It has been shown recently, that lack of BECLIN-1 leads to downregulation of the BECLIN-1 complex binding partners.38 Indeed, Beclin-1 deficient, BCR-ABL expressing MEF showed downregula- tion of BECLIN-1 binding partners, which could be res- cued by re-expression of either wt Beclin-1 or both phos- pho-mutants ((BEC FF and EE). Furthermore, expression of the Beclin-1 Y233E/Y352E mutant leads to decreased UVRAG and ATG14 levels, whereas Rubicon levels were increased compared to phosphorylation-deficient BECLIN-1 cells (Figure 4D).
From our results we hypothesized that the phosphory- lation status of BECLIN-1 is important for the stabilization and recruitment of the different binding partners to the BECLIN-1 complex. Interestingly, co-immunoprecipita- tion of BECLIN-1 complex components revealed that autophagy activating proteins like VPS15, VPS34 and ATG14 were recruited less to the phospho-mimic BECLIN-1 (BEC EE) complex compared to the phospho- deficient BECLIN-1 (BEC FF) complex (Figure 4E, Online Supplementary Figure S4C). These results indicate that the altered autophagy by the two mutants is due to altered binding capacities of positive regulators to the BECLIN-1 core complex and thereby alters BECLIN-1 complex activ- ity.
Next we asked, whether expression of the phospho- mimic Beclin-1 mutant could overcome the TKI-induced BCR-ABL inhibition-mediated autophagy induction and indeed, expression of Beclin-1 EE Y233E/Y352E impaired nilotinib-induced autophagy measured by LC3-II expres- sion (Figure 4F) and LC3 puncta accumulation (Figure 4G- H). BCR-ABL inhibition by nilotinib was not able to enhance the autophagy-stimulatory effect of the phospho- rylation-deficient BECLIN-1 Y233F/Y352F mutant com-
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