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FEBS J
2013 Nov 01;28021:5269-82. doi: 10.1111/febs.12453.
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Phosphoproteomic analysis of anaplastic lymphoma kinase (ALK) downstream signaling pathways identifies signal transducer and activator of transcription 3 as a functional target of activated ALK in neuroblastoma cells.
Sattu K
,
Hochgräfe F
,
Wu J
,
Umapathy G
,
Schönherr C
,
Ruuth K
,
Chand D
,
Witek B
,
Fuchs J
,
Li PK
,
Hugosson F
,
Daly RJ
,
Palmer RH
,
Hallberg B
.
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Activation of the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase is a key oncogenic mechanism in a growing number of tumor types. In the majority of cases, ALK is activated by fusion with a dimerizing partner protein as a result of chromosomal translocation events, most studied in the case of the nucleophosmin-ALK and echinoderm microtubule-associated protein-like 4-ALK oncoproteins. It is now also appreciated that the full-length ALK receptor can be activated by point mutations and by deletions within the extracellular domain, such as those observed in neuroblastoma. Several studies have employed phosphoproteomics approaches to find substrates of ALK fusion proteins. In this study, we used MS-based phosphotyrosine profiling to characterize phosphotyrosine signaling events associated with the full-length ALK receptor. A number of previously identified and novel targets were identified. One of these, signal transducer and activator of transcription 3 (STAT3), has previously been observed to be activated in response to oncogenic ALK signaling, but the significance of this in signaling from the full-length ALK receptor has not been explored further. We show here that activated ALK robustly activates STAT3 on Tyr705 in a number of independent neuroblastoma cell lines. Furthermore, knockdown of STAT3 by RNA interference resulted in a reduction in myelocytomatosis neuroblastom (MYCN) protein levels downstream of ALK signaling. These observations, together with a decreased level of MYCN and inhibition of neuroblastoma cell growth in the presence of STAT3 inhibitors, suggest that activation of STAT3 is important for ALK signaling activity in neuroblastoma.
Figure 1. (A) Tyrosine residues phosphorylated in the kinase domain of ALK. The intracellular domain of ALK containing the protein kinase domain (PKD) (red) and potential autophosphorylation sites were searched with phosphomotif (http://www.hprd.org/PhosphoMotif_finder) as indicated. Presented and compared side-by-side with our phosphotyrosine mapping of activated full-length ALK are the global surveys of phosphotyrosine peptides identified in EML4–ALK and NPM–ALK 29,30. The critical tyrosines in the activation loop of the kinase domain of ALK are boxed 45. (B) Protein–protein interactions of human orthologs of the phosphoproteins identified in ALK-expressing PC12 cells. In the network, proteins with upregulated phosphorylation sites in activated ALK-expressing PC12 cells as compared with control cells are in red, and proteins with downregulated phosphorylation sites are in green. Blue edges indicate protein–protein interactions, and orange edges indicate kinase–substrate relationships. Only the network including ALK is shown. The pale blue balls indicate (human orthologs of) PC12 proteins with identified/mapped phosphotyrosines that were not found to be significantly regulated.
Figure 2. STAT3 phosphorylation and interaction with ALK on ALK activation. (A) Tet-on-inducible PC12 cell clones expressing either wild-type ALK or the ALKF1174S mutant receptor were employed. Protein expression was induced with 1 μg·mL−1 doxycycline, and cells were serum-starved for 24 h prior to stimulation with 1 μg·mL−1 ALK-activating mAb (mAb31) for 30 min or 24 h. Whole cell lysates were analyzed by SDS/PAGE, and this was followed by immunoblotting with antibodies against p-ALKY1278, ALK, p-STAT3Y705, and p-ERK. Pan-ERK and pan-STAT3 antibodies were employed as loading controls. (B, C) PC12 cells were transfected with either wild-type ALK or the ALKF1174S mutant together with FLAG-tagged STAT3 prior to stimulation with 1 μg·mL−1 ALK-activating mAb (mAb46) for 24 h, in the presence or absence of 250 nm crizotinib, as indicated. Lysates were immunoprecipitated (IP) with either antibody against FLAG (M2) (B) or with antibody against ALK (mAb31) (C), and this was followed by immunoblotting for ALK, FLAG, and STAT3, as indicated. WCL, whole cell lysate.
Figure 3. Loss of STAT3 results in reduced MYCN levels. Two independent STAT3 siRNAs (#1 or #2) were employed to downregulate STAT3 levels in CLB-GE (A), CLB-BAR (B), Kelly (C) and CLB-GA (D) neuroblastoma cell lines. Cells were transfected with either control scrambled siRNA, STAT3 siRNA#1 or STAT3 siRNA#2 prior to cell lysis 48 h post-transfection. Whole cell lysates were subsequently immunoblotted for STAT3, MYCN, and pan-ERK (as loading control), as indicated. Lfm, lipofectamine; scC, scramble control.
Figure 4. STAT3 activity is required for regulation of MYCN expression by ALK. Neuroblastoma cell lines CLB-GE (A), CLB-BAR (B), Kelly (C) and CLB-GA (D) were starved and treated with either 250 nm crizotinib (24 h), 5 μm FLLL32 (8 h), 5 μm STATTIC (8 h), or control, as indicated. After cell lysis, samples were immunoblotted with antibodies against p-ALKY1278, MYCN, p-STAT3Y705, and p-ERK. Pan-ERK, ALK and STAT3 antibodies were employed as loading controls. Three independent experiments with similar results were performed, and representative blots are shown.
Figure 5. Inhibition of STAT3 reduces MYCN transcription. (A) Luciferase assay of neuroblastoma cell lines transfected with MYCNP–luciferase or empty pGL2 vector as a control (ctrl). The neuroblastoma cell lines CLB-GE and CLB-BAR were transfected with empty pGL2 (ctrl) or MYCNP–luciferase. Cells were then serum-starved, and STAT3 was inhibited with 2.5 μm FLLL32 or STATTIC for 12 h. White bars: untreated neuroblastoma cells. Gray bars: cells treated with FLLL32. Black bars: cells treated with STATTIC. Results are presented as relative luciferase activity, where untreated samples transfected with empty pGL2 vector were set to 1. (B) qRT-PCR of MYCN mRNA in neuroblastoma cell lines. The neuroblastoma cell lines CLB-GE and CLB-BAR were starved and treated with 2.5 μm FLLL32 or STATTIC for 12 h. Primers amplifying part of the coding sequence of RPL19 (B) or RPL29 (data not shown) were used to control for differences in cDNA input. Relative expression was calculated according to the ΔΔCt relative quantification method. Each sample within an experiment was analyzed in duplicate, and the experiment was carried out at least three times. White bars: untreated cells. Gray bars: cells treated with FLLL32. Black bars: cells treated with STATTIC.
Figure 6. Loss of STAT3 function decreases neuroblastoma cell proliferation. (A, B) Neuroblastoma cell lines CLB-GE (▪), CLB-BAR (▴), Kelly (‐) and CLB-GA (•) were treated with 250 nm crizotinib (A) and 1.5 μm FLLL32 (B) for 5 days. Proliferation was analyzed with the resazurin cell proliferation assay. Values are reported as fold relative fluorescence from FLLL32-treated cells versus relative fluorescence from untreated cells (♦). Results are from three representative experiments, with each experiment performed in triplicate. (C) Neuroblastoma cell lines CLB-BAR, CLB-GE, CLB-GA and Kelly were grown on six-well plates with complete growth medium, starved, and treated with 250 nm crizotinib and 1.5 μm FLLL32 for 6 h. Cell lysates were immunoblotted with antibodies against p-STAT3 and PARP. Tubulin was used as a loading control. (D–G) CLB-BAR (D), CLB-GA (E) CLB-GE (F) and Kelly (G) cell lines were transfected with scrambled siRNA (SiC) (▪), STAT3 siRNA#1 (Si1) (▴) or STAT3 siRNA#2 (Si2) (▴) at 0 and 24 h. Cell viability was assessed at 0, 3, 4 and 5 days post-transfection, with the resazurin assay. Values are reported as fold relative fluorescence from siRNA-transfected cells versus relative fluorescence from control mock-transfected cells. Results are from one of three representative experiments, with each experiment performed in triplicate.
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