Volume 36, Issue 2, Pages 326-339 (October 2009) Mislocalized Activation of Oncogenic RTKs Switches Downstream Signaling Outcomes  Chunaram Choudhary, Jesper V. Olsen, Christian Brandts, Jürgen Cox, Pavankumar N.G. Reddy, Frank D. Böhmer, Volker Gerke, Dirk- E. Schmidt-Arras, Wolfgang E. Berdel, Carsten Müller-Tidow, Matthias Mann, Hubert Serve  Molecular Cell  Volume 36, Issue 2, Pages 326-339 (October 2009) DOI: 10.1016/j.molcel.2009.09.019 Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 1 Tunicamycin or BFA Inhibit Flt3 Surface Expression and Flt3-ITD-Induced MAPK and PI3K but Not STAT5a/b Activation (A) Tunicamycin and BFA inhibit MAPK and PI3K but augment STAT5 activation. Activation of signaling was analyzed in 32D cells overexpressing Flt3-ITD using phospho-specific antibodies against Flt3, STAT5a/b, Erk1/2, and Akt. STAT5 activity was also analyzed by determining the protein expression of the known STAT target genes Pim-1 and Pim-2. PKC412 is an inhibitor of the Flt3 kinase. The results are representative of four independent experiments. The differentially glycosylated forms of Flt3 receptor are indicated by the arrow signs: the upper arrow shows the mature form and the lower arrow indicates immature form of the receptor. p = phospho, t = total. (B) Flt3 surface expression is inhibited by tunicamycin or BFA. The surface expression of Flt3-ITD was analyzed using the indicated antibodies by flow cytometry. (C) Tunicamycin or BFA induces ER retention of Flt3-ITD. To demonstrate the colocalization of Flt3-ITD and PDI, HeLa cells transfected with Flt3-ITD were immunostained with the indicated antibodies and imaged using immunofluorescence microscopy. Flt3 staining indicates expression of total Flt3-ITD receptor. (D) STAT5 is not phosphorylated upon tunicamycin and BFA treatment in Flt3-WT-expressing cells. The 32D-Flt3-WT cells were deprived of growth factors and cultured in the absence or presence of tunicamycin or BFA for 8 hr. Cells were stimulated with Flt3 ligand for 10 min to activate Flt3-WT receptors. Lysates from Flt3-ITD cells were used as positive control to show phosphorylation of Flt3 and STAT5. Molecular Cell 2009 36, 326-339DOI: (10.1016/j.molcel.2009.09.019) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 2 Flt3-ITD-ΔECD and Flt3-ITD-myr Show Differential Sensitivity to Tunicamycin or BFA (A) Surface expression of Flt3-ITD-ΔECD is inhibited by BFA but not by tunicamycin. Flow cytometric analyses of 32D-Flt3-ITD-ΔECD cells showed opposite effects of tunicamycin and BFA on the surface expression of Flt3-ITD-ΔECD. (B) Flt3-ITD-ΔECD strongly activates MAPK and Akt in the presence of tunicamycin. Signaling analyses of 32D-Flt3-ITD-ΔECD showed an increased activation of Akt and Erk1/2 in the presence of tunicamycin. In contrast, treatment with BFA diminished Erk1/2 and Akt activation and increased STAT5a/b phosphorylation. (C) Flt3-ITD-myr is compromised in STAT5a/b activation. Comparative signaling analyses of Flt3-ITD versus Flt3-ITD-myr in 32D cells revealed diminished STAT5a/b but strong Akt and Erk1/2 activation by Flt3-ITD-myr in a tunicamycin- and BFA-resistant manner. The data are representative of three independent experiments. (D) Intracellularly activated Flt3-ITD provides antiapoptotic signals. The 32D cells expressing the indicated Flt3 receptors were cultured in the presence of the indicated growth factors and/or inhibitors, and apoptotic cells were measured by flow cytometry. Molecular Cell 2009 36, 326-339DOI: (10.1016/j.molcel.2009.09.019) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 3 Compartment-Dependent Activation of Signaling by c-Kit-ITD and Endogenous Flt3-ITD (A and B) BFA inhibits the surface expression of Flt3-ITD and c-Kit-ITD on MV4-11 and 32D cells, respectively. Surface expression of Flt3 and c-Kit was analyzed by flow cytometry. (C) Spatial regulation of Flt3-ITD signaling in MV4-11 cells. Activation of signaling was analyzed by employing phospho-specific antibodies for Flt3, STAT5a/b, and Erk1/2. (D) Location-dependent activation of signaling by c-Kit-ITD. Activation of signaling was analyzed in 32D cells expressing c-Kit-ITD using the indicated phospho-specific antibodies. Molecular Cell 2009 36, 326-339DOI: (10.1016/j.molcel.2009.09.019) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figures 4 SILAC-Based Quantitative Phosphoproteomics of Flt3-ITD (A) The 32D vector control or 32D-Flt3-ITD cells were triple SILAC labeled and treated overnight as indicated. Phosphopeptides were enriched and analyzed by MS. (B) Typical examples of peptides demonstrating compartment-dependent phosphorylation. The upper panels show full-scan mass spectra of phosphopeptides displaying SILAC triplets that are not regulated by Flt3-ITD (LQPEQGpSPKK) or phosphorylated by cell-surface-bound Flt3-ITD (KLPpSTTL) or by ER-retained Flt3-ITD (HLpSHPEPEQQHVIQR). The lower panels depict fragmentation spectra (MS/MS) of the corresponding peptides from upper panels. The sequence-specific N-terminal and C-terminal ions are indicated as b and y ions. ∗ indicates loss (−98 Da) of phosphoric acid. Molecular Cell 2009 36, 326-339DOI: (10.1016/j.molcel.2009.09.019) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 5 Compartment-Specific Phosphorylation of Flt3-ITD Tyrosines and Spatial Activation of Downstream Signaling (A and B) Compartment-dependent phosphorylation of Flt3-ITD activates MAPK and PI3K pathways from the cell surface. Phosphorylation of MAPK and PI3K cascade components was measured by SILAC-based quantitative phosphoproteomics. The ratios represent the fold change of phosphorylation in 32D-Flt3-ITD cells in the presence of BFA compared to 32D vector control cells or 32D-Flt3-ITD treated with PKC412. (C) Activation of STAT5 pathway by ER-retained Flt3-ITD. Activation of STAT5 pathway components was quantified as described above. (D) Spatial regulation of Flt3-ITD tyrosine phosphorylation. Tyrosines Y591 and Y842 show differential phosphorylation on the surface-bound versus ER-retained Flt3-ITD, and they are also differentially phosphorylated by ligand-activated Flt3-WT. (E) Schematic presentation of Flt3-ITD signaling compartmentalization. Activation of Flt3-ITD on the cell surface activates MAPK and PI3K pathways. In contrast, mislocalized activation of Flt3-ITD on the ER aberrantly activates the STAT5 pathway and upregulates Pim-1/2, which in turn phosphorylates further-downstream targets such as eIF4B and BAD. Molecular Cell 2009 36, 326-339DOI: (10.1016/j.molcel.2009.09.019) Copyright © 2009 Elsevier Inc. Terms and Conditions