Analysis of ALK2 interactions in U2O Scells

  • To characterize interactions involving ALK2 in U2OS cells, transient transfection was used to express wild type or R206H variants of ALK2
  • Transiently expressing U2OS were either used unstimulated, or were stimulated with BMP4
  • Datasets from unstimulated and BMP4 stimulated cells were compared to identify changes in the protein interaction network after activation of the BMP pathway

Pilot purification experiments using U2OS cells

  • Cells were transiently transfected with ALK2 wild type expression constructs
  • Transiently expressing cells were either used as non-stimulated control or stimulated with BMP4
  • Small scale pilot purifications were carried out according to the optimized protocol developed with Hek293 cells


fop-41

  • Cells were transiently transfected with ALK2 R206H expression constructs
  • Transiently expressing cells were either used as non-stimulated control or stimulated with BMP4
  • Small scale pilot purifications were carried out according to the optimized protocol developed with Hek293 cells

 

fop-42

Large scale purification of ALK2 from unstimulated U2OS cells

  • Volcano-Plot of final merged datasets for ALK2 R206H and control
  • Significantly enriched complex partners for ALK2 R206H are boxed

fop-43

  • Primary dataset of large scale purifications for ALK2 wild type
  • Triplicate purifications are shown for ALK2 wild type and control
  • Color coding shows variations in relative abundance of complex partners identified

fop-44

  • Volcano-Plot of final merged datasets for ALK2 wild type and control
  • Significantly enriched complex partners for ALK2 wild type are boxed

fop-45

  • Scatter plots of triplicate purifications are shown
  • Control purifications and ALK2 R206H purifications are shown to demonstrate reproducibility

fop-49

  • Primary dataset of large scale purifications for ALK2 R206H
  • Triplicate purifications are shown for ALK2 R206H and control
  • Color coding shows variations in relative abundance of complex partners identified

  • Volcano-Plot of final merged datasets for ALK2 R206H and control
  • Significantly enriched complex partners for ALK2 R206H are boxed

fop-48-1

  • Scatter plots of triplicate purifications from BMP4 stimulated cells are shown
  • Control purifications and ALK2 wild type purifications are shown to demonstrate reproducibility

fop-49

  • Primary dataset of large scale purifications for ALK2 wild type from BMP4 stimulated cells
  • Triplicate purifications are shown for ALK2 wild type and control
  • Color coding shows variations in relative abundance of complex partners identified

fop-50

  • Volcano-Plot of final merged datasets for ALK2 wild type and control BMP4 stimulated cells
  • Significantly enriched complex partners for ALK2 wild type are boxed

fop-51

  • Scatter plots of triplicate purifications from BMP4 stimulated cells are shown
  • Control purifications and ALK2 R206H purifications are shown to demonstrate reproducibility

 

fop-52

  • Primary dataset of large scale purifications for ALK2 R206H from BMP4 stimulated cells
  • Triplicate purifications are shown for ALK2 R206H and control
  • Color coding shows variations in relative abundance of complex partners identified

fop-53

  • Volcano-Plot of final merged datasets for ALK2 R206H and control BMP4 stimulated cells
  • Significantly enriched complex partners for ALK2 R206H are boxed

 

fop-54 Results: Purification of ALK2 complexes from U2OS cells

  • For ALK2 wild type purified from unstimulated U2OS cells, 8 interactors were identified
  • Interactors were mainly involved in receptor sorting, recycling and degradation
  • Stimulation of U2OS cells with BMP4 led to the identification of 15 interactors
  • 9 additional interactors were identified upon BMP4 stimulation, one interaction was lost
  • The ALK2 R206H mutant showed a strongly reduced interaction network: only one interactor was identified
  • Stimulation with BMP4 did not change the interaction network of ALK2 R206H

 Comparison of ALK2 wild type and R206H interactors identified

fop-56

Graphical representation of ALK2 interaction networks

  • Merged interaction network of all ALK2 complexes identified (wild type vs R206H; unstimulated vs stimulated)
  • Graphical representation was done using Cytoscape

fop57

Summary: ALK2interaction network in U2OScells

  • The interaction network of ALK2 in U2OS cells shows a large number of proteins involved in receptor sorting and turnover
  • Interactors include chaperones, components of the ubiquitination pathway and components of the ERAD pathway
  • Stimulation with BMP4 leads to additional interaction of ALK2 with several lysosomal proteins and E3 ubiquitin ligases
  • In contrast, the mutant ALK2 R206H does not show interactions with components of either the ubiquitination or ERAD pathways
  • The only identified interaction involving ALK2 R206H is with the calcium binding protein calnexin (CANX)