Dissection of the BCR-ABL signaling network using highly specific monobody inhibitors to the SHP2 SH2 domains

Proc Natl Acad Sci U S A. 2013 Sep 10;110(37):14924-9. doi: 10.1073/pnas.1303640110. Epub 2013 Aug 26.

Abstract

The dysregulated tyrosine kinase BCR-ABL causes chronic myelogenous leukemia in humans and forms a large multiprotein complex that includes the Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2). The expression of SHP2 is necessary for BCR-ABL-dependent oncogenic transformation, but the precise signaling mechanisms of SHP2 are not well understood. We have developed binding proteins, termed monobodies, for the N- and C-terminal SH2 domains of SHP2. Intracellular expression followed by interactome analysis showed that the monobodies are essentially monospecific to SHP2. Two crystal structures revealed that the monobodies occupy the phosphopeptide-binding sites of the SH2 domains and thus can serve as competitors of SH2-phosphotyrosine interactions. Surprisingly, the segments of both monobodies that bind to the peptide-binding grooves run in the opposite direction to that of canonical phosphotyrosine peptides, which may contribute to their exquisite specificity. When expressed in cells, monobodies targeting the N-SH2 domain disrupted the interaction of SHP2 with its upstream activator, the Grb2-associated binder 2 adaptor protein, suggesting decoupling of SHP2 from the BCR-ABL protein complex. Inhibition of either N-SH2 or C-SH2 was sufficient to inhibit two tyrosine phosphorylation events that are critical for SHP2 catalytic activity and to block ERK activation. In contrast, targeting the N-SH2 or C-SH2 revealed distinct roles of the two SH2 domains in downstream signaling, such as the phosphorylation of paxillin and signal transducer and activator of transcription 5. Our results delineate a hierarchy of function for the SH2 domains of SHP2 and validate monobodies as potent and specific antagonists of protein-protein interactions in cancer cells.

Keywords: engineered proteins; interaction proteomics; protein interaction inhibitor; tyrosine phosphatase.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Adaptor Proteins, Signal Transducing / metabolism
  • Amino Acid Sequence
  • Cell Transformation, Neoplastic
  • Crystallography, X-Ray
  • Enzyme Inhibitors / chemistry
  • Enzyme Inhibitors / pharmacology
  • Fusion Proteins, bcr-abl / antagonists & inhibitors*
  • Fusion Proteins, bcr-abl / chemistry
  • Fusion Proteins, bcr-abl / genetics
  • HEK293 Cells
  • Humans
  • K562 Cells
  • Models, Molecular
  • Peptide Library
  • Peptides / chemistry
  • Peptides / genetics
  • Peptides / pharmacology
  • Protein Binding
  • Protein Interaction Domains and Motifs
  • Protein Tyrosine Phosphatase, Non-Receptor Type 11 / antagonists & inhibitors*
  • Protein Tyrosine Phosphatase, Non-Receptor Type 11 / chemistry
  • Protein Tyrosine Phosphatase, Non-Receptor Type 11 / genetics
  • Signal Transduction
  • src Homology Domains

Substances

  • Adaptor Proteins, Signal Transducing
  • Enzyme Inhibitors
  • GAB2 protein, human
  • Peptide Library
  • Peptides
  • Fusion Proteins, bcr-abl
  • PTPN11 protein, human
  • Protein Tyrosine Phosphatase, Non-Receptor Type 11

Associated data

  • PDB/4JE4
  • PDB/4JEG