Dissection of the BCR-ABL signaling network using highly specific monobody inhibitors to the SHP2 SH2 domains.
Publication Type:Journal Article
Source:Proc Natl Acad Sci U S A, Volume 110, Issue 37, p.14924-9 (2013)
Keywords:Adaptor Proteins, Signal Transducing, Amino Acid Sequence, Cell Transformation, Neoplastic, Crystallography, X-Ray, Enzyme Inhibitors, Fusion Proteins, bcr-abl, HEK293 Cells, Humans, K562 Cells, Models, Molecular, Peptide Library, Peptides, Protein Binding, Protein Interaction Domains and Motifs, Protein Tyrosine Phosphatase, Non-Receptor Type 11, Signal Transduction, src Homology Domains
<p>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. </p>