title: Dissection of the BCR-ABL signaling network using highly specific monobody inhibitors to the SHP2 SH2 domains.
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.
authors: Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637 and Swiss Institute for Experimental Cancer Research, School of Life Sciences, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.
authors: Fern
authors: F
authors: Sha
authors: Emel Basak
authors: EB
authors: Gencer
authors: Sandrine
authors: S
authors: Georgeon
authors: Akiko
authors: A
authors: Koide
authors: Norihisa
authors: N
authors: Yasui
authors: Shohei
authors: S
authors: Koide
authors: Oliver
authors: O
authors: Hantschel
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000595:Amino Acid Sequence; D002471:Cell Transformation, Neoplastic; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D016044:Fusion Proteins, bcr-abl / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D020014:K562 Cells; D008958:Models, Molecular; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11 / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D015398:Signal Transduction; D018909:src Homology Domains
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D004791:Enzyme Inhibitors; C490895:GAB2 protein, human; D019151:Peptide Library; D010455:Peptides; D016044:Fusion Proteins, bcr-abl; C516724:PTPN11 protein, human; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11
keywords: engineered proteins; interaction proteomics; protein interaction inhibitor; tyrosine phosphatase
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