Targeting Oncogenic Src Homology 2 Domain-Containing Phosphatase 2 (SHP2) by Inhibiting its Protein-Protein Interactions

AbstractWe developed a new class of inhibitors of protein-protein interactions of the SHP2 phosphatase, which is pivotal in multiple signaling pathways and a central target in the therapy of cancer and rare diseases. Currently available SHP2 inhibitors target the catalytic site or an allosteric pocket, but lack specificity or are ineffective on disease-associated SHP2 mutants. Based on the consideration that pathogenic lesions cause signaling hyperactivation due to increased SHP2 association with cognate proteins, we developed peptide-based molecules with low nM affinity for the N-terminal Src homology domain of SHP2, good selectivity, stability to degradation and an affinity for pathogenic variants of SHP2 up to 20 times higher than for the wild-type protein. The best peptide reverted the effects of a pathogenic variant (D61G) in zebrafish embryos. Our results provide a novel route for SHP2-targeted therapies and a tool to investigate the role of protein-protein interactions in the function of SHP2.

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Targeting Oncogenic Src Homology 2 Domain-Containing Phosphatase 2 (SHP2) by Inhibiting Its Protein–Protein Interactions

Journal of Medicinal Chemistry ◽

10.1021/acs.jmedchem.1c01371 ◽

2021 ◽

Author(s):

Sara Bobone ◽

Luca Pannone ◽

Barbara Biondi ◽

Maja Solman ◽

Elisabetta Flex ◽

...

Keyword(s):

Protein Interactions ◽

Protein Protein Interactions ◽

Src Homology 2 ◽

Src Homology ◽

Src Homology 2 Domain

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Multiple stimuli induce tyrosine phosphorylation of the Crk-binding sites of paxillin

Biochemical Journal ◽

10.1042/bj3600057 ◽

2001 ◽

Vol 360 (1) ◽

pp. 57-66 ◽

Cited By ~ 19

Author(s):

Michael D. SCHALLER ◽

Erik M. SCHAEFER

Keyword(s):

Cell Adhesion ◽

Tyrosine Phosphorylation ◽

Binding Sites ◽

Signalling Molecules ◽

Src Homology 2 ◽

Dependent Cell ◽

Src Homology ◽

Tyrosine Phosphorylated Protein ◽

Src Homology 2 Domain

Paxillin is a focal-adhesion-associated, tyrosine-phosphorylated protein. In cells transformed by the src, crk or BCR-Abl oncogenes, the phosphotyrosine content of paxillin is elevated. In normal cells paxillin functions in signalling following integrin-dependent cell adhesion or exposure to a number of stimuli, including growth factors and neuropeptides. These stimuli induce tyrosine phosphorylation of paxillin, regulating the association of Src homology 2 domain-containing signalling molecules with paxillin. There are multiple sites of tyrosine phosphorylation on paxillin. To elucidate the role of paxillin in transducing signals in response to various stimuli, it is essential to identify all of the sites of phosphorylation on paxillin and to define which residues are phosphorylated in response to distinct stimuli. We describe two new sites of tyrosine phosphorylation on paxillin, residues at positions 40 and 88. Using paxillin variants with phenylalanine substitutions at phosphorylation sites and phospho-specific paxillin antibodies, tyrosine phosphorylation of paxillin in response to distinct stimuli was examined. The results demonstrate that Tyr31 and Tyr118, which are binding sites for Crk, are major sites of tyrosine phosphorylation following cell adhesion or stimulation with platelet-derived growth factor or angiotensin II. Thus multiple stimuli may elicit similar signalling events downstream of paxillin.

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A Regulatory Role for Src Homology 2 Domain–Containing Inositol 5′-Phosphatase (Ship) in Phagocytosis Mediated by Fcγ Receptors and Complement Receptor 3 (αMβ2; Cd11b/Cd18)

Journal of Experimental Medicine ◽

10.1084/jem.193.1.61 ◽

2000 ◽

Vol 193 (1) ◽

pp. 61-72 ◽

Cited By ~ 109

Author(s):

Dianne Cox ◽

Benjamin M. Dale ◽

Masaki Kashiwada ◽

Cheryl D. Helgason ◽

Steven Greenberg

Keyword(s):

Cell Model ◽

Complement Receptor ◽

Phosphatidylinositol 3 Kinase ◽

Src Homology 2 ◽

Catalytically Active ◽

Src Homology ◽

Complement Receptor 3 ◽

Phagocytic Cups ◽

Src Homology 2 Domain

The Src homology 2 domain–containing inositol 5′-phosphatase (SHIP) is recruited to immunoreceptor tyrosine-based inhibition motif (ITIM)–containing proteins, thereby suppressing phosphatidylinositol 3-kinase (PI 3-kinase)–dependent pathways. The role of SHIP in phagocytosis, a PI 3-kinase–dependent pathway, is unknown. Overexpression of SHIP in macrophages led to an inhibition of phagocytosis mediated by receptors for the Fc portion of IgG (FcγRs). In contrast, macrophages expressing catalytically inactive SHIP or lacking SHIP expression demonstrated enhanced phagocytosis. To determine whether SHIP regulates phagocytosis mediated by receptors that are not known to recruit ITIMs, we determined the effect of SHIP expression on complement receptor 3 (CR3; CD11b/CD18; αMβ2)–dependent phagocytosis. Macrophages overexpressing SHIP demonstrated impaired CR3-mediated phagocytosis, whereas macrophages expressing catalytically inactive SHIP demonstrated enhanced phagocytosis. CR3-mediated phagocytosis in macrophages derived from SHIP−/− mice was up to 2.5 times as efficient as that observed in macrophages derived from littermate controls. SHIP was localized to FcγR- and CR3-containing phagocytic cups and was recruited to the cytoskeleton upon clustering of CR3. In a transfected COS cell model of activation-independent CR3-mediated phagocytosis, catalytically active but not inactive SHIP also inhibited phagocytosis. We conclude that PI 3-kinase(s) and SHIP regulate multiple forms of phagocytosis and that endogenous SHIP plays a role in modulating β2 integrin outside-in signaling.

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Role of Src homology 2 domain‐containing protein B in the regulation of GFR and renal afferent arteriole responsiveness (1088.12)

The FASEB Journal ◽

10.1096/fasebj.28.1_supplement.1088.12 ◽

2014 ◽

Vol 28 (S1) ◽

Author(s):

Xiang Gao ◽

Johan Sällström ◽

Zufu Ma ◽

Michael Welsh ◽

A. Erik Persson

Keyword(s):

Afferent Arteriole ◽

Src Homology 2 ◽

Src Homology ◽

Src Homology 2 Domain ◽

Protein B

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Distinct Role of ZAP-70 and Src Homology 2 Domain-Containing Leukocyte Protein of 76 kDa in the Prolonged Activation of Extracellular Signal-Regulated Protein Kinase by the Stromal Cell-Derived Factor-1α/CXCL12 Chemokine

The Journal of Immunology ◽

10.4049/jimmunol.171.1.360 ◽

2003 ◽

Vol 171 (1) ◽

pp. 360-367 ◽

Cited By ~ 53

Author(s):

Kimberly N. Kremer ◽

Troy D. Humphreys ◽

Ashok Kumar ◽

Nan-Xin Qian ◽

Karen E. Hedin

Keyword(s):

Protein Kinase ◽

Stromal Cell ◽

Src Homology 2 ◽

Extracellular Signal ◽

Src Homology ◽

Stromal Cell Derived Factor ◽

Src Homology 2 Domain ◽

Distinct Role

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Role of SHPS-1 in the Regulation of Insulin-like Growth Factor I–stimulated Shc and Mitogen-activated Protein Kinase Activation in Vascular Smooth Muscle Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e04-10-0918 ◽

2005 ◽

Vol 16 (7) ◽

pp. 3353-3364 ◽

Cited By ~ 48

Author(s):

Yan Ling ◽

Laura A. Maile ◽

Jaroslava Lieskovska ◽

Jane Badley-Clarke ◽

David R. Clemmons

Keyword(s):

Tyrosine Phosphatase ◽

Mitogen Activated Protein Kinase ◽

Mutant Form ◽

Mapk Phosphorylation ◽

Src Homology 2 ◽

Igf I ◽

Mitogen Activated Protein ◽

Src Homology ◽

Src Homology 2 Domain

Insulin-like growth factor I (IGF-I) stimulates smooth muscle cell (SMC) proliferation, and the mitogen-activated protein kinase (MAPK) pathway plays an important role in mediating IGF-I–induced mitogenic signaling. Our prior studies have shown that recruitment of Src homology 2 domain tyrosine phosphatase (SHP-2) to the membrane scaffolding protein Src homology 2 domain–containing protein tyrosine phosphatase substrate-1 (SHPS-1) is required for IGF-I–dependent MAPK activation. The current studies were undertaken to define the upstream signaling components that are required for IGF-I–stimulated MAPK activation and the role of SHPS-1 in regulating this process. The results show that IGF-I–induced Shc phosphorylation and its subsequent binding to Grb2 is required for sustained phosphorylation of MAPK and increased cell proliferation in SMCs. Furthermore, for Shc to be phosphorylated in response to IGF-I requires that Shc must associate with SHPS-1 and this association is mediated in part by SHP-2. Preincubation of cells with a peptide that contains a phospho-tyrosine binding motif sequence derived from SHPS-1 inhibited IGF-I–stimulated SHP-2 transfer to SHPS-1, the association of Shc with SHPS-1, and IGF-I–dependent Shc phosphorylation. Expression of an SHPS-1 mutant that did not bind to Shc or SHP-2 resulted in decreased Shc and MAPK phosphorylation in response to IGF-I. In addition, SMCs expressing a mutant form of the β3 subunit of the αVβ3, which results in impairment of SHP-2 transfer to SHPS-1, also showed attenuated IGF-I–dependent Shc and MAPK phosphorylation. Further analysis showed that Shc and SHP-2 can be coimmunoprecipitated after IGF-I stimulation. A cell-permeable peptide that contained a polyproline sequence from Shc selectively inhibited Shc/SHP-2 association and impaired Shc but not SHP-2 binding to SHPS-1. Exposure to this peptide also inhibited IGF-I–stimulated Shc and MAPK phosphorylation. Cells expressing a mutant form of Shc with the four prolines substituted with alanines showed no Shc/SHPS-1 association in response to IGF-I. We conclude that SHPS-1 functions as an anchor protein that recruits both Shc and SHP-2 and that their recruitment is necessary for IGF-I–dependent Shc phosphorylation, which is required for an optimal mitogenic response in SMCs.

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