Journal: bioRxiv

Article Title: The pathogenic T42A mutation in SHP2 rewires interaction specificity and enhances signaling

doi: 10.1101/2023.07.10.548257

Figure Lengend Snippet: ( A ) Domain architecture diagram of SHP2. SHP2 consists of two SH2 domains (yellow and pink) and a phosphatase domain (green). Relevant mutations and the catalytic cysteine (C459) are indicated. ( B ) SHP2 is kept in its auto-inhibited state by interactions between the N-SH2 and PTP domain (PDB: 4DGP). In its active state, the N-SH2 domain is pulled away, and the catalytic cysteine is accessible. Although the structure of SHP2 E76K (PDB: 6CRF) is used to represent the active state, multiple active states likely exist. ( C ) SHP2 is activated by upstream stimuli. The SH2 domains bind to tyrosinephosphorylated upstream proteins, such as transmembrane receptors, inducing a conformational change that activates SHP2. ( D ) Disease-associated mutations cluster largely, but not exclusively, on the interdomain interface between the N-SH2 and the PTP domain (PDB: 4DGP). Highlighted unlabeled mutation sites include: N58, G60, Y62, E69, F71, A72, E76, Q79, D106, E110, Q256, G268, Y279, I282, F285, N308, I309, T411, A461, G464, T468, R498, R501, M504, Q510. ( E ) Mutations in or near the N-SH2 binding pocket (PDB: 6ROY). T42 is engaging the phosphotyrosine of the phosphopeptide ligand, whereas L43 is facing into the SH2 domain core. T52 is near the residues surrounding the phosphotyrosine. ( F ) Mutations in or near the C-SH2 binding pocket (PDB: 6R5G). R138 is engaged with the phosphotyrosine of the phosphopeptide ligand, whereas E139 is facing away from the binding pocket.

Article Snippet: The SHP2 full-length, wild-type gene used as the template for all SHP2 constructs in this study was cloned from the pGEX-4TI SHP2 WT plasmid, which was a generous gift from Ben Neel (Addgene plasmid #8322).

Techniques: Mutagenesis, Binding Assay, Phospho-proteomics

Journal: bioRxiv

Article Title: The pathogenic T42A mutation in SHP2 rewires interaction specificity and enhances signaling

doi: 10.1101/2023.07.10.548257

Figure Lengend Snippet: ( A ) Measured binding affinities of N-SH2 WT against peptides derived from various known SHP2 interactors. ( B ) Fold-change in K D for N-SH2 T42A compared to N-SH2 WT , for each of the peptides shown in panel (A). ( C ) Same as (B), but for N-SH2 L43F . ( D ) Same as (B), but for N-SH2 T52S . Source data can be found in Table S2.

Article Snippet: The SHP2 full-length, wild-type gene used as the template for all SHP2 constructs in this study was cloned from the pGEX-4TI SHP2 WT plasmid, which was a generous gift from Ben Neel (Addgene plasmid #8322).

Techniques: Binding Assay, Derivative Assay

Journal: bioRxiv

Article Title: The pathogenic T42A mutation in SHP2 rewires interaction specificity and enhances signaling

doi: 10.1101/2023.07.10.548257

Figure Lengend Snippet: ( A ) Hydrogen bonding of Thr 42 in SHP2 N-SH2 WT to the phosphoryl group of phosphopeptide ligands, as seen in several crystal structures (PDB codes: 6ROY, 1AYA, 1AYB, 3TL0, 5DF6, 5X94, and 5X7B). ( B ) Structure of N-SH2 WT bound to the PD-1 pTyr 223 (ITIM) peptide at the end of a 1 μs MD simulation, highlighting a hydrogen bond network and other key interactions around the phosphotyrosine residue. ( C ) Structure of N-SH2 T42A bound to the PD-1 pTyr 223 (ITIM) peptide at the end of a 1 μs MD simulation, highlighting a distinct hydrogen bond network around the phosphotyrosine residues, relative to that seen for N-SH2 WT . ( D ) Overlay of the states shown in panels B and C, highlighting a change in position for the phosphotyrosine residue and peptide main chain upon T42A mutation. The N-SH2 WT state is in yellow with a dark-gray ligand. The N-SH2 T42A state is in light gray, with a light gray ligand. ( E ) Distribution of distances between the Lys 55 Nζ atom and the phosphotyrosine phosphorus atοm in simulations of the PD-1 pTyr 223 peptide bound to N-SH2 WT (black) or N-SH2 T42A (red). ( F ) Distribution of distances between the Lys 55 Nζ atom and the +2 Glu Cδ atom in simulations of the PD-1 pTyr 223 peptide bound to N-SH2 WT (black) or N-SH2 T42A (red). ( G ) An ion pair between Lys 55 and the +2 Glu residue (Glu 225) in the PD-1 pTyr 223 (ITIM) peptide, frequently observed in N-SH2 T42A simulations. ( H ) Effects of the T42A mutation in the context of the K55R mutation. The enhancement in binding affinity by the T42A mutation is attenuated by the K55R mutation for some peptides (CagA-D, PD-1 pTyr 223, and MILR1 pTyr 338) but not others (IRS1 pTyr 1179 and Imhof-9).

Article Snippet: The SHP2 full-length, wild-type gene used as the template for all SHP2 constructs in this study was cloned from the pGEX-4TI SHP2 WT plasmid, which was a generous gift from Ben Neel (Addgene plasmid #8322).

Techniques: Phospho-proteomics, Residue, Mutagenesis, Binding Assay

Journal: bioRxiv

Article Title: The pathogenic T42A mutation in SHP2 rewires interaction specificity and enhances signaling

doi: 10.1101/2023.07.10.548257

Figure Lengend Snippet: ( A ) SHP2 activation is measured by incubation with phosphopeptide ligands, followed by monitoring dephosphorylation of the small-molecule substrate DiFMUP to generate fluorescent DiFMU. ( B ) Representative activation curves for SHP2 WT , highlighting peptide-dependent changes in EC 50 and amplitude. ( C ) Correlation between the EC 50 of SHP2 WT activation by phosphopeptides and the K D of those phosphopeptides for the N-SH2 WT domain. ( D ) Correlation between activation EC 50 values for SHP2 WT and SHP2 R 138 Q , which has weakened C-SH2 binding capacity. ( E ) Comparison of SHP2 WT and SHP2 T42A activation curves for the PD-1 pTyr 248 peptide, highlighting a significant impact on both EC 50 and amplitude. ( F ) Comparison of SHP2 WT and SHP2 T42A activation curves for the Imhof-9 peptide, highlighting a minor change in EC 50 and amplitude. ( G ) Bubble plot juxtaposing the EC 50 values for activation of SHP2 WT and SHP2 T42A by nine peptides, alongside the fold-change in K D for binding of those peptides to N-SH2 WT vs N-SH2 T42A . The dotted line indicates where EC 50 values would be equivalent for SHP2 WT and SHP2 T42A . The graph shows that peptides with a large fold-change in binding affinity (larger bubble) have a large fold-change in EC 50 values for SHP2 T42A over SHP2 WT (distance from dotted line). All EC 50 values can be found in Table S5.

Article Snippet: The SHP2 full-length, wild-type gene used as the template for all SHP2 constructs in this study was cloned from the pGEX-4TI SHP2 WT plasmid, which was a generous gift from Ben Neel (Addgene plasmid #8322).

Techniques: Activation Assay, Incubation, Phospho-proteomics, De-Phosphorylation Assay, Binding Assay, Comparison

Journal: bioRxiv

Article Title: The pathogenic T42A mutation in SHP2 rewires interaction specificity and enhances signaling

doi: 10.1101/2023.07.10.548257

Figure Lengend Snippet: ( A ) Schematic diagram depicting the co-immunoprecipitation (co-IP) experiments with SHP2 and either Gab1, Gab2, or PD-1 in HEK293 cells. The interactor proteins are phosphorylated by a hyperactive form of c-Src kinase. SHP2 co-immunoprecipitation experiments with ( B ) Gab1, ( C ) Gab2, and ( D ) PD-1, demonstrating that SHP2 T42A binds tighter to these phosphoproteins than SHP2 WT . In each case, SHP2 was immunoprecipitated via its myc-tag. Co-immunoprecipitation of the interacting protein was detected using an α-FLAG antibody for Gab1/Gab2 and a PD-1-specific antibody for PD-1. For PD-1, the experiment was also conducted by immunoprecipitating PD-1 and detecting co-immunoprecipitation of SHP2 using an α-myc antibody. ( E ) Schematic depiction of EGF stimulation and phospho-Erk signaling experiments in the presence of co-expressed SHP2 and either Gab1 or Gab2. ( F ) Comparison of phospho-Erk levels in response to EGF stimulation in cells expressing Gab1 and either SHP2 WT or SHP2 T42A . ( G ) Comparison of phospho-Erk levels in response to EGF stimulation in cells expressing Gab2 and either SHP2 WT or SHP2 T42A . For panels (F) and (G), the numbers below the blots indicate phospho-Erk levels relative to the 0 minute sample with SHP2 WT .

Article Snippet: The SHP2 full-length, wild-type gene used as the template for all SHP2 constructs in this study was cloned from the pGEX-4TI SHP2 WT plasmid, which was a generous gift from Ben Neel (Addgene plasmid #8322).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Comparison, Expressing

Journal: Journal of Biological Chemistry

Article Title: A cellular target engagement assay for the characterization of SHP2 (PTPN11) phosphatase inhibitors

doi: 10.1074/jbc.ra119.010838

Figure Lengend Snippet: Figure 1. Allosteric control mechanisms for SHP2 are the basis for drug discovery. (A) In normal cells under basal conditions, SHP2 adopts an autoinhibited closed conformation in which its N-terminal SH2 domain binds and blocks the PTP active site. Cell stimulation leads to Tyr-phosphorylation of SHP2 binding proteins that then recruit SHP2 via its SH2 domains, causing SHP2 to open into its active conformation; tyrosine phosphorylation within the C-terminal tail (pY542 and pY580) further enhances SHP2 activity. (B) In solid tumors, overexpression or aberrant phosphorylation of RTKs or scaffolding adapters result in hyperactivation of SHP2. (C) In leukemias, somatic mutations located at the interface between the N-SH2 and PTP domains prevent SHP2 from closing, resulting in a constitutively active SHP2. (D) Crystal structure of the SHP2:SHP099 complex (PDB accession number 5EHR) with the N-SH2 (blue), C-SH2 (green), and phosphatase domain (orange) in the closed, autoinhibited conformation. The allosteric inhibitor SHP099 binds in a "tunnel" formed at an interface of the three domains and stabilizes SHP2 in its inactive conformation. (D) Allosteric SHP2 inhibitors such as SHP099 or RMC-4550 compete with SHP2 activation, as the allosteric binding site only exists in the closed conformation. The effect of SHP2 gain-of- function mutations is to destabilize the autoinhibited confirmation of SHP2. Therefore, many oncogenic SHP2 mutants are resistant to inhibition by the SHP099 class of compounds.

Article Snippet: For recombinant expression of the full-length SHP2 (1-594), a glutathione S-transferase (GST) fusion construct in pGEX-4T1 was used to produce a thrombin-cleavable construct (Addgene plasmid #8322).

Techniques: Control, Drug discovery, Cell Stimulation, Phospho-proteomics, Binding Assay, Activity Assay, Over Expression, Scaffolding, Activation Assay, Inhibition

Journal: Journal of Biological Chemistry

Article Title: Adapter Protein Shc Regulates Janus Kinase 3 Phosphorylation

doi: 10.1074/jbc.c113.527523

Figure Lengend Snippet: FIGURE 2. Adapter protein Shc regulates Jak3 activation through regulating Jak3 interactions with tyrosine phosphatases. A, Western analysis for the expression of FLAG-tagged ShcA-wt or its mutants was done using lysates from stably transfected HT-29 Cl-19A cells and FLAG antibody. B, Western analysis of co-immunoprecipitates from cell lysates of stably transfected cells from A treated with IL-2 (50 units/ml) were done using indicated antibodies for phos- photyrosine (pY), phospho-Jak3 (pJak3), and tyrosine phosphatase SHP2 and PTP1B using previously reported protocols (8). C, dephosphorylation of phospho- Jak3 was determined using in vitro phosphatase assay using P-Jak3-immunoprecipitated Jak3 from cell lysates of IL-2-treated HT-29 Cl-19A cells as described under “Experimental Procedures”. pY20, phosphotyrosine antibody from clone PY20. D, direct interactions between recombinant purified proteins of FLAG- tagged-ShcA-wt or mutants and indicated phosphatases were determined by pairwise binding assay as in Fig. 1, and protein complexes were immunopre- cipitated using the indicated antibodies followed by IB by FLAG antibodies. For input controls IB was done using the indicated antibodies for phosphatases. E, susceptibility toward staurosporine-induced apoptosis was determined in stably transfected cells from A using a GFP-certified apoptosis assay kit as reported before (7). Yellow color in the Merged panels shows GFP and annexin V co-localized apoptotic cells, whereas green color indicates nonapoptotic healthy cells. F, immunofluorescence microscopy on control or staurosporine-treated cells (Stauro) from E was done using Alexa Fluor 488-conjugated phalloidin to stain F-actin. Right panels, P-Jak3 was assessed in cells from F and G using IP and IB with the indicated antibodies. G, wound closure was measured as a percentage of the original wound area as reported before (8). H, model for Shc-mediated Jak3 dephosphorylation. G, values are mean S.E. * indicates statistically significant differences from IL-2, p 0.05, n 3 experiments. E and F, images were stacked and processed using NIS Element software (Nikon). Representative blots (A–D and F) or images (E and F) are shown from n 3 experiments. Scale bar, 14 M.

Article Snippet: Constructs for full-length SHP1, SHP2, and PTP1B cloned in pGEX2T were from Addgene.

Techniques: Activation Assay, Western Blot, Expressing, Stable Transfection, Transfection, De-Phosphorylation Assay, In Vitro, Phosphatase Assay, Immunoprecipitation, Recombinant, Purification, Binding Assay, Apoptosis Assay, Immunofluorescence, Microscopy, Control, Staining, Software