N-SH2 Domain Research Articles

Binding Mechanism between CrkII and cAbl Kinase

The interaction between non-receptor tyrosine kinase cAbl and signaling adaptor protein CrkII is implicated in diverse cellular processes, such as cell migration, proliferation, and bacterial infection. Because of the modular architecture of their structures, the interaction between cAbl and CrkII can undergo multiple alternative binding modes. This series of interactions starts with the binding of the N-terminal Src homology 3 (nSH3) domain of CrkII to proline-rich motifs (PRMs) in the intrinsically disordered region of cAbl kinase. Despite their importance, the detailed binding mechanism and functional significance of these interactions remains elusive. In this presentation, we report the detailed structure, thermodynamics, and kinetics of the interactions between the nSH3 domain of CrkII and PRMs of cAbl kinase. Our dynamics and structural studies, using NMR spectroscopy and crystallography, indicate that these modular interactions act as a binding catalyst that accelerates the association between CrkII and cAbl kinase. We also present our recent finding of a potent inhibitor of the cAbl-CrkII interaction.

Thermodynamic contribution of backbone conformational entropy in the binding between SH3 domain and proline-rich motif

Biological functions of intrinsically disordered proteins (IDPs), and proteins containing intrinsically disordered regions (IDRs) are often mediated by short linear motifs, like proline-rich motifs (PRMs). Upon binding to their target proteins, IDPs undergo a disorder-to-order transition which is accompanied by a large conformational entropy penalty. Hence, the molecular mechanisms underlying control of conformational entropy are critical for understanding the binding affinity and selectivity of IDPs-mediated protein-protein interactions (PPIs). Here, we investigated the backbone conformational entropy change accompanied by binding of the N-terminal SH3 domain (nSH3) of CrkII and PRM derived from guanine nucleotide exchange factor 1 (C3G). In particular, we focused on the estimation of conformational entropy change of disordered PRM upon binding to the nSH3 domain. Quantitative characterization of conformational dynamics of disordered peptides like PRMs is limited. Hence, we combined various methods, including NMR model-free analysis, δ2D, DynaMine, and structure-based calculation of entropy loss. This study demonstrates that the contribution of backbone conformational entropy change is significant in the PPIs mediated by IDPs/IDRs.

Interaction of Both SH2 Domains of SHP-2 with a PD-1 Homodimer Is Required for PD-1-Mediated Inhibition of T Cell Responses

Programmed death-1 (PD-1) is a checkpoint receptor expressed on activated T-cells. PD-1 has a key role in maintenance of peripheral tolerance but also restrains anti-viral and anti-tumor immunity. Although PD-1 blockade leads to durable clinical responses in a significant fraction of patients, the majority of patients have only transient responses, emphasizing the need for better understanding of the mechanism of PD-1-mediated T cell inhibition. PD-1 consists of a single N-terminal IgV-like domain, a 20 amino acid stalk separating the IgV domain from the plasma membrane, a transmembrane domain, and a cytoplasmic tail containing two tyrosine-based structural motifs, an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). SHP-2 tyrosine phosphatase interacts with the ITSM and has a critical role in PD-1-mediated inhibition but the precise mechanism is poorly understood. We sought to determine how PD-1: SHP-2 interaction leads to inhibition of T-cell responses. SHP-2 contains two SH2 domains, a phosphatase (PTP) domain and a C-terminus tail (C-tail), forming a structure of N-SH2-C-SH2-PTP-C-tail. We generated five GST-fusion proteins in which GST was fused with either SHP-2 full length, N-SH2, C-SH2 C-SH2-PTP (lacking the N-terminus SH2 domain), or PTP. Pull-down assays using lysates from human T cells revealed that PD-1 interacted with GST-SHP-2 fusion protein only after TCR/CD3-mediated activation with simultaneous PD-1 ligation, and the interaction of PD-1 with SHP-2 was mediated via the SH2 domains of SHP-2. The SH2 domains of SHP-2 have a crucial and distinct role in regulating SHP-2 PTPase activity. In the absence of a tyrosine-phosphorylated ligand, N-SH2 binds the PTP domain leading to an auto-inhibitory closed conformation that blocks the PTP site. Phosphorylation of Y542 in the SHP-2 C-tail leads to an intramolecular interaction of Y542 with the N-SH2 domain that relieves N-SH2 binding to the PTP domain and thereby reverses basal inhibition of the PTPase. Phosphorylation of Y580 in the SHP-2 C-tail relieves the auto-inhibitory closed conformation by interaction with the C-SH2 domain. Subsequent high affinity intermolecular interaction of the N-SH2 with a phosphorylated protein partner completely disrupts its PTP recognition surface, reversing the auto-inhibitory conformation and activating the PTPase activity, whereas the C-SH2 domain contributes to binding energy and specificity. We found that in activated T cells, PD-1-associated SHP-2 was phosphorylated in the tyrosines of the C-tail. To determine whether PD-1 selectively interacts with a specific SH2 domain of SHP-2, we mutagenized the functional sites of N-SH2 and C-SH2 domains at arginines 32 and 138, respectively, to alanine (R32A and R138A) and transfected COS cells with cDNA of SHP-2 wild type or each SH2 mutant together with PD-1 and TCR proximal kinase Fyn, which is required for PD-1 phosphorylation. Immunoprecipitation and immunoblot showed that mutagenesis of either SH2 domain abrogated interaction of SHP-2 with PD-1, indicating that both SH2 domains of SHP-2 are involved in the interaction with PD-1. Surprisingly, each SH2 domain of SHP-2 interacted with tyrosine phosphorylated ITSM of PD-1, as determined by immunoblot with a phopho-PD-1 antibody specific for the phosphorylated tyrosine residue Y245 and by disruption of both N-SH2:PD-1 and C-SH2:PD-1 interaction by mutation of PD-1 ITSM tyrosine residue Y245. These results indicate that SHP-2 brings together two tyrosine phosphorylated PD-1 molecules by interaction with N-SH2 and C-SH2 domains. To determine the functional implications of PD-1 homodimerization, we cultured human T cells in the presence of a soluble dimeric PD-L1 or a monomeric PD-L1. Although dimeric PD-L1 inhibited T cell proliferation and IFN-g production, monomeric PD-L1 had the opposite effect. Our results reveal a previously unidentified mechanism of PD-1: SHP-2 interaction and have implications for the development of PD-1-binding compounds to selectively suppress T cell responses by dimerizing PD-1 or to enhance T cell activation by disrupting PD-1 homodimerization. Our findings open new avenues for the development of selective PD-1-binding compounds in order to augment T cell responses for the induction of antitumor immunity or to suppress aberrant T cell activation in autoimmunity and graft versus host disease. DisclosuresNo relevant conflicts of interest to declare.

Kinetic Insights into the Binding between the nSH3 Domain of CrkII and Proline-Rich Motifs in cAbl

The interaction between CrkII and cAbl is implicated in diverse cellular processes. This interaction starts with the binding of the N-terminal Src homology 3 (nSH3) domain of CrkII to the proline-rich motifs of cAbl (PRMscAbl). Despite its critical importance, the detailed binding mechanism between the nSH3 domain and PRMs remains elusive. In this study, we used nuclear magnetic resonance Carr-Purcell-Meiboom-Gill relaxation dispersion experiment to study the binding kinetics between the nSH3 domain of CrkII and PRMscAbl. Our results highlight that the nSH3 domain binds to three PRMscAbl with very high on- and off-rate constants, indicating the transient nature of the binding. To further characterize the binding transition state, we conducted the Eyring and linear free energy relationship analyses using temperature-dependent kinetic data. These data indicate that the binding transition state of the nSH3 domain and PRM is accompanied by small activation enthalpy, owing to partial desolvation of the transition state. These results also highlight the similarity between the transition and free states, in terms of structure and energetics. Although the binding of the nSH3 domain and PRM displays the features consistent with a diffusion-limited process within our experimental conditions, further tests are necessary to determine if the binding is a true diffusion-limited process.

Study of SHP-2 (PTPN11) allosterism on structural movement using solution perturbed molecular dynamics simulation

Src-homology2 (SH2) domain-containing protein tyrosine phosphatase-2 (SHP-2) is a ubiquitously expressed protein-tyrosine phosphatase. SHP-2 is linked to human diseases and plays broad roles in cancer pathogenesis. Activity of the SHP-2 catalytic domain is auto-inhibited by an N-SH2 domain, maintaining SHP-2 in an inactive state. N-SH2 conformation switches to expose the catalytic domain, forming an active state. However, the switch mechanism is not clearly described. Although structural and kinetic studies in silicon have simulated the biological catalytic processes, Molecular Dynamics (MD) simulation has not provided sufficient information to explain the allosteric mechanism of SHP2. To further understand the conformational process, we created a novel method, solvent perturbed MD (SPMD), with high kinetic energy solvent to study SHP2 allosterism. We also illustrated the putative factor at the mouth of the opening pocket of SHP-2. We found, based on SPMD simulations, the distance between ASP61, of N-SH2 blocker, and ARG465 at the bottom of the pocket, reached 1nm. Moreover, SPMD did not denature SHP2; the torsion angle and position of residue ARG465 in the bottom of the pocket were not significantly perturbed. Our SPMD simulation also demonstrated the periodic opening and closing of the blocker. Thus, our findings on the conformational alteration of SHP-2 in a solution interrupted system furthers our understanding of allosterism.

Abstract 1261: Nintedanib induces antitumor effects in triple-negative breast cancer cells by targeting SHP-1/p-STAT3-mediated signaling pathway

Abstract Background: Triple-negative breast cancer (TNBC) is a heterogeneous and aggressive subtype that currently lack of specific targeted therapy. Previous studies have suggested that disturbing the oncogenic STAT3 signaling by activating a protein tyrosine phosphatase SHP-1 as a possible anti-cancer strategy. Our previous study also found that nintedanib (BIBF1120), a multi-kinase inhibitor that blocks mainly anti-angiogenic pathways and has been approved for idiopathic pulmonary fibrosis and for non-small cell lung cancer (in Europe), inhibits hepatocellular carcinoma cell growth independent of its anti-angiokinase activity (J Hepatology 2015). In this study, we tested nintedanib in TNBC cells and examined the underlying molecular mechanism by which nintedanib shows anti-TNBC effects. Methods: TNBC cell lines (MDA-MB-468, MDA-MB-231, HCC-1395) were used for in vitro studies. Cell viability was examined by MTT assay. Apoptosis was examined by flow cytometry and western blot. Signal transduction pathways in cells were assessed by western blot. Purified SHP-1 proteins or cells expressing deletion N-SH2 domain or D61A point mutants (SHP-1 mutants with constant open form conformations) were used to investigate the potential effects of nintedanib on SHP-1. In vivo efficacy of nintedanib was tested in nude mice bearing orthotopic breast cancer cells xenografts. Results: MTT assays revealed nintedanib induced anti-proliferation in the 3 TNBC cell lines. Sub-G1 flow cytometric and western blot analysis showed that nintedanib induced apoptosis in association with downregulation of p-STAT3 and its downstream proteins such as cyclin D1 in both dose and time-dependent manners. Overexpression of STAT3 suppressed nintedanib-mediated apoptosis in MDA-MB-231 cells. Nintedanib further increased SHP-1 activity in purified SHP-1 proteins, suggesting that nintedanib may directly affects SHP-1 activity for p-STAT3 inhibition. Moreover, either specific SHP-1 activity inhibitor or SHP-1 siRNA reduced the apoptotic effects of nintedanib on MDA-MB-231 and MDA-MB-468 cells, validating the role of SHP-1 activation in nintedanib-induced apoptosis. Furthermore, nintedanib-induced apoptosis was attenuated in TNBC cells expressing SHP-1 mutants with constant open form conformations, including deletion mutants of N-SH2 domain or D61A mutants, suggesting that the auto-inhibition structure of SHP-1 mediated the effect of nintedanib. Importantly, intraperitoneal injection of nintedanib showed antitumor activity, increased SHP-1 activity and p-STAT3 downregulation in MDA-MB-231 xenograft tumors. Conclusions Our results suggest that nintedanib induced anti-tumor effect by SHP-1 dependent STAT3 inhibition in human TNBC cells. These findings may provide new signaling pathway insight into the inhibition of TNBC growth by nintedanib. Citation Format: Chun-Yu Liu, Chia-Han Lee, Chun-Teng Huang, Pei-Yi Chu, Wan-Lun Wang, Ling-Ming Tseng, Chung-Wai Shiau, Kuen-Feng Chen. Nintedanib induces antitumor effects in triple-negative breast cancer cells by targeting SHP-1/p-STAT3-mediated signaling pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1261.

Abstract 3772: SHP-1 determines the radiosensitivity of liver cancer cell and dovitinib acts as a novel radiosensitizer in hepatocellular carcinoma via targeting SHP-1/STAT3 signaling

Abstract Background Hepatocellular carcinoma (HCC) is one of the most lethal human malignancies and curative therapy is not an option for most patients. There is growing interest in the potential benefit of radiotherapy (RT)-integrated therapy. This study aimed to investigate the biological impacts of a novel tumor suppressor Src homology 2 (SH2) domain-containing phosphatase 1 (SHP-1) and its downstream effecter, STAT3, in regulating the radiosensitivity of HCC cells. Furthermore, we explored the efficacy and mechanism of an investigational drug, dovitinib, used in combination with RT. Material and methods To understand the impacts of SHP-1/STAT3 signaling affects radiosensitivity, HCC cells with ectopic expression of STAT3, SHP-1 and a catalytic mutant SHP-1 were treated with or without radiotherapy and analyzed by flow cytometry and colony formation assay. Furthermore, five HCC cell lines (PLC5, Hep3B, SK-Hep1, HA59T and Huh-7) were treated with dovitnib, RT or both. The in vitro and in vivo effects of above-mentioned treatments were analyzed. Results By sub-G1 analysis and colony formation, we found that HCC cell with ectopic expression of SHP-1 was much more sensitive to RT-induced apoptotic effects, while overexpression of STAT3 or catalytic-dead mutant SHP-1 restored RT-induced reduction of HCC cell survival. Next, we investigated the effects of dovitinib, which showed that dovitinib treatment resulted in SHP-1-mediated downregulation of p-STAT3 and promoted potent apoptosis of HCC cells. Ectopic expression of STAT3, or inhibition of SHP-1 diminished the effects of dovitinib on HCC cells. Furthermore, by ectopic expression and purified recombinant proteins of various mutant forms of SHP-1, the N-SH2 domain of SHP-1 was found to be required for dovitinib treatment. Importantly, we found that dovitinib potentiated the in vitor and in vivo effects of RT in HCC cells through affecting the SHP-1/STAT3 signaling. Conclusions SHP-1/STAT3 signaling is critically associated with the radiosensitivity of HCC cells. A combination therapy with RT and the SHP-1 agonist, such as dovitinib, resulted in enhanced in vitro and in vivo anti-HCC effects. Citation Format: Man-Hsin Hung, Chao-Yuan Huang, Wei-Tien Tai, Ming-Hsien Tsai, Chih-Ting Shin, Szu-Yuan Wu, Chung-Wai Shiau, Kuen-Feng Chen. SHP-1 determines the radiosensitivity of liver cancer cell and dovitinib acts as a novel radiosensitizer in hepatocellular carcinoma via targeting SHP-1/STAT3 signaling. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3772.

Binding Mechanism of the N-Terminal SH3 Domain of CrkII and Proline-Rich Motifs in cAbl

The N-terminal Src homology 3 (nSH3) domain of a signaling adaptor protein, CT-10 regulator of kinase II (CrkII), recognizes proline-rich motifs (PRMs) of binding partners, such as cAbl kinase. The interaction between CrkII and cAbl kinase is involved in the regulation of cell spreading, microbial pathogenesis, and cancer metastasis. Here, we report the detailed biophysical characterizations of the interactions between the nSH3 domain of CrkII and PRMs in cAbl. We identified that the nSH3 domain of CrkII binds to three PRMs in cAbl with virtually identical affinities. Structural studies, by using x-ray crystallography and NMR spectroscopy, revealed that the binding modes of all three nSH3:PRM complexes are highly similar to each other. Van ’t Hoff analysis revealed that nSH3:PRM interaction is associated with favorable enthalpy and unfavorable entropy change. The combination of experimentally determined thermodynamic parameters, structure-based calculations, and 15N NMR relaxation analysis highlights the energetic contribution of conformational entropy change upon the complex formation, and water molecules structured in the binding interface of the nSH3:PRM complex. Understanding the molecular basis of nSH3:PRM interaction will provide, to our knowledge, new insights for the rational design of small molecules targeting the interaction between CrkII and cAbl.

Role of Electrostatic Interactions in Binding of Peptides and Intrinsically Disordered Proteins to Their Folded Targets: 2. The Model of Encounter Complex Involving the Double Mutant of the c-Crk N-SH3 Domain and Peptide Sos.

In the first part of this work (paper 1, Xue, Y. et al. Biochemistry 2014 , 53 , 6473 ), we have studied the complex between the 10-residue peptide Sos and N-terminal SH3 domain from adaptor protein c-Crk. In the second part (this paper), we designed the double mutant of the c-Crk N-SH3 domain, W169F/Y186L, with the intention to eliminate the interactions responsible for tight peptide-protein binding, while retaining the interactions that create the initial electrostatic encounter complex. The resulting system was characterized experimentally by measuring the backbone and side-chain (15)N relaxation rates, as well as binding shifts and (1)H(N) temperature coefficients. In addition, it was also modeled via a series of ∼5 μs molecular dynamics (MD) simulations recorded in a large water box under an Amber ff99SB*-ILDN force field. Similar to paper 1, we have found that the strength of arginine-aspartate and arginine-glutamate salt bridges is overestimated in the original force field. To address this problem we have applied the empirical force-field correction described in paper 1. Specifically, the Lennard-Jones equilibrium distance for the nitrogen-oxygen pair across Arg-to-Asp/Glu salt bridges has been increased by 3%. This modification led to MD models in good agreement with the experimental data. The emerging picture is that of a fuzzy complex, where the peptide "dances" over the surface of the protein, making transient contacts via salt-bridge interactions. Every once in a while the peptide assumes a certain more stable binding pose, assisted by a number of adventitious polar and nonpolar contacts. On the other hand, occasionally Sos flies off the protein surface; it is then guided by electrostatic steering to quickly reconnect with the protein. The dynamic interaction between Sos and the double mutant of c-Crk N-SH3 gives rise to only small binding shifts. The peptide retains a high degree of conformational mobility, although it is appreciably slowed down due to its (loose) association with the protein. Note that spin relaxation data are indispensable in determining the dynamic status of the peptide. Such data can be properly modeled only on a basis of bona fide MD simulations, as shown in our study. We anticipate that in future the field will move away from the ensemble view of protein disorder and toward more sophisticated MD models. This will require further optimization of force fields, aimed specifically at disordered systems. Efforts in this direction have been recently initiated by several research groups; the empirical salt-bridge correction proposed in our work falls in the same category. MD models obtained with the help of suitably refined force fields and guided by experimental NMR data will provide a powerful insight into an intricate world of disordered biomolecules.

Dovitinib Acts As a Novel Radiosensitizer in Hepatocellular Carcinoma by Targeting SHP-1/STAT3 Signaling

Hepatocellular carcinoma (HCC) is among the most lethal human malignancies, and curative therapy is not an option for most patients. There is growing interest in the potential benefit of combining targeted therapies with radiation therapy (RT). This study aimed to characterize the efficacy and mechanism of an investigational drug, dovitinib, used in combination with RT. HCC cell lines (PLC5, Hep3B, SK-Hep1, HA59T, and Huh-7) were treated with dovitinib, RT, or both, and apoptosis and signal transduction were analyzed. Dovitinib treatment resulted in Src homology region 2 (SH2) domain-containing phosphatase 1 (SHP-1)-mediated downregulation of p-STAT3 and promoted potent apoptosis of HCC cells. Ectopic expression of STAT3, or inhibition of SHP-1, diminished the effects of dovitinib on HCC cells. By ectopic expression and purified recombinant proteins of various mutant forms of SHP-1, the N-SH2 domain of SHP-1 was found to be required for dovitinib treatment. Overexpression of STAT3 or catalytic-dead mutant SHP-1 restored RT-induced reduction of HCC cell survival. Conversely, ectopic expression of SHP-1 or activation of SHP-1 by dovitinib enhanced the effects of RT against HCC invitro and invivo. SHP-1/STAT3 signaling is critically associated with the radiosensitivity of HCC cells. Combination therapy with RT and the SHP-1 agonist, such as dovitinib, resulted in enhanced invitro and invivo anti-HCC effects.

Focal adhesion kinase-dependent focal adhesion recruitment of SH2 domains directs SRC into focal adhesions to regulate cell adhesion and migration.

Directed cell migration requires dynamical control of the protein complex within focal adhesions (FAs) and this control is regulated by signaling events involving tyrosine phosphorylation. We screened the SH2 domains present in tyrosine-specific kinases and phosphatases found within FAs, including SRC, SHP1 and SHP2, and examined whether these enzymes transiently target FAs via their SH2 domains. We found that the SRC_SH2 domain and the SHP2_N-SH2 domain are associated with FAs, but only the SRC_SH2 domain is able to be regulated by focal adhesion kinase (FAK). The FAK-dependent association of the SRC_SH2 domain is necessary and sufficient for SRC FA targeting. When the targeting of SRC into FAs is inhibited, there is significant suppression of SRC-mediated phosphorylation of paxillin and FAK; this results in an inhibition of FA formation and maturation and a reduction in cell migration. This study reveals an association between FAs and the SRC_SH2 domain as well as between FAs and the SHP2_N-SH2 domains. This supports the hypothesis that the FAK-regulated SRC_SH2 domain plays an important role in directing SRC into FAs and that this SRC-mediated FA signaling drives cell migration.

New and Unexpected Biological Functions for the Src-Homology 2 Domain-Containing Phosphatase SHP-2 in the Gastrointestinal Tract.

SHP-2 is a tyrosine phosphatase expressed in most embryonic and adult tissues. SHP-2 regulates many cellular functions including growth, differentiation, migration, and survival. Genetic and biochemical evidence show that SHP-2 is required for rat sarcoma viral oncogene/extracellular signal-regulated kinases mitogen-activated protein kinase pathway activation by most tyrosine kinase receptors, as well as by G-protein–coupled and cytokine receptors. In addition, SHP-2 can regulate the Janus kinase/signal transducers and activators of transcription, nuclear factor-κB, phosphatidyl-inositol 3-kinase/Akt, RhoA, Hippo, and Wnt/β-catenin signaling pathways. Emerging evidence has shown that SHP-2 dysfunction represents a key factor in the pathogenesis of gastrointestinal diseases, in particular in chronic inflammation and cancer. Variations within the gene locus encoding SHP-2 have been associated with increased susceptibility to develop ulcerative colitis and gastric atrophy. Furthermore, mice with conditional deletion of SHP-2 in intestinal epithelial cells rapidly develop severe colitis. Similarly, hepatocyte-specific deletion of SHP-2 induces hepatic inflammation, resulting in regenerative hyperplasia and development of tumors in aged mice. However, the SHP-2 gene initially was suggested to be a proto-oncogene because activating mutations of this gene were found in pediatric leukemias and certain forms of liver and colon cancers. Moreover, SHP-2 expression is up-regulated in gastric and hepatocellular cancers. Notably, SHP-2 functions downstream of cytotoxin-associated antigen A (CagA), the major virulence factor of Helicobacter pylori, and is associated with increased risks of gastric cancer. Further compounding this complexity, most recent findings suggest that SHP-2 also coordinates carbohydrate, lipid, and bile acid synthesis in the liver and pancreas. This review aims to summarize current knowledge and recent data regarding the biological functions of SHP-2 in the gastrointestinal tract.

Pharmacological Targeting SHP-1-STAT3 Signaling Is a Promising Therapeutic Approach for the Treatment of Colorectal Cancer

STAT3 activation is associated with poor prognosis in human colorectal cancer (CRC). Our previous data demonstrated that regorafenib (Stivarga) is a pharmacological agonist of SH2 domain-containing phosphatase 1 (SHP-1) that enhances SHP-1 activity and induces apoptosis by targeting STAT3 signals in CRC. This study aimed to find a therapeutic drug that is more effective than regorafenib for CRC treatment. Here, we showed that SC-43 was more effective than regorafenib at inducing apoptosis in vitro and suppressing tumorigenesis in vivo. SC-43 significantly increased SHP-1 activity, downregulated p-STAT3Tyr705 level, and induced apoptosis in CRC cells. An SHP-1 inhibitor or knockdown of SHP-1 by siRNA both significantly rescued the SC-43–induced apoptosis and decreased p-STAT3Tyr705 level. Conversely, SHP-1 overexpression increased the effects of SC-43 on apoptosis and p-STAT3Tyr705 level. These data suggest that SC-43–induced apoptosis mediated through the loss of p-STAT3Tyr705 was dependent on SHP-1 function. Importantly, SC-43–enhanced SHP-1 activity was because of the docking potential of SC-43, which relieved the autoinhibited N-SH2 domain of SHP-1 and inhibited p-STAT3Tyr705 signals. Importantly, we observed that a significant negative correlation existed between SHP-1 and p-STAT3Tyr705expression in CRC patients (P = .038). Patients with strong SHP-1 and weak p-STAT3Tyr705 expression had significantly higher overall survival compared with patients with weak SHP-1 and strong p-STAT3Tyr705 expression (P = .029). In conclusion, SHP-1 is suitable to be a useful prognostic marker and a pharmacological target for CRC treatment. Targeting SHP-1-STAT3 signaling by SC-43 may serve as a promising pharmacotherapy for CRC.

Oncogenic mutations weaken the interactions that stabilize the p110α-p85α heterodimer in phosphatidylinositol 3-kinase α.

Phosphatidylinositol 3-kinase (PI3K) α is a heterodimeric lipid kinase that catalyzes the conversion of phosphoinositol-4,5-bisphosphate to phosphoinositol-3,4,5-trisphosphate. The PI3Kα signaling pathway plays an important role in cell growth, proliferation, and survival. This pathway is activated in numerous cancers, where the PI3KCA gene, which encodes for the p110α PI3Kα subunit, is mutated. Its mutation often results in gain of enzymatic activity; however, the mechanism of activation by oncogenic mutations remains unknown. Here, using computational methods, we show that oncogenic mutations that are far from the catalytic site and increase the enzymatic affinity destabilize the p110α-p85α dimer. By affecting the dynamics of the protein, these mutations favor the conformations that reduce the autoinhibitory effect of the p85α nSH2 domain. For example, we determined that, in all of the mutants, the nSH2 domain shows increased positional heterogeneity as compared with the wild-type, as demonstrated by changes in the fluctuation profiles computed by normal mode analysis of coarse-grained elastic network models. Analysis of the interdomain interactions of the wild-type and mutants at the p110α-p85α interface obtained with molecular dynamics simulations suggest that all of the tumor-associated mutations effectively weaken the interactions between p110α and p85α by disrupting key stabilizing interactions. These findings have important implications for understanding how oncogenic mutations change the conformational multiplicity of PI3Kα and lead to increased enzymatic activity. This mechanism may apply to other enzymes and/or macromolecular complexes that play a key role in cell signaling.

Abstract PR02: Targeting PI3K: The PIP2 binding site

Abstract Structural biology has yielded many significant insights into inhibitor binding and isoform selectivity, directing the design of many important clinical candidates. To date, structural studies have focused on the ATP-binding site and ATP-competitive inhibitors. We have recently determined the structure of PI3Kα; in complex with the soluble lipid substrate mimetic, diC4-PIP2. The substrate binds in a positively charged pocket, defined by the activation and P-loops of the kinase domain, and the iSH2 domain of p85. The positively charged residues responsible for binding PIP2 that are present on the activation loop of Class I PI3K are missing in Class II and III PI3K. This explains why these two classes have lower affinity for PIP2. The complex structure provides insights into the catalysis and regulation of PI3K. Key interactions between the activation loop and nSH2 domain may modulate active and inactive conformations of the enzyme upon the binding of phosphorylated receptor tyrosine kinases. In addition, a second lipid substrate was identified in the structure. It binds in a hydrophobic pocket between the Adaptor Binding Domain (ABD), kinase domain and iSH2 domain of p85. Fluorescence quenching studies confirm the ability of PI3K to bind an additional PIP2 molecule. This abstract is also being presented as Poster B17. Citation Format: Michelle Miller, Oleg Schmidt-Kittler, David M. Bolduc, Evan T. Brower, Daniele Chaves-Moreira, Marc Allaire, Kenneth W. Kinzler, Ian G. Jennings, Philip E. Thompson, Philip A. Cole, L. Mario Amzel, Bert Vogelstein, Sandra B. Gabelli. Targeting PI3K: The PIP2 binding site. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr PR02.

Direct Chemical Activation of a Rationally Engineered Signaling Enzyme.

Few chemical strategies for activating enzymes have been developed. Here we show that a biarsenical compound (FlAsH) can directly activate a rationally engineered protein tyrosine phosphatase (Shp2 PTP) by disrupting autoinhibitory interactions between Shp2's N-terminal SH2 domain and its PTP domain. We found that introducing a tricysteine motif at a loop of Shp2's N-SH2 domain confers affinity for FlAsH; binding of FlAsH to the cysteine-enriched loop relieves Shp2's inhibitory interdomain interaction and substantially increases the enzyme's PTP activity. Activation of engineered Shp2 is substrate independent and is observed in the contexts of both purified enzyme and complex proteomes. A chemical means for activating Shp2 could be useful for investigating its roles in signaling and oncogenesis, and the loop-targeting strategy described herein could provide a blueprint for the development of target-specific activators of other autoinhibited enzymes.

Tyrosine phosphatase SHP-2 mediates C-type lectin receptor-induced activation of the kinase Syk and anti-fungal TH17 responses.

SUMMARYFungal infection stimulates the canonical C-type lectin receptors (CLRs) signaling pathway via Syk activation. Here we show that SHP-2 plays a crucial role in mediating CLRs-induced Syk activation. Genetic ablation of Shp-2 (Ptpn11) in dendritic cells (DCs) and macrophages impaired Syk-mediated signaling and abrogated pro-inflammatory gene expression following fungal stimulation. Mechanistically, SHP-2 operates as a scaffold facilitating the recruitment of Syk to dectin-1 or FcRγ, through its N-SH2 domain and a previously unrecognized C-terminal ITAM motif. We demonstrate that DC-derived SHP-2 is crucial for the induction of IL-1β, IL-6 and IL-23, and anti-fungal TH17 cell responses to control Candida albicans infection. Together, these data reveal a mechanism by which SHP-2 mediates Syk activation in response to fungal infections

Shp2 regulates leukemic stem cell frequency in MLL-rearranged acute myeloid leukemia.

Shp2 is a non-receptor protein tyrosine phosphatase (PTP) encoded by the PTPN11 gene. It contains two SH2 domains, a PTP domain, and a carboxyl-terminal region with tyrosine phosphorylation sites. The Shp2 PTP activity is autoinhibited by its N-SH2 domain. Shp2 is activated by docking to specific tyrosine phosphorylated sites via SH2 domains or by gain-of-function (GOF) mutations that disrupt the autoinhibition. GOF Shp2 mutations are most frequently detected in juvenile myelomonocytic leukemia (JMML), representing 35% of cases. Animal model studies using conditional knockin of GOF Shp2 E76K or Shp2 D61G in the hematopoietic compartments have shown that these Shp2 mutants cause JMML-like myeloproliferative disease (MPD), albeit after ~6 months of latency (1,2).

Changing the topology of protein backbone: the effect of backbone cyclization on the structure and dynamics of a SH3 domain.

Understanding of the effects of the backbone cyclization on the structure and dynamics of a protein is essential for using protein topology engineering to alter protein stability and function. Here we have determined, for the first time, the structure and dynamics of the linear and various circular constructs of the N-SH3 domain from protein c-Crk. These constructs differ in the length and amino acid composition of the cyclization region. The backbone cyclization was carried out using intein-mediated intramolecular chemical ligation between the juxtaposed N- and the C-termini. The structure and backbone dynamics studies were performed using solution NMR. Our data suggest that the backbone cyclization has little effect on the overall three-dimensional structure of the SH3 domain: besides the termini, only minor structural changes were found in the proximity of the cyclization region. In contrast to the structure, backbone dynamics are significantly affected by the cyclization. On the subnanosecond time scale, the backbone of all circular constructs on average appears more rigid than that of the linear SH3 domain; this effect is observed over the entire backbone and is not limited to the cyclization site. The backbone mobility of the circular constructs becomes less restricted with increasing length of the circularization loop. In addition, significant conformational exchange motions (on the sub-millisecond time scale) were found in the N-Src loop and in the adjacent β-strands in all circular constructs studied in this work. These effects of backbone cyclization on protein dynamics have potential implications for the stability of the protein fold and for ligand binding.

Protein-protein interaction between caveolin-1 and SHP-2 is dependent on the N-SH2 domain of SHP-2.

Src homology 2-containing protein tyrosine phosphatase 2 (SHP-2) is known to protect neurons from neurodegeneration during ischemia/reperfusion injury. We recently reported that ROS-mediated oxidative stress promotes phosphorylation of endogenous SHP-2 in astrocytes and complex formation between caveolin-1 and SHP-2 in response to oxidative stress. To examine the region of SHP-2 participating in complex formation with caveolin-1, we generated three deletion mutant constructs and six point mutation constructs of SHP-2. Compared with wild-type SHP-2, binding of the N-SH2 domain deletion mutant of SHP-2 to p-caveolin-1 was reduced greatly, using flow cytometric competitive binding assays and surface plasmon resonance (SPR). Moreover, deletion of the N-SH2 domain of SHP-2 affected H2O2-mediated ERK phosphorylation and Src phosphorylation at Tyr 419 in primary astrocytes, suggesting that N-SH2 domain of SHP-2 is responsible for the binding of caveolin-1 and contributes to the regulation of Src phosphorylation and activation following ROS-induced oxidative stress in brain astrocytes. [BMB Reports 2015; 48(3): 184-189]