N-terminal SH3 Domain Research Articles

Crystal structures of p120RasGAP N-terminal SH2 domain in its apo form and in complex with a p190RhoGAP phosphotyrosine peptide.

The Rho and Ras pathways play vital roles in cell growth, division and motility. Cross-talk between the pathways amplifies their roles in cell proliferation and motility and its dysregulation is involved in disease pathogenesis. One important interaction for cross-talk occurs between p120RasGAP (RASA1), a GTPase activating protein (GAP) for Ras, and p190RhoGAP (p190RhoGAP-A, ARHGAP35), a GAP for Rho. The binding of these proteins is primarily mediated by two SH2 domains within p120RasGAP engaging phosphorylated tyrosines of p190RhoGAP, of which the best studied is pTyr-1105. To better understand the interaction between p120RasGAP and p190RhoGAP, we determined the 1.75 Å X-ray crystal structure of the N-terminal SH2 domain of p120RasGAP in the unliganded form, and its 1.6 Å co-crystal structure in complex with a synthesized phosphotyrosine peptide, EEENI(p-Tyr)SVPHDST, corresponding to residues 1100–1112 of p190RhoGAP. We find that the N-terminal SH2 domain of p120RhoGAP has the characteristic SH2 fold encompassing a central beta-sheet flanked by two alpha-helices, and that peptide binding stabilizes specific conformations of the βE-βF loop and arginine residues R212 and R231. Site-directed mutagenesis and native gel shifts confirm phosphotyrosine binding through the conserved FLVR motif arginine residue R207, and isothermal titration calorimetry finds a dissociation constant of 0.3 ± 0.1 μM between the phosphopeptide and SH2 domain. These results demonstrate that the major interaction between two important GAP proteins, p120RasGAP and p190RhoGAP, is mediated by a canonical SH2-pTyr interaction.

Molecular Dynamics model of peptide-protein conjugation: case study of covalent complex between Sos1 peptide and N-terminal SH3 domain from Grb2

We have investigated covalent conjugation of VPPPVPPRRRX′ peptide (where X′ denotes Nε-chloroacetyl lysine) to N-terminal SH3 domain from adapter protein Grb2. Our experimental results confirmed that the peptide first binds to the SH3 domain noncovalently before establishing a covalent linkage through reaction of X′ with the target cysteine residue C32. We have also confirmed that this reaction involves a thiolate-anion form of C32 and follows the SN2 mechanism. For this system, we have developed a new MD-based protocol to model the formation of covalent conjugate. The simulation starts with the known coordinates of the noncovalent complex. When two reactive groups come into contact during the course of the simulation, the reaction is initiated. The reaction is modeled via gradual interpolation between the two sets of force field parameters that are representative of the noncovalent and covalent complexes. The simulation proceeds smoothly, with no appreciable perturbations to temperature, pressure or volume, and results in a high-quality MD model of the covalent complex. The validity of this model is confirmed using the experimental chemical shift data. The new MD-based approach offers a valuable tool to explore the mechanics of protein-peptide conjugation and build accurate models of covalent complexes.

SPIN90, an adaptor protein, alters the proximity between Rab5 and Gapex5 and facilitates Rab5 activation during EGF endocytosis

During ligand-mediated receptor endocytosis, the small GTPase Rab5 functions in vesicle fusion and trafficking. Rab5 activation is known to require interactions with its guanine nucleotide-exchange factors (GEFs); however, the mechanism regulating Rab5 interactions with GEFs remains unclear. Here, we show that the SH3-adapter protein SPIN90 participates in the activation of Rab5 through the recruitment of both Rab5 and its GEF, Gapex5, to endosomal membranes during epidermal growth factor (EGF)-mediated endocytosis. SPIN90 strongly interacts with the inactive Rab5/GDI2 complex through its C-terminus. In response to EGF signaling, extracellular signal-regulated kinase (ERK)-mediated phosphorylation of SPIN90 at Thr-242 enables SPIN90 to bind Gapex5 through its N-terminal SH3 domain. Gapex5 is a determinant of Rab5 membrane targeting, while SPIN90 mediates the interaction between Gapex5 and Rab5 in a phosphorylation-dependent manner. Collectively, our findings suggest that SPIN90, as an adaptor protein, simultaneously binds inactive Rab5 and Gapex5, thereby altering their spatial proximity and facilitating Rab5 activation.

1880-P: The Protein-Tyrosine Phosphatase Shp1 Interacts with PPAR Gamma and Regulates Its Transcriptional Activity through Dephosphorylation of Selected Tyrosine Sites

Src homology region 2 domain-containing phosphatase-1 (Shp1) plays an important role in modulating insulin signaling and thereby glucose homeostasis. Our laboratory has demonstrated that despite being more prone to hepatic steatosis, mice with a liver specific Shp1 knock out (Shp1H-KO) on high-fat diet (HFD) exhibit improved fasting glycaemia, reduced insulin resistance and lower inflammation than their flox/flox littermates. Microarray analysis of HFD-fed Shp1H-KO versus Shp1fl/fl livers revealed elevated levels of peroxisome proliferator-activated receptor gamma (PPARγ), an important transcription factor controlling adipogenesis. In addition, overexpression of Shp1 decreased PPARγ activity in vitro. The aim of this study was to investigate the mechanism of regulation of PPARγ by Shp1. Molecular modeling analyses suggested that Shp1 interaction with PPARγ depends on the tyrosine phosphorylation status of PPARγ. We found, by co-immunoprecipitation, that Shp1 N-terminal SH2-domains binds to PPARγ. We confirmed PPARγ tyrosine-phosphorylation and demonstrated that this phosphorylation could be reduced by Shp1 in vitro. Using mass spectrometry, we identified 5 phosphorylated tyrosine residues in PPARγ. Mutagenesis experiments showed that PPARγ significantly lost its activity when two of these sites were selectively and independently mutated to the non-phosphorylatable phenylalanine but did not interfere with the nuclear localization of PPARγ. Additionally, we generated Shp1 knockout HepG2 cells using CRISPR-Cas9 technology. We found that expression of several PPARγ regulated transcripts was upregulated in Shp1-KO cells as compared to control cells. Altogether, we show that PPARγ is a novel substrate of Shp1. Our results reveal a fine regulation of PPARγ tyrosine phosphorylation by Shp1, which might lead to novel therapeutic approaches for treating metabolic disorders related to obesity. Disclosure A. Kumar: None. M. Lavallee-Bourget: None. M. Forest: None. M. Schwab: None. K. Bellmann: None. V. Houde: None. H. Maaroufi: None. N. Beauchemin: None. A. Marette: Advisory Panel; Self; Plexus, Valbiotis. Consultant; Self; Danone Nutricia Research.

SUN-007 Discovery of Novel Signaling Pathways Downstream of PTK6 in TNBC Models

Triple negative breast cancer (TNBC) patients have a worse prognosis relative to other breast cancer subtypes. The glucocorticoid receptor (GR) is a ubiquitous steroid receptor that mediates homeostatic responses to life-induced stressors via glucocorticoids synthesized in the adrenal cortex. High GR expression predicts poor outcome in TNBC. The molecular mechanisms for how stress and GR contribute to TNBC progression are largely unknown. We previously described overexpression of protein tyrosine kinase 6 (PTK6) in response to both cellular and endocrine stress, coordinated by GR/HIF complexes in TNBC models. PTK6-induced signaling further amplified phosphorylation of GR on Ser134. PTK6 knock-down impaired TNBC motility, and blocked outgrowth of cancer stem-like cells (CSC) as measured by tumorsphere assays. Furthermore, PTK6 expression was integral for cancer cell survival upon chemotherapy treatment (Taxol, 5-FU). Interestingly, kinase activity of PTK6 was not required for TNBC cell motility or CSC outgrowth. This finding is of critical importance for drug design, given that previous efforts have primarily focused on targeting the PTK6 kinase domain. To probe the mechanisms of PTK6 signaling, we have created kinase-intact domain structure mutants of PTK6 via in frame deletions of the N-terminal SH3 or SH2 domain. MDA-MB-231 cells expressing PTK6 which lacks an SH2 domain (SH2-del PTK6) exhibited increased formation of tumorspheres relative to wt or kinase-dead (KM) full-length PTK6. Surprisingly, SH2-del PTK6+ cells were strikingly less responsive to both serum- and growth factor-stimulated cell motility relative to wt or KM controls. To track signal transduction pathways activated in TNBC cells harboring PTK6 domain mutants (wt, KM, SH2-del PTK6, PTK6 null), we used Reverse Phase Protein Array (RPPA). Our data suggests that the SH2 domain of PTK6 mediates TNBC motility via activation of the Rho and/or AhR signaling pathways, while CSC outgrowth primarily occurs via activation of the p38 and/or canonical Wnt signaling pathways. Together, these studies nominate PTK6 as an effector of advanced cancer phenotypes in GR+ TNBC cells and identify novel therapeutic targets (Rho, AhR) for treatment of TNBC tumors. Additionally, our work defines PTK6 domain-specific functions and reveals new opportunities for pharmacologically targeting the PTK6 SH2-domain in TNBC.

Mutation p.R356Q in the Collybistin Phosphoinositide Binding Site Is Associated With Mild Intellectual Disability.

The recruitment of inhibitory GABAA receptors to neuronal synapses requires a complex interplay between receptors, neuroligins, the scaffolding protein gephyrin and the GDP-GTP exchange factor collybistin (CB). Collybistin is regulated by protein-protein interactions at the N-terminal SH3 domain, which can bind neuroligins 2/4 and the GABAAR α2 subunit. Collybistin also harbors a RhoGEF domain which mediates interactions with gephyrin and catalyzes GDP-GTP exchange on Cdc42. Lastly, collybistin has a pleckstrin homology (PH) domain, which binds phosphoinositides, such as phosphatidylinositol 3-phosphate (PI3P/PtdIns3P) and phosphatidylinositol 4-monophosphate (PI4P/PtdIns4P). PI3P located in early/sorting endosomes has recently been shown to regulate the postsynaptic clustering of gephyrin and GABAA receptors and consequently the strength of inhibitory synapses in cultured hippocampal neurons. This process is disrupted by mutations in the collybistin gene (ARHGEF9), which cause X-linked intellectual disability (XLID) by a variety of mechanisms converging on disrupted gephyrin and GABAA receptor clustering at central synapses. Here we report a novel missense mutation (chrX:62875607C>T, p.R356Q) in ARHGEF9 that affects one of the two paired arginine residues in the PH domain that were predicted to be vital for binding phosphoinositides. Functional assays revealed that recombinant collybistin CB3SH3-R356Q was deficient in PI3P binding and was not able to translocate EGFP-gephyrin to submembrane microaggregates in an in vitro clustering assay. Expression of the PI3P-binding mutants CB3SH3-R356Q and CB3SH3-R356N/R357N in cultured hippocampal neurones revealed that the mutant proteins did not accumulate at inhibitory synapses, but instead resulted in a clear decrease in the overall number of synaptic gephyrin clusters compared to controls. Molecular dynamics simulations suggest that the p.R356Q substitution influences PI3P binding by altering the range of structural conformations adopted by collybistin. Taken together, these results suggest that the p.R356Q mutation in ARHGEF9 is the underlying cause of XLID in the probands, disrupting gephyrin clustering at inhibitory GABAergic synapses via loss of collybistin PH domain phosphoinositide binding.

EGF Stimulates Rab35 Activation and Gastric Cancer Cell Migration by Regulating DENND1A-Grb2 Complex Formation

Aims: The aim of this study was to reveal the specific molecular mechanisms by which DENND1A accepts EGF signaling and activates Rab35 in gastric cancer.Methods: The expression of proteins related to DENND1A was examined by western blot analysis. Activation of Rab35 was assessed by GST-pulldown. The interaction of DENND1A and Grb2 was assessed by GST-pulldown and co-immunoprecipitation assays. The relationship between DENND1A and cell migration and invasion was detected using wound healing and transwell by gene overexpression and RNA interference.Results: EGF stimulation significantly promoted cell migration, whereas transfection with siRab35 partially inhibited EGF-promoted cell migration. DENND1A is also involved in these processes and active Rab35. Moreover, DENND1A binds to the N-terminal and C-terminal SH3 domains of Grb2 through PRD. Of special interest is the observation that EGFR can recruit Grb2-DENND1A complex under EGF stimulation. Further results reveal that the higher the expression of DENND1A, the shorter progression-free survival of gastric cancer patients.Conclusion: In summary, we confirmed that EGF-Grb2-DENND1A-Rab35 signaling pathway with the interaction of DENND1A and Grb2 as a regulatory center could regulate gastric cancer cell migration and invasion. Ultimately, the expression level of DENND1A predicts the survival status of gastric cancer patients and may become one of the important targets for the treatment of gastric cancer.

Mechanism of Folding and Binding of the N-Terminal SH2 Domain from SHP2.

SHP2 is a phosphatase protein, involved in many cellular pathways, comprising two SH2 domains (namely N-SH2 and C-SH2) and a phosphatase domain. Among others, the interaction between SHP2 and Gab2 (Grb2 associated binder) is critical in cell death and differentiation. SHP2 binds to Gab2 through its SH2 domains, which recognize specific regions of Gab2 characterized by the presence of a phosphorylated tyrosine. In order to shed light on the dynamic and functional properties of this protein-protein interaction, we studied the mechanism of folding of N-SH2 and the binding process to a peptide mimicking a region of Gab2. The data presented represent the first description by stopped-flow of the kinetics of binding of an SH2 domain in solution. By performing experiments at different ionic strengths, we elucidate the electrostatic nature of the interaction, highlighting a key role of the negative charge of the phosphotyrosine in the recognition event of the reaction. Furthermore, by analyzing the equilibrium and kinetics of folding of N-SH2 folding we demonstrate the presence of an intermediate along the folding pathway. These results are discussed in the light of previous works on another SH2 domain.

Abstract 3492: Wnt5a induces ROR1 to associate with DOCK1 and promote growth of breast cancer cells

Abstract Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncoembryonic protein expressed on breast cancer, but not on virtually all normal adult tissues. Studies have found that high-level expression of ROR1 in tumors of breast cancer patients associated with enhanced cell growth, aggressive disease, and shorter overall survival compared to that of patients with tumors that had low expression of ROR1 (Zhang S., et al., PLOS ONE, 7(3): e31127, 2012). Analysis of ROR1-immune precipitates of primary breast cancer patient-derived-xenograft (PDX) lysates via immunoblot analyses revealed that ROR1 was associated with DOCK1 (Dedicator of Cytokinesis 1, also known as DOCK180) in breast cancer PDX tumors. DOCK1 is a member of the DOCK-A subfamily of guanine exchange factors (GEFs) specific for Rac1 that is expressed in breast cancer PDX tumors or cell lines. DOCK1 also contains a N-terminal SH3 domain. We found that ROR1 dissociated from DOCK1 in PDX cells cultured in serum-free medium, unless the cells were stimulated with exogenous Wnt5a. Wnt5a could induce ROR1 to complex with DOCK1 and cause activation of Rac1; this effect could be inhibited by silencing DOCK1 in PDX cells. Furthermore, these effects of Wnt5a on PDX cells also could be blocked by treatment of the PDX cells with cirmtuzumab, a humanized anti-ROR1 mAb, which is undergoing clinical evaluation in patients with cancer. We corroborated these findings using the breast cancer cell-line MCF7, which also expresses DOCK1, but does not express ROR1. MCF7 cells can be transduced to express ROR1 or various mutant forms of ROR1, allowing us to examine the structure-function relationships required for ROR1-DOCK1 interactions. We confirmed that DOCK1 complexes with ROR1 in response to Wnt5a in MCF7-ROR1 cells. We find that silencing DOCK1 specifically impaired the capacity of Wnt5a to enhance growth of MCF7-ROR1 cells in vitro. We generated truncated forms of ROR1 and found the cytoplastmic proline-rich domain (PRD) of ROR1 was required for ROR1 to complex with DOCK1 and activate Rac1 upon stimulation with Wnt5a. We next introduced single amino-acid substitutions of proline (P) to alanine (A) in the ROR1-PRD at positions 784, 808, 826, or 841 in potential SH3-binding sites. In contrast to wild-type ROR1, or other ROR1P->A mutants, ROR1P(808)A had impaired capacity to recruit DOCK1 to ROR1 in response to Wnt5a. Moreover, unlike MCF7 cells transfected with wild-type ROR1 or ROR1 with P->A substitutions at positions 784, 826, or 841, MCF7 cells transfected to express ROR1P(808)A did not have a growth advantage over that of MCF7 cells that do not express ROR1. This study reveals that the recruitment of DOCK1 may be critical for the capacity of Wnt5a to enhance breast cancer cell growth, which may contribute to the observed increased tendency for disease progression in breast cancer patients who have tumors that express high-levels of ROR1. Citation Format: Md Kamrul Hasan, Victoria Tripple, George F. Widhopf, Suping Zhang, Barbara A. Parker, Thomas J. Kipps. Wnt5a induces ROR1 to associate with DOCK1 and promote growth of breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3492.

NEDD9 stimulated MMP9 secretion is required for invadopodia formation in oral squamous cell carcinoma.

Neural precursor cell expressed developmentally downregulated 9 (NEDD9) is a component of the metastatic signatures of melanoma, breast cancer, glioblastoma, lung cancer and head and neck squamous cell carcinoma (HNSCC). Here we tested the efficacy of NEDD9's domains in stimulating matrix metalloproteinase (MMP) secretion and invadopodia formation in cells stably expressing various NEDD9 mutants. Replacement of the 13 YxxP motif substrate domain (SD) tyrosines and the C-terminal Y629 with phenylalanines (F14NEDD9) eliminated tyrosine phosphorylation, MMP9 secretion and loss of invadopodia formation. Mutation of the N-terminal SH3 domain Y12 to glutamic acid (Y12ENEDD9) or phenylalanine (Y12FNEDD9) reduced MMP9 secretion and inhibited invadopodia formation. SH3 domain deletion (∆SH3NEDD9) resulted in the loss of MMP9 secretion and a lack of invadopodia formation. The SH3–SD domain (SSNEDD9) construct exhibited tyrosine phosphorylation and stimulated MMP9 secretion, as did ∆CTNEDD9 which lacked the C-terminus (∆C-terminal; ∆CT). E13NEDD9 expression blocked MMP9 secretion and invadopodia formation. MICAL1 (molecule interacting with Cas-L1) silencing with a short hairpin RNA reduced MMP9 secretion, vimentin and E-cadherin levels while increasing N-cadherin and Rab6 levels, consistent with reduced invasive behavior. These findings indicate that NEDD9 SD phosphorylation and SH3 domain interactions are necessary for increasing MMP9 secretion and invadopodia formation.

EGFR-Phosphorylated Platelet Isoform of Phosphofructokinase 1 Promotes PI3K Activation

EGFR activates phosphatidylinositide 3-kinase (PI3K), but the mechanism underlying this activation is not completely understood. We demonstrated here that EGFR activation resulted in lysine acetyltransferase 5 (KAT5)-mediated K395 acetylation of the platelet isoform of phosphofructokinase 1 (PFKP) and subsequent translocation of PFKP to the plasma membrane, where the PFKP was phosphorylated at Y64 by EGFR. Phosphorylated PFKP binds to the N-terminal SH2 domain of p85α, which is distinct from binding of Gab1 to the C-terminal SH2 domain of p85α, and recruited p85α to the plasma membrane resulting in PI3K activation. PI3K-dependent AKT activation results in enhanced phosphofructokinase 2 (PFK2) phosphorylation and production of fructose-2,6-bisphosphate, which inturn promotes PFK1 activation. PFKP Y64 phosphorylation-enhanced PI3K/AKT-dependent PFK1 activation and GLUT1 expression promoted the Warburg effect, tumor cell proliferation, and brain tumorigenesis. These findings underscore the instrumental role of PFKP in PI3K activation and enhanced glycolysis through PI3K/AKT-dependent positive-feedback regulation.

An Allosteric Mechanism of Abl Kinase Activation and Catalysis

c-Abl kinase plays a critical role in coordinating responses to growth factors, cytokines, cell motility, DNA damage responses and oxidative stress. Bcr-Abl, a fusion oncoprotein and the genetic basis of chronic myeloid leukemia (CML) is constitutively active and despite intense research the basis for this activation remains unclear. c-Abl activity is regulated by the N-terminal SH3 and SH2 domains working in concert to lock the kinase domain in an inhibited conformation. However, mechanistic insight into how regulated inhibition and activation is achieved is missing. Moreover, the effect of the regulatory domain on the conformation of the kinase domain has not been elucidated. Using high resolution NMR and other biophysical techniques we characterize allosteric mechanism of regulation of Abl kinase and identify a dynamic role of the regulatory module in the activation process.

Dual Allosteric Inhibition of SHP2 Phosphatase.

SHP2 is a cytoplasmic protein tyrosine phosphatase encoded by the PTPN11 gene and is involved in cell proliferation, differentiation, and survival. Recently, we reported an allosteric mechanism of inhibition that stabilizes the auto-inhibited conformation of SHP2. SHP099 (1) was identified and characterized as a moderately potent, orally bioavailable, allosteric small molecule inhibitor, which binds to a tunnel-like pocket formed by the confluence of three domains of SHP2. In this report, we describe further screening strategies that enabled the identification of a second, distinct small molecule allosteric site. SHP244 (2) was identified as a weak inhibitor of SHP2 with modest thermal stabilization of the enzyme. X-ray crystallography revealed that 2 binds and stabilizes the inactive, closed conformation of SHP2, at a distinct, previously unexplored binding site-a cleft formed at the interface of the N-terminal SH2 and PTP domains. Derivatization of 2 using structure-based design resulted in an increase in SHP2 thermal stabilization, biochemical inhibition, and subsequent MAPK pathway modulation. Downregulation of DUSP6 mRNA, a downstream MAPK pathway marker, was observed in KYSE-520 cancer cells. Remarkably, simultaneous occupation of both allosteric sites by 1 and 2 was possible, as characterized by cooperative biochemical inhibition experiments and X-ray crystallography. Combining an allosteric site 1 inhibitor with an allosteric site 2 inhibitor led to enhanced pharmacological pathway inhibition in cells. This work illustrates a rare example of dual allosteric targeted protein inhibition, demonstrates screening methodology and tactics to identify allosteric inhibitors, and enables further interrogation of SHP2 in cancer and related pathologies.

Allosteric Inhibitors of SHP2 with Therapeutic Potential for Cancer Treatment.

SHP2, a cytoplasmic protein-tyrosine phosphatase encoded by the PTPN11 gene, is involved in multiple cell signaling processes including Ras/MAPK and Hippo/YAP pathways. SHP2 has been shown to contribute to the progression of a number of cancer types including leukemia, gastric, and breast cancers. It also regulates T-cell activation by interacting with inhibitory immune checkpoint receptors such as the programmed cell death 1 (PD-1) and B- and T-lymphocyte attenuator (BTLA). Thus, SHP2 inhibitors have drawn great attention by both inhibiting tumor cell proliferation and activating T cell immune responses toward cancer cells. In this study, we report the identification of an allosteric SHP2 inhibitor 1-(4-(6-bromonaphthalen-2-yl)thiazol-2-yl)-4-methylpiperidin-4-amine (23) that locks SHP2 in a closed conformation by binding to the interface of the N-terminal SH2, C-terminal SH2, and phosphatase domains. Compound 23 suppresses MAPK signaling pathway and YAP transcriptional activity and shows antitumor activity in vivo. The results indicate that allosteric inhibition of SHP2 could be a feasible approach for cancer therapy.

Structural and functional insights into the interaction between the Cas family scaffolding protein p130Cas and the focal adhesion-associated protein paxillin

The Cas family scaffolding protein p130Cas is a Src substrate localized in focal adhesions (FAs) and functions in integrin signaling to promote cell motility, invasion, proliferation, and survival. p130Cas targeting to FAs is essential for its tyrosine phosphorylation and downstream signaling. Although the N-terminal SH3 domain is important for p130Cas localization, it has also been reported that the C-terminal region is involved in p130Cas FA targeting. The C-terminal region of p130Cas or Cas family homology domain (CCHD) has been reported to adopt a structure similar to that of the focal adhesion kinase C-terminal focal adhesion-targeting domain. The mechanism by which the CCHD promotes FA targeting of p130Cas, however, remains unclear. In this study, using a calorimetry approach, we identified the first LD motif (LD1) of the FA-associated protein paxillin as the binding partner of the p130Cas CCHD (in a 1:1 stoichiometry with a Kd ∼4.2 μm) and elucidated the structure of the p130Cas CCHD in complex with the paxillin LD1 motif by X-ray crystallography. Of note, a comparison of the CCHD/LD1 complex with a previously solved structure of CCHD in complex with the SH2-containing protein NSP3 revealed that LD1 had almost identical positioning of key hydrophobic and acidic residues relative to NSP3. Because paxillin is one of the key scaffold molecules in FAs, we propose that the interaction between the p130Cas CCHD and the LD1 motif of paxillin plays an important role in p130Cas FA targeting.

Abstract 2084: Conformational activation and allosteric inhibition of SHP2 in RTK-driven cancers

Abstract The non-receptor protein tyrosine phosphatase (PTP) SHP2 is an important component of RTK signaling in response to growth factor stimulus and sits just upstream of the RAS-MAPK signaling cascade. The first oncogenic phosphatase to be identified, SHP2 is dysregulated in multiple human diseases including the developmental disorders Noonan and Leopard syndromes, as well as leukemia, lung cancer and neuroblastoma where aberrant activity of SHP2 leads to uncontrolled MAPK signaling. Cancer-associated activating mutations in SHP2 impart an “auto-on” state of the enzyme, boosting basal activity by shifting the equilibrium away from the auto-inhibited state. Reduction of SHP2 activity through genetic knockdown suppresses tumor growth, validating SHP2 as a target for cancer therapy. SHP099, a recently reported potent and selective allosteric inhibitor of SHP2, stabilizes the auto-inhibited form of SHP2 through interactions with the N-terminal SH2 and C-terminal PTP domains of the protein. SHP099 suppresses MAPK signaling in RTK amplified cancers resulting in suppressed proliferation in vitro and inhibition of tumor growth in mouse tumor xenograft models. Together, these data demonstrate the therapeutic potential of SHP2 inhibition in the treatment of cancer and other RAS/MAPK-linked diseases. Citation Format: Michael G. Acker, Ying-Nan P. Chen, Matthew J. LaMarche, Ho Man Chan, Peter Fekkes, Jorge Garcia-Fortanet, Jonathan R. LaRochelle, Brandon Antonakos, Christine Hiu-Tung Chen, Zhuoliang Chen, Vesselina G. Cooke, Jason R. Dobson, Zhan Deng, Fei Feng, Brant Firestone, Michelle Fodor, Cary Fridrich, Hui Gao, Huai-Xiang Hao, Jaison Jacob, Samuel Ho, Kathy Hsiao, Zhao B. Kang, Rajesh Karki, Mitsunori Kato, Jay Larrow, Laura R. La Bonte, Gang Liu, Shumei Liu, Dyuti Majumdar, Matthew J. Meyer, Mark Palermo, Minying Pu, Edmund Price, Subarna Shakya, Michael D. Shultz, Kavitha Venkatesan, Ping Wang, Markus Warmuth, Sarah Williams, Guizhi Yang, Jing Yuan, Ji-Hu Zhang, Ping Zhu, Stephen C. Blacklow, Timothy Ramsey, Nicholas J. Keen, William R. Sellers, Travis Stams, Pascal D. Fortin. Conformational activation and allosteric inhibition of SHP2 in RTK-driven cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2084. doi:10.1158/1538-7445.AM2017-2084

Cosolutes, Crowding, and Protein Folding Kinetics.

Long accepted as the most important interaction, recent work shows that steric repulsions alone cannot explain the effects of macromolecular cosolutes on the equilibrium thermodynamics of protein stability. Instead, chemical interactions have been shown to modulate, and even dominate, crowding-induced steric repulsions. Here, we use 19F NMR to examine the effects of small and large cosolutes on the kinetics of protein folding and unfolding using the metastable 7 kDa N-terminal SH3 domain of the Drosophila signaling protein drk (SH3), which folds by a two-state mechanism. The small cosolutes consist of trimethylamine N-oxide and sucrose, which increase equilibrium protein stability, and urea, which destabilizes proteins. The macromolecules comprise the stabilizing sucrose polymer, Ficoll, and the destabilizing globular protein, lysozyme. We assessed the effects of these cosolutes on the differences in free energy between the folded state and the transition state and between the unfolded ensemble and the transition state. We then examined the temperature dependence to assess changes in activation enthalpy and entropy. The enthalpically mediated effects are more complicated than suggested by equilibrium measurements. We also observed enthalpic effects with the supposedly inert sucrose polymer, Ficoll, that arise from its macromolecular nature. Assessment of activation entropies shows important contributions from solvent and cosolute, in addition to the configurational entropy of the protein that, again, cannot be gleaned from equilibrium data. Comparing the effects of Ficoll to those of the more physiologically relevant cosolute lysozyme reveals that synthetic polymers are not appropriate models for understanding the kinetics of protein folding in cells.

Grb2 carboxyl-terminal SH3 domain can bivalently associate with two ligands, in an SH3 dependent manner

Src homology domain containing leukocyte protein of 65 kDa (SLP65), the growth factor receptor binding protein 2 (Grb2), and the guanine nucleotide exchange factor for the Rho family GTPases (Vav), self associate in unstimulated B cells as components of the preformed B cell receptor transducer module, in an SH3-dependent manner. The complex enables the B cell to promptly respond to BCR aggregation, resulting in signal amplification. It also facilitates Vav translocation to the membrane rafts, for activation. Here we uncover the molecular mechanism by which the complex may be formed in the B cell. The C-terminal SH3 domain (SH3C) of Grb2 bivalently interacts with the atypical non-PxxP proline rich region of SLP65, and the N-terminal SH3 domain (SH3N) of Vav, both the interactions crucial for the proper functioning of the B cell. Most surprisingly, the two ligands bind the same ligand binding site on the surface of Grb2 SH3C. Addition of SLP65 peptide to the Grb2-Vav complex abrogates the interaction completely, displacing Vav. However, the addition of Vav SH3N to the SLP65-Grb2 binary complex, results in a trimeric complex. Extrapolating these results to the in vivo conditions, Grb2 should bind the SLP65 transducer module first, and then Vav should associate.

The Janus Kinase (JAK) FERM and SH2 Domains: Bringing Specificity to JAK-Receptor Interactions.

The Janus kinases (JAKs) are non-receptor tyrosine kinases essential for signaling in response to cytokines and interferons and thereby control many essential functions in growth, development, and immune regulation. JAKs are unique among tyrosine kinases for their constitutive yet non-covalent association with class I and II cytokine receptors, which upon cytokine binding bring together two JAKs to create an active signaling complex. JAK association with cytokine receptors is facilitated by N-terminal FERM and SH2 domains, both of which are classical mediators of peptide interactions. Together, the JAK FERM and SH2 domains mediate a bipartite interaction with two distinct receptor peptide motifs, the proline-rich “Box1” and hydrophobic “Box2,” which are present in the intracellular domain of cytokine receptors. While the general sidechain chemistry of Box1 and Box2 peptides is conserved between receptors, they share very weak primary sequence homology, making it impossible to posit why certain JAKs preferentially interact with and signal through specific subsets of cytokine receptors. Here, we review the structure and function of the JAK FERM and SH2 domains in light of several recent studies that reveal their atomic structure and elucidate interaction mechanisms with both the Box1 and Box2 receptor motifs. These crystal structures demonstrate how evolution has repurposed the JAK FERM and SH2 domains into a receptor-binding module that facilitates interactions with multiple receptors possessing diverse primary sequences.

The Molecular Mechanisms Underlying the Hijack of Host Proteins by the 1918 Spanish Influenza Virus.

The 1918 Spanish influenza A virus (IAV) caused one of the most serious pandemics in history. The nonstructural protein 1 (NS1) of the 1918 IAV hijacks the interaction between human CrkII and JNK1. Little is, however, known about its molecular mechanism. Here, we performed X-ray crystallography, NMR relaxation dispersion experiment, and fluorescence spectroscopy to determine the structural, kinetic, and thermodynamic mechanisms underlying the hijacking of CrkII by 1918 IAV NS1. We observed that the interaction between a proline-rich motif in NS1 and the N-terminal SH3 domain of CrkII displays strikingly rapid kinetics and exceptionally high affinity with 100-fold faster kon and 3300-fold lower Kd compared to those for the CrkII-JNK1 interaction. These results provide molecular insight into the mechanism by which 1918 IAV NS1 hijacks CrkII and disrupts its interactions with critical cellular signaling proteins.