CCDC6-RET fusion protein regulates Ras/MAPK signaling through the fusion- GRB2-SHC1 signal niche.

Rearranged during transfection (RET) rearrangement oncoprotein-mediated Ras/MAPK signaling cascade is constitutively activated in cancers. Here, we demonstrate a unique signal niche. The niche is a ternary complex based on the chimeric RET liquid-liquid phase separation. The complex comprises the rearranged kinase (RET fusion); the adaptor (GRB2), and the effector (SHC1). Together, they orchestrate the Ras/MAPK signal cascade, which is dependent on tyrosine kinase. CCDC6-RET fusion undergoes LLPS requiring its kinase domain and its fusion partner. The CCDC6-RET fusion LLPS promotes the autophosphorylation of RET fusion, with enhanced kinase activity, which is necessary for the formation of the signaling niche. Within the signal niche, the interactions among the constituent components are reinforced, and the signal transduction efficiency is amplified. The specific RET fusion-related signal niche elucidates the mechanism of the constitutive activation of the Ras/MAPK signaling pathway. Beyond just focusing on RET fusion itself, exploration of the ternary complex potentially unveils a promising avenue for devising therapeutic strategies aimed at treating RET fusion-driven diseases.

GRB2 stabilizes RAD51 at reversed replication forks suppressing genomic instability and innate immunity against cancer.

Growth factor receptor-bound protein 2 (GRB2) is a cytoplasmic adapter for tyrosine kinase signaling and a nuclear adapter for homology-directed-DNA repair. Here we find nuclear GRB2 protects DNA at stalled replication forks from MRE11-mediated degradation in the BRCA2 replication fork protection axis. Mechanistically, GRB2 binds and inhibits RAD51 ATPase activity to stabilize RAD51 on stalled replication forks. In GRB2-depleted cells, PARP inhibitor (PARPi) treatment releases DNA fragments from stalled forks into the cytoplasm that activate the cGAS-STING pathway to trigger pro-inflammatory cytokine production. Moreover in a syngeneic mouse metastatic ovarian cancer model, GRB2 depletion in the context of PARPi treatment reduced tumor burden and enabled high survival consistent with immune suppression of cancer growth. Collective findings unveil GRB2 function and mechanism for fork protection in the BRCA2-RAD51-MRE11 axis and suggest GRB2 as a potential therapeutic target and an enabling predictive biomarker for patient selection for PARPi and immunotherapy combination.

GRB2 is a BECN1 interacting protein that regulates autophagy.

GRB2 is an adaptor protein of HER2 (and several other tyrosine kinases), which we identified as a novel BECN1 (Beclin 1) interacting partner. GRB2 co-immunoprecipitated with BECN1 in several breast cancer cell lines and regulates autophagy through a mechanism involving the modulation of the class III PI3Kinase VPS34 activity. In ovo studies in a CAM (Chicken Chorioallantoic Membrane) model indicated that GRB2 knockdown, as well as overexpression of GRB2 loss-of-function mutants (Y52A and S86A-R88A) compromised tumor growth. These differences in tumor growth correlated with differential autophagy activity, indicating that autophagy effects might be related to the effects on tumorigenesis. Our data highlight a novel function of GRB2 as a BECN1 binding protein and a regulator of autophagy.

Functional Classification and Interaction Selectivity Landscape of the Human SH3 Domain Superfamily.

SRC homology 3 (SH3) domains are critical interaction modules that orchestrate the assembly of protein complexes involved in diverse biological processes. They facilitate transient protein-protein interactions by selectively interacting with proline-rich motifs (PRMs). A database search revealed 298 SH3 domains in 221 human proteins. Multiple sequence alignment of human SH3 domains is useful for phylogenetic analysis and determination of their selectivity towards PRM-containing peptides (PRPs). However, a more precise functional classification of SH3 domains is achieved by constructing a phylogenetic tree only from PRM-binding residues and using existing SH3 domain-PRP structures and biochemical data to determine the specificity within each of the 10 families for particular PRPs. In addition, the C-terminal proline-rich domain of the RAS activator SOS1 covers 13 of the 14 recognized proline-rich consensus sequence motifs, encompassing differential PRP pattern selectivity among all SH3 families. To evaluate the binding capabilities and affinities, we conducted fluorescence dot blot and polarization experiments using 25 representative SH3 domains and various PRPs derived from SOS1. Our analysis has identified 45 interacting pairs, with binding affinities ranging from 0.2 to 125 micromolar, out of 300 tested and potential new SH3 domain-SOS1 interactions. Furthermore, it establishes a framework to bridge the gap between SH3 and PRP interactions and provides predictive insights into the potential interactions of SH3 domains with PRMs based on sequence specifications. This novel framework has the potential to enhance the understanding of protein networks mediated by SH3 domain-PRM interactions and be utilized as a general approach for other domain-peptide interactions.

Preosteoclast plays a pathogenic role in syndesmophyte formation of ankylosing spondylitis through the secreted PDGFB - GRB2/ERK/RUNX2 pathway.

OBJECTIVES: Ankylosing spondylitis (AS) is a chronic inflammatory disease that mainly affects the sacroiliac joint and spine. However, the real mechanisms of immune cells acting on syndesmophyte formation in AS are not well identified. We aimed to find the key AS-associated cytokine and assess its pathogenic role in AS. METHODS: A protein array with 1000 cytokines was performed in five AS patients with the first diagnosis and five age- and gender-matched healthy controls to discover the differentially expressed cytokines. The candidate differentially expressed cytokines were further quantified by multiplex protein quantitation (3 AS-associated cytokines and 3 PDGF-pathway cytokines) and ELISA (PDGFB) in independent samples (a total of 140 AS patients vs 140 healthy controls). The effects of PDGFB, the candidate cytokine, were examined by using adipose-derived stem cells (ADSCs) and human fetal osteoblast cell line (hFOB1.19) as in vitro mesenchymal cell and preosteoblast models, respectively. Furthermore, whole-transcriptome sequencing and enrichment of phosphorylated peptides were performed by using cell models to explore the underlying mechanisms of PDGFB. The xCELLigence system was applied to examine the proliferation, chemotaxis, and migration abilities of PDGFB-stimulated or PDGFB-unstimulated cells. RESULTS: The PDGF pathway was observed to have abnormal expression in the protein array, and PDGFB expression was further found to be up-regulated in 140 Chinese AS patients. Importantly, PDGFB expression was significantly correlated with BASFI (Pearson coefficient/p value = 0.62/6.70E - 8) and with the variance of the mSASSS score (mSASSS 2 years - baseline, Pearson coefficient/p value = 0.76/8.75E - 10). In AS patients, preosteoclasts secreted more PDGFB than the healthy controls (p value = 1.16E - 2), which could promote ADSCs osteogenesis and enhance collagen synthesis (COLI and COLIII) of osteoblasts (hFOB 1.19). In addition, PDGFB promoted the proliferation, chemotaxis, and migration of ADSCs. Mechanismly, in ADSCs, PDGFB stimulated ERK phosphorylation by upregulating GRB2 expression and then increased the expression of RUNX2 to promote osteoblastogenesis of ADSCs. CONCLUSION: PDGFB stimulates the GRB2/ERK/RUNX2 pathway in ADSCs, promotes osteoblastogenesis of ADSCs, and enhances the extracellular matrix of osteoblasts, which may contribute to pathological bone formation in AS.

Regulation of microRNA expression by the adaptor protein GRB2.

Protein interactions with the microRNA (miRNA)-mediated gene silencing protein Argonaute 2 (AGO2) control miRNA expression. miRNA biogenesis starts with the production of precursor transcripts and culminates with the loading of mature miRNA onto AGO2 by DICER1. Here we reveal an additional component to the regulatory mechanism for miRNA biogenesis involving the adaptor protein, growth factor receptor-bound protein 2 (GRB2). The N-terminal SH3 domain of GRB2 is recruited to the PAZ domain of AGO2 forming a ternary complex containing GRB2, AGO2 and DICER1. Using small-RNA sequencing we identified two groups of miRNAs which are regulated by the binding of GRB2. First, mature and precursor transcripts of mir-17~92 and mir-221 miRNAs are enhanced. Second, mature, but not precursor, let-7 family miRNAs are diminished suggesting that GRB2 directly affects loading of these miRNAs. Notably, the resulting loss of let-7 augments expression of oncogenic targets such as RAS. Thus, a new role for GRB2 is established with implications for cancer pathogenesis through regulation of miRNA biogenesis and oncogene expression.

Grb2-related adaptor protein GRAP is a novel regulator of liver fibrosis.

AIMS: Excessive liver fibrosis is frequently observed in chronic liver diseases and associated with decline of liver functions. Hepatic stellate cells (HSCs) are considered the principal mediator of liver fibrosis by trans-differentiating into myofibroblasts. In the present study we investigated the role of Grb2-related adaptor protein (GRAP) in HSC activation and liver fibrosis. METHODS AND MATERIALS: Liver fibrosis was induced by carbon tetrachloride (CCl4) injection. Gene expression was examined by quantitative PCR. Cell proliferation was evaluated by EdU incorporation. DNA-protein interaction was examined by chromatin immunoprecipitation (ChIP). KEY FINDINGS: GRAP expression was up-regulated during HSC-myofibroblast transition both in vivo and in vitro. Mechanistically, serum response factor (SRF) and myocardin-related transcription factor A (MRTF-A) formed a complex to bind to the GRAP promoter and activate GRAP transcription. Small interfering RNA (siRNA) mediated GRAP silencing blocked HSC-myofibroblast transition in vitro. Importantly, adeno-associated virus 6 (AAV6) mediated GRAP knockdown in myofibroblasts attenuated liver fibrosis in mice. Of note, inhibition of ERK signaling abrogated enhancement of HSC-myofibroblast transition by GRAP over-expression. SIGNIFICANCE: Our data suggest that GRAP, possibly via ERK activation, regulates HSC-myofibroblast transition and contributes to liver fibrosis. Screening for small-molecule GRAP inhibitors may yield novel therapeutic solutions against liver fibrosis.

Targeting the Grb2 cSH3 domain: Design, synthesis and biological evaluation of the first series of modulators.

Growth factor receptor bound protein 2 (Grb2) is an adaptor protein featured by a nSH3-SH2-cSH3 domains. Grb2 finely regulates important cellular pathways such as growth, proliferation and metabolism and a minor lapse of this tight control may totally change the entire pathway to the oncogenic. Indeed, Grb2 is found overexpressed in many tumours type. Consequently, Grb2 is an attractive therapeutic target for the development of new anticancer drug. Herein, we reported the synthesis and the biological evaluation of a series of Grb2 inhibitors, developed starting from a hit-compound already reported by this research unit. The newly synthesized compounds were evaluated by kinetic binding experiments, and the most promising derivatives were assayed in a short panel of cancer cells. Five of the newly synthesized derivatives proved to be able to bind the targeted protein with valuable inhibitory concentration in one-digit micromolar concentration. The most active compound of this series, derivative 12, showed an inhibitory concentration of about 6 µM for glioblastoma and ovarian cancer cells, and an IC50 of 1.67 for lung cancer cell. For derivative 12, the metabolic stability and the ROS production was also evaluated. The biological data together with the docking studies led to rationalize an early structure activity relationship.

GRB2 promotes thymocyte positive selection by facilitating THEMIS-mediated inactivation of SHP1.

The T-lineage restricted protein THEMIS has been shown to play a critical role in T cell development. THEMIS, via its distinctive CABIT domains, inhibits the catalytic activity of the tyrosine phosphatase SHP1 (PTPN6). SHP1 and THEMIS bind to the ubiquitous cytosolic adapter GRB2, and the purported formation of a tri-molecular THEMIS-GRB2-SHP1 complex facilitates inactivation of SHP1 by THEMIS. The importance of this function of GRB2 among its numerous documented activities is unclear as GRB2 binds to multiple proteins and participates in several signaling responses in thymocytes. Here, we show that similar to Themis-/- thymocytes, the primary molecular defect in GRB2-deficient thymocytes is increased catalytically active SHP1 and the developmental block in GRB2-deficient thymocytes is alleviated by deletion or inhibition of SHP1 and is exacerbated by SHP1 overexpression. Thus, the principal role of GRB2 during T cell development is to promote THEMIS-mediated inactivation of SHP1 thereby enhancing the sensitivity of TCR signaling in CD4+CD8+ thymocytes to low affinity positively selecting self-ligands.

Measurement of FGFR3 signaling at the cell membrane via total internal reflection fluorescence microscopy to compare the activation of FGFR3 mutants.

Fibroblast growth factor receptors (FGFRs) initiate signal transduction via the RAS/mitogen-activated protein kinase pathway by their tyrosine kinase activation known to determine cell growth, tissue differentiation, and apoptosis. Recently, many missense mutations have been reported for FGFR3, but we only know the functional effect for a handful of them. Some mutations result in aberrant FGFR3 signaling and are associated with various genetic disorders and oncogenic conditions. Here, we employed micropatterned surfaces to specifically enrich fluorophore-tagged FGFR3 (monomeric GFP [mGFP]-FGFR3) in certain areas of the plasma membrane of living cells. We quantified receptor activation via total internal reflection fluorescence microscopy of FGFR3 signaling at the cell membrane that captured the recruitment of the downstream signal transducer growth factor receptor-bound 2 (GRB2) tagged with mScarlet (GRB2-mScarlet) to FGFR3 micropatterns. With this system, we tested the activation of FGFR3 upon ligand addition (fgf1 and fgf2) for WT and four FGFR3 mutants associated with congenital disorders (G380R, Y373C, K650Q, and K650E). Our data showed that ligand addition increased GRB2 recruitment to WT FGFR3, with fgf1 having a stronger effect than fgf2. For all mutants, we found an increased basal receptor activity, and only for two of the four mutants (G380R and K650Q), activity was further increased upon ligand addition. Compared with previous reports, two mutant receptors (K650Q and K650E) had either an unexpectedly high or low activation state, respectively. This can be attributed to the different methodology, since micropatterning specifically captures signaling events at the plasma membrane. Collectively, our results provide further insight into the functional effects of mutations to FGFR3.

The Role of the LINC01234/miR-433-3p/GRB2 ceRNA Network in NSCLC Cell Malignant Proliferation.

BACKGROUND: Non-small cell lung cancer (NSCLC) is associated with high morbidity and mortality. Dysregulation of lncRNAs leads to NSCLC progression. OBJECTIVE: This study aims to explore the regulatory mechanism of lncRNA LINC01234 in NSCLC. MATERIALS AND METHODS: LINC01234 expression in NSCLC cells was determined. Cell proliferation was detected using CCK-8, colony formation, and EDU assays after transfection of siRNA LINC01234 into H1299 cells and transfection of pcDNA3.1-LINC01234 into H1975 cells. Subcellular localization of LINC01234 was predicted and the binding relations between LINC01234 and miR-433-3p as well as miR-433-3p and GRB2 were verified. The expression levels of miR-433-3p and GRB2 in NSCLC cells were determined. Joint experiments of miR-433-3p inhibitor + si- LINC01234-1 or oe-GRB2 + si-LINC01234-1 were conducted to verify the role of miR-433-3p and GRB2 in NSCLC cell malignant proliferation. RESULTS: LINC01234 was abundantly expressed in NSCLC cells. LINC01234 silencing reduced NSCLC cell proliferation while LINC01234 overexpression enhanced cell proliferation. LINC01234 competitively bound to miR-433-3p and miR-433-3p directly targeted GRB2. miR- 433-3p knockdown or GRB2 overexpression counteracted the repressive effect of LINC01234 silencing on NSCLC cell malignant proliferation. CONCLUSION: LINC01234 competitively bound to miR-433-3p and promoted GRB2 transcription to augment NSCLC cell malignant proliferation.

High expression of GRB2 associated binding protein 3 mRNA predicts positive prognosis in melanoma.

Malignant melanoma is the most aggressive form of skin cancer, and it is characterized by poor prognosis in patients with metastatic diseases. Accurate prediction of prognosis is crucial for therapeutic decisions. In this study, bioinformatics analysis was used to explore the prognostic value of growth factor receptor-bound protein 2-associated binding protein 3 (GAB3) mRNA. RNA transcriptome sequencing data and clinical data from The Cancer Genome Atlas and genotype-tissue expression (GTEx) were analyzed for differentially expressed genes in high and low GAB3 mRNA expression groups in melanoma. Performing gene enrichment analysis and constructing protein-protein interaction networks. High expression of GAB3 was significantly correlated with a lower T stage, melanoma Clark level, Breslow depth, and melanoma ulceration. And high GAB3 expression was also associated with better progression-free interval in T1 and T2 stages and N0 stage and longer overall survival in T1 and T2 stages, N0 stage, and N1 stage. GAB3 promoted high levels of infiltration of macrophages and activated natural killer cells in melanoma. High expression of GAB3 predicted a positive prognosis in early-stage melanoma that may be mediated by the anticancer immune response.

Anti-c-MET Fab-Grb2-Gab1 Fusion Protein-Mediated Interference of c-MET Signaling Pathway Induces Methuosis in Tumor Cells.

Bio-macromolecules have potential applications in cancer treatment due to their high selectivity and efficiency in hitting therapeutic targets. However, poor cell membrane permeability has limited their broad-spectrum application in cancer treatment. The current study developed highly internalizable anti-c-MET antibody Fab fusion proteins with intracellular epitope peptide chimera to achieve the dual intervention from the extracellular to intracellular targets in tumor therapy. In vitro experiments demonstrated that the fusion proteins could interfere with the disease-associated intracellular signaling pathways and inhibit the uncontrolled proliferation of tumor cells. Importantly, investigation of the underlying mechanism revealed that these protein chimeras could induce vacuolation in treated cells, thus interfering with the normal extension and arrangement of microtubules as well as the mitosis, leading to the induction of methuosis-mediated cell death. Furthermore, in vivo tumor models indicated that certain doses of fusion proteins could inhibit the A549 xenograft tumors in NOD SCID mice. This study thus provides new ideas for the intracellular delivery of bio-macromolecules and the dual intervention against tumor cell signaling pathways.

The Grb2 splice variant, Grb3-3, is a negative regulator of RAS activation.

Activation of RAS is crucial in driving cellular outcomes including proliferation, differentiation, migration and apoptosis via the MAPK pathway. This is initiated on recruitment of Grb2, as part of a Grb2-Sos complex, to an up-regulated receptor tyrosine kinase (RTK), enabling subsequent interaction of Sos with the plasma membrane-localised RAS. Aberrant regulation at this convergence point for RTKs in MAPK signalling is a key driver of multiple cancers. Splicing of the GRB2 gene produces a deletion variant, Grb3-3, that is incapable of binding to RTKs. We show that, despite maintaining the ability to bind to Sos, the Grb3-3-Sos complex remains in the cytoplasm, unable to engage with RAS. Competition between Grb2 and Grb3-3 for binding to C-terminal proline-rich sequences on Sos modulates MAPK signalling. Additionally, we demonstrate that splicing is regulated by heterogenous nuclear riboproteins C1/C2, and that normal and malignant colon tissue show differential Grb3-3 expression.

Insight into the C-terminal SH3 domain mediated binding of Drosophila Drk to Sos and Dos.

Drk, a Drosophila homologue of human GRB2, interacts with Sevenless (Sev) receptor via its SH2 domain, while the N- and C-terminal SH3 domains (Drk-NSH3 and Drk-CSH3, respectively) are responsible for the interaction with proline-rich motifs (PRMs) of Son of sevenless (Sos) or Daughter of Sevenless (Dos). Drk-NSH3 on its own has a conformational equilibrium between folded and unfolded states, and the folded state is stabilised by the association with a Sos-derived proline-rich peptide with PxxPxR motif. In contrast, Drk-CSH3 is supposed to bind PxxxRxxKP motifs in Dos. Aiming at clarifying the structural and functional differences between the two SH3 domains, we performed NMR studies of Drk-CSH3. The resulting solution structure and the 15N-relaxation data showed that Drk-CSH3 consists of a stable domain. Large chemical shift perturbation was commonly found around the RT loop and the hydrophobic patch, while there were also changes that occur characteristically for Sos- or Dos-derived peptides. Sos-derived two peptides with PxxPxR motif showed stronger affinity to Drk-CSH3, indicating that the Sos PRMs can bind both N- and C-SH3 domains. Dos-derived two peptides could also bind Drk-CSH3, but with much weaker affinity, suggesting a possibility that any cooperative binding of Dos-PRMs may strengthen the Drk-Dos interaction. The NMR studies as well as the docking simulations provide valuable insights into the biological and biophysical functions of two SH3 domains in Drk.

Evaluation of the relationship between miR-1271 and GRB2 gene in endometriosis.

BACKGROUND: Endometriosis is a common gynecological condition with a substantial economic burden on society. It is known that both genetic and environmental factors are contributing to the phenotypic development of the disease. MicroRNAs have a vital role in the pathogenesis of endometriosis. miR-1271 and its direct target gene, GRB2 (growth factor receptor-bound protein 2), expression have been studied in gynecologic cancers, while their role in endometriosis has not been studied. OBJECTIVE: We measured miR-1271 and GRB2 gene expression in the eutopic and ectopic tissues of patients (endometrial tissues) in contrast to the control samples from healthy women. MATERIALS AND METHODS: In this study, a total of 45 samples (15 control samples, 15 eutopic samples and 15 ectopic samples) were collected. We used qRT-PCR (quantitative polymerase chain reaction) to evaluate the expression levels of the miR-1271 and GRB2 gene. RESULTS: We observed inverse expression of miR-1271 and GRB2 gene. MiR-1271 expression was significantly reduced in patients with endometriosis compared with healthy women. While there was a noticeable increase in the expression level of its target gene, GRB2, in tissues of endometriosis patients compared with normal control samples. CONCLUSION: We have shown an inverse relationship between the reduction of miR-1271 expression level and increase in the expression level of GRB2, therefore, increased GRB2 expression in endometriosis tissues can be due to decreased expression of this microRNA. Our findings suggested that miR-1271 maybe play a role as a biomarker in the diagnosis of patients with endometriosis.

Macrophage SAMSN1 protects against sepsis-induced acute lung injury in mice.

OBJECTIVE: Inflammation and oxidative stress contribute to the progression of sepsis-induced acute lung injury (ALI). SAM domain, SH3 domain and nuclear localization signals 1 (SAMSN1) is a signaling adaptor protein, and mainly regulates inflammatory response of various immune cells. The present study generates macrophage-specific SAMSN1-knockout (Samsn1MKO) and SAMSN1-transgenic (Samsn1MTG) mice to investigate its role and mechanism in sepsis-induced ALI. METHODS: Samsn1MKO and Samsn1MTG mice were exposed to lipopolysaccharide (LPS) instillation or cecal ligation and puncture (CLP) surgery to induce sepsis-induced ALI. Bone marrow transplantation, cellular depletion and non-invasive adoptive transfer of bone marrow-derived macrophages (BMDMs) were performed to validate the role of macrophage SAMSN1 in sepsis-induced ALI in vivo. Meanwhile, BMDMs were isolated from Samsn1MKO or Samsn1MTG mice to further clarify the role of SAMSN1 in vitro. RESULTS: Macrophage SAMSN1 expression was increased in response to LPS stimulation, and negatively correlated with LPS-induced ALI in mice. Macrophage SAMSN1 deficiency exacerbated, while macrophage SAMSN1 overexpression ameliorated LPS-induced inflammation, oxidative stress and ALI in mice and in BMDMs. Mechanistically, we found that macrophage SAMSN1 overexpression prevented LPS-induced ALI though activating AMP-activated protein kinase α2 (AMPKα2) in vivo and in vitro. Further studies revealed that SAMSN1 directly bound to growth factor receptor bound protein 2-associated protein 1 (GAB1) to prevent its protein degradation, and subsequently enhanced protein kinase A (PKA)/AMPKα2 activation in a protein tyrosine phosphatase, non-receptor type 11 (PTPN11, also known as SHP2)-dependent manner. Moreover, we observed that macrophage SAMSN1 overexpression diminished CLP-induced ALI in mice. CONCLUSION: Our study documents the protective role of macrophage SAMSN1 against sepsis-induced inflammation, oxidative stress and ALI through activating AMPKα2 in a GAB1/SHP2/PKA pathway, and defines it as a promising biomarker and therapeutic target to treat sepsis-induced ALI.

The structural basis of BCR-ABL recruitment of GRB2 in chronic myelogenous leukemia.

BCR-ABL drives chronic myeloid leukemia (CML). BCR binding to GRB2 transduces signaling via the Ras/MAPK pathway. Despite considerable data confirming the binding, molecular-level understanding of exactly how the two proteins interact, and, especially, what are the determinants of the specificity of the SH2GRB2 domain-phosphorylated BCR (pBCR) recognition are still open questions. Yet, this is vastly important for understanding binding selectivity, and for predicting the phosphorylated receptors, or peptides, that are likely to bind. Here, we uncover these determinants and ascertain to what extent they relate to the affinity of the interaction. Toward this end, we modeled the complexes of the pBCR and SH2GRB2 and other pY/Y-peptide-SH2 complexes and compared their specificity and affinity. We observed that pBCR's 176FpYVNV180 motif is favorable and specific to SH2GRB2, similar to pEGFR, but not other complexes. SH2GRB2 contains two binding pockets: pY-binding recognition pocket triggers binding, and the specificity pocket whose interaction is governed by N179 in pBCR and W121 in SH2GRB2. Our proposed motif with optimal affinity to SH2GRB2 is E/D-pY-E/V-N-I/L. Collectively, we provide the structural basis of BCR-ABL recruitment of GRB2, outline its specificity hallmarks, and delineate a blueprint for prediction of BCR-binding scaffolds and for therapeutic peptide design.

Allosteric regulation of GRB2 modulates RAS activation.

RAS activation is a multiple-step process in which linkage of the extracellular stimuli to the RAS activator SOS1 is the main step in RAS activation. GRB2 adaptor protein is the main modulator in SOS1 recruitment to the plasma membrane and its activation. This interaction is well studied but the exact mechanism of GRB2-SOS1 complex formation and SOS1 activation has yet remained obscure. Here, a new allosteric mechanism for the GRB2 regulation is described as a prerequisite for the modulation of SOS1 activation. This regulatory mechanism comprises a series of intramolecular interactions which are potentiated by GRB2 interaction with upstream ligands.Abbreviations: GRB2, growth factor receptor-bound protein 2; SOS1, son of sevenless 1; RAS, Rat Sarcoma; GEF, guanine nucleotide exchange factor; GAP, GTPase-activating protein; HER2, human epidermal growth factor receptor; SH3, SRC Homology 3; SH2, SRC Homology 2; PRD, proline-rich domain; PRM, proline-rich motif; PRP, proline-rich peptide; RTK, receptor tyrosine kinases.

TGF-ß1/SH2B3 axis regulates anoikis resistance and EMT of lung cancer cells by modulating JAK2/STAT3 and SHP2/Grb2 signaling pathways.

The pathogenesis of lung cancer, the most common cancer, is complex and unclear, leading to limited treatment options and poor prognosis. To provide molecular insights into lung cancer development, we investigated the function and underlying mechanism of SH2B3 in the regulation of lung cancer. We indicated SH2B3 was diminished while TGF-ß1 was elevated in lung cancer tissues and cells. Low SH2B3 level was correlated with poor prognosis of lung cancer patients. SH2B3 overexpression suppressed cancer cell anoikis resistance, proliferation, migration, invasion, and EMT, while TGF-ß1 promoted those processes via reducing SH2B3. SH2B3 bound to JAK2 and SHP2 to repress JAK2/STAT3 and SHP2/Grb2/PI3K/AKT signaling pathways, respectively, resulting in reduced cancer cell anoikis resistance, proliferation, migration, invasion, and EMT. Overexpression of SH2B3 suppressed lung cancer growth and metastasis in vivo. In conclusion, SH2B3 restrained the development of anoikis resistance and EMT of lung cancer cells via suppressing JAK2/STAT3 and SHP2/Grb2/PI3K/AKT signaling cascades, leading to decreased cancer cell proliferation, migration, and invasion.