SH3-binding Motif Research Articles

The Toxoplasma secreted effector TgWIP modulates dendritic cell motility by activating host tyrosine phosphatases Shp1 and Shp2

The obligate intracellular parasite Toxoplasma gondii causes life-threatening toxoplasmosis to immunocompromised individuals. The pathogenesis of Toxoplasma relies on its swift dissemination to the central nervous system through a ‘Trojan Horse’ mechanism using infected leukocytes as carriers. Previous work found TgWIP, a protein secreted from Toxoplasma, played a role in altering the actin cytoskeleton and promoting cell migration in infected dendritic cells (DCs). However, the mechanism behind these changes was unknown. Here, we report that TgWIP harbors two SH2-binding motifs that interact with tyrosine phosphatases Shp1 and Shp2, leading to phosphatase activation. DCs infected with Toxoplasma exhibited hypermigration, accompanying enhanced F-actin stress fibers and increased membrane protrusions such as filopodia and pseudopodia. By contrast, these phenotypes were abrogated in DCs infected with Toxoplasma expressing a mutant TgWIP lacking the SH2-binding motifs. We further demonstrated that the Rho-associated kinase (Rock) is involved in the induction of these phenotypes, in a TgWIP-Shp1/2 dependent manner. Collectively, the data uncover a molecular mechanism by which TgWIP modulates the migration dynamics of infected DCs in vitro.

The Toxoplasma secreted effector TgWIP modulates dendritic cell motility by activating host tyrosine phosphatases Shp1 and Shp2.

The obligate intracellular parasite Toxoplasma gondii causes life-threatening toxoplasmosis to immunocompromised individuals. The pathogenesis of Toxoplasma relies on its swift dissemination to the central nervous system through a 'Trojan Horse' mechanism using infected leukocytes as carriers. Previous work found TgWIP, a protein secreted from Toxoplasma, played a role in altering the actin cytoskeleton and promoting cell migration in infected dendritic cells (DCs). However, the mechanism behind these changes was unknown. Here, we report that TgWIP harbors two SH2-binding motifs that interact with tyrosine phosphatases Shp1 and Shp2, leading to phosphatase activation. DCs infected with Toxoplasma exhibited hypermigration, accompanying enhanced F-actin stress fibers and increased membrane protrusions such as filopodia and pseudopodia. By contrast, these phenotypes were abrogated in DCs infected with Toxoplasma expressing a mutant TgWIP lacking the SH2-binding motifs. We further demonstrated that the Rho-associated kinase (Rock) is involved in the induction of these phenotypes, in a TgWIP-Shp1/2 dependent manner. Collectively, the data uncover a molecular mechanism by which TgWIP modulates the migration dynamics of infected DCs in vitro.

Comprehensive phenotypes of patients with SYNGAP1-related disorder reveals high rates of epilepsy and autism.

To delineate the comprehensive phenotypic spectrum of SYNGAP1-related disorder in a large patient cohort aggregated through a digital registry. We obtained de-identified patient data from an online registry. Data were extracted from uploaded medical records. We reclassified all SYNGAP1 variants using American College of Medical Genetics criteria and included patients with pathogenic/likely pathogenic (P/LP) single nucleotide variants or microdeletions incorporating SYNGAP1. We analyzed neurodevelopmental phenotypes, including epilepsy, intellectual disability (ID), autism spectrum disorder (ASD), behavioral disorders, and gait dysfunction for all patients with respect to variant type and location within the SynGAP1 protein. We identified 147 patients (50% male, median age 8 years) with P/LP SYNGAP1 variants from 151 individuals with data available through the database. One hundred nine were truncating variants and 22 were missense. All patients were diagnosed with global developmental delay (GDD) and/or ID, and 123 patients (84%) were diagnosed with epilepsy. Of those with epilepsy, 73% of patients had GDD diagnosed before epilepsy was diagnosed. Other prominent features included autistic traits (n = 100, 68%), behavioral problems (n = 100, 68%), sleep problems (n = 90, 61%), anxiety (n = 35, 24%), ataxia or abnormal gait (n = 69, 47%), sensory problems (n = 32, 22%), and feeding difficulties (n = 69, 47%). Behavioral problems were more likely in those patients diagnosed with anxiety (odds ratio [OR] 3.6, p = .014) and sleep problems (OR 2.41, p = .015) but not necessarily those with autistic traits. Patients with variants in exons 1-4 were more likely to have the ability to speak in phrases vs those with variants in exons 5-19, and epilepsy occurred less frequently in patients with variants in the SH3 binding motif. We demonstrate that the data obtained from a digital registry recapitulate earlier but smaller studies of SYNGAP1-related disorder and add additional genotype-phenotype relationships, validating the use of the digital registry. Access to data through digital registries broadens the possibilities for efficient data collection in rare diseases.

Defining Signaling Interactomes Involved in Non-Canonical Wnt Signaling By ROR1 or ROR2 in Hairy Cell Leukemia That Can Influence Cell Morphology and Migration

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) and 2 (ROR2) are highly conserved oncoembryonic surface proteins expressed on neoplastic cells of patients with a variety of human cancers, but not on most normal postpartum tissues. ROR1 and ROR2 are each a receptor for non-canonical Wnt factors that direct migration and planar cell polarity, which is essential for organ development. We developed humanized monoclonal antibodies (mAbs) each specific for ROR1 (UC-961) or ROR2 (6E6-70) and found the leukemia cells of patients with classic hairy cell leukemia (cHCL) or variant HCL (vHCL) express both ROR1 and ROR2 and that these receptors function in HCL non-canonical Wnt signaling (Widhopf, G. et al). We hypothesized that ROR1 and ROR2 each may associate with overlapping and distinctive sets of proteins, which may contribute to ROR1/2, ROR1, or ROR2 signaling in HCL. To test this hypothesis, we optimized conditions for generating immune precipitates (i.p.) with either UC-961 or 6E6-70, and examined the proteins of each i.p. via data dependent acquisition mass spectrometry. Consistent with the assumed capacity of each of these mAb to disrupt ROR1/2 heteroligomerization, we identified ROR1, but not ROR2, in i.p. generated using UC-961 and ROR2, but not ROR1, in i.p. generated using 6E6-70 on cell lysates of leukemia cells isolated from a patient with cHCL. In the anti-ROR1 i.p. we identified 1,414 proteins, 849 (60%) of which also were found in the anti-ROR2 i.p.. In the anti-ROR2 i.p. we identified 2,523 proteins, a third of which also were found in the ROR1 i.p.. Among proteins common to both anti-ROR1 and anti-ROR2 i.p. we noted hematopoietic cell-specific protein-1 (HS1 or HCLS1), which we found associated a SH3-binding motif that was dependent on a proline residue at position 841 of activated ROR1 in chronic lymphocytic leukemia (CLL); we found this SH3-binding motif was critical for ROR1-dependent Wnt5a-induced chemokine-directed migration ( Leukemia 12:2615, 2017). Also found common to both ROR1 and ROR2 in HCL were proteins that drive actin polymerization and cytoskeletal dynamics that influence cell morphology and migration. To validate that ROR2 itself was involved in binding HS1 and in enhancing migration, we generated mutant forms of ROR2, each harboring a proline-to-alanine mutation in one of several proline residues found in the proline-rich domain of ROR2. We transduced MEC1 cells, which lack ROR1 or ROR2, with lentivirus vectors encoding wild-type ROR2 or each one of several P-A mutants of ROR2. Flow cytometry confirmed that each of the transduced cells had comparable expression of surface ROR2. We generated anti-ROR2 i.p. using lysates of each and found that all but one of the mutant forms of ROR2 could bind to HS1 by immunoblot analysis. In contrast to wild-type ROR2, or other mutant forms of ROR2, expression of this mutant form of ROR2 could not enhance chemokine-directed migration relative to that of wild-type MEC1 cells. Collectively, our studies define signaling interactomes involved in non-canonical Wnt signaling by ROR1 and/or ROR2 in hairy cell leukemia and define a residue within ROR2 that is necessary for binding HS1 and facilitating cellular migration.

Signaling Role of Viral Protein Motif and Its Application in CAR T Cell Therapy

Adoptive cell therapy using Chimeric Antigen Receptor (CAR) T cells, such as Tisagenlecleucel and Axicabtagene Ciloleucel, has revolutionized cancer immunotherapy and become a game-changer in cancer care. However, several challenges, including durable remission and limited expansion after infusion, persist. Recent work from the Riddell lab (PMID: 34429382) demonstrated that T Cell Receptors (TCRs) are more sensitive than CARs due to the presence of T cell signaling proteins like CD3-, CD3ε, and CD3-, which facilitate the recruitment of Lck to phosphorylate immunoreceptor tyrosine-based activation motifs (ITAMs). Although CARs were designed to mimic TCR signaling, they still lack the full sensitivity. In this study, we hypothesized that enhancing tyrosine phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) presented on CD3 in CAR T cells amplifies the CAR-mediated signaling as well as improves sensitivity of CAR co-stimulatory signaling. To achieve this, we developed a novel co-stimulation system where anti-CD19 CAR T cells co-expressed Tip37, a 37-amino acid segment of the Herpesvirus saimiri (HVS) tyrosine-protein kinase-interacting protein (Tip). This specific segment interacts exclusively with the major T cell kinase, Lck, effectively regulating its kinase activation. The critical region for this interaction lies within 37 amino acids (146-182) in the central part of Tip, which contains two motifs: the SH3 binding motif (SH3B; 174-182) and the C-terminal Src-related kinase homology motif (CSKH; 146-155). The 37 amino acid peptide fragments alone form a stable complex with Lck. Lck is crucial in the initial stages of T cell activation, not only when triggered by the native TCR stimulation but also in response to CAR-antigen stimulation. Following antigen-induced T cell activation, Lck initiates the phosphorylation of tyrosines in the ITAMs present in CD3ζ domains. This leads to the recruitment of ZAP70 kinases to the ITAMs of CD3ζ, facilitating the formation of signaling protein complexes. The results of our in vitro experiments using the immortalized T cell line, Jurkat cells, demonstrate that the inclusion of the Tip37 component in the CAR leads to the formation of the Lck-CAR complex. This, in turn, significantly enhances the phosphorylation of ITAMs in CARs and triggers downstream signaling. Importantly, the CAR T cells with the Tip37 co-stimulatory domain do not secrete cytokines when in an unstimulated state. This suggests that the heightened Lck phosphorylation does not lead to the auto-activation of T cells. As a result, CAR T cells engineered with Tip37 can be considered safe and hold promise for future applications. We engineered CAR19 T cells co-expressing Tip (Tip-CART19 cells) with a co-stimulatory domain of 4-1BB and CD3 (Tip-CART19 cells). As a control, we generated CART19 cells lacking the TIP domain (WT CART19 cells). We confirmed that Tip expression induced enhanced CD3 phosphorylation in CART19 T cells. Additionally, we observed that a significantly augmented outgrowth of CD8+ cells engineered with the TIP-containing CAR compared with canonical CAR T cells. Further, TIP-CAR T cells displayed increased expression of CD38 and Ki67. Interestingly, we also noted an expansion of CCR7+CD45RA- central memory subsets in CD4+ CART19 cells and CCR7-CD45RA- effector memory subsets in CD8+ CART19 cells. Finally, as evidenced by apparent changes in phenotype, Tip-CART19 cells exhibited improved cytotoxic ability and significant outgrowth of CART19 cells with in vitro repeated CAR stimulation using CD19-expressing tumor cells, as previously described in our study (Fraietta et al., Nature 2018). This study highlights that amplification of the constitutive Lck recruitment and amplified CD3 phosphorylation in CART19 cells via the introduction of Tip37 domain results in enhanced anti-leukemia cytotoxic ability and preservation of proliferative function in CART19 cells. Our discovery introduces a novel approach for amplifying CAR signaling to improve CAR T cell therapy.

Molecular characterization and expression analysis of a QM protein gene in Chinese mitten crab Eriocheir sinensis.

QM protein was previously discovered as a tumor suppressor, and numerous studies have shown that QM protein also played important roles in the immune responses. To investigate the potential roles of the QM protein gene in Eriocheir sinensis, the QM protein gene (designated as EsQM) has been cloned from E. sinensis using the rapid amplification of cDNA ends (RACE) technique. The cDNA of EsQM is 781 bp in length, consisting of a 654 bp open reading frame (ORF), encoding 219 amino acids, a 27 bp 5' untranslated region (UTR) and a 94 bp 3' UTR. The EsQM protein has a calculated molecular weight of 25.4 kDa and a theoretical isoelectric point of 10.10. The deduced protein sequence of EsQM contains a Ribosomal_L16 domain, an SH3-binding motif, an N-acylation site, two putative antibiotic binding sites, two putative protein kinase C phosphorylation sites, and two amidation sites. EsQM is extremely conserved and exhibits more than 85% similarities to previously identified arthropod QM protein genes. By real-time quantitative PCR (qPCR) analysis, we found that EsQM mRNA transcripts were detectable in all the examined tissues, with the highest expression in hemocytes. The mRNA expression of EsQM in hemocytes was significantly upregulated after the stimulation of Aeromonas hydrophila or polybrominated diphenyl ether-47 (BDE-47). Moreover, EsQM mRNA expression in hemocytes responded more quickly and lasted longer when stimulated by A.hydrophila than BDE-47. Thus, EsQM can respond to bacterial infection and environmental pollution, and might be involved in the defense mechanism to both biological and non-biological stimulation of arthropods.

RhoGAP6 interacts with COPI to regulate protein transport.

RhoGAP6 is the most highly expressed GTPase-activating protein (GAP) in platelets specific for RhoA. Structurally RhoGAP6 contains a central catalytic GAP domain surrounded by large, disordered N- and C-termini of unknown function. Sequence analysis revealed three conserved consecutive overlapping di-tryptophan motifs close to the RhoGAP6 C-terminus which were predicted to bind to the mu homology domain (MHD) of δ-COP, a component of the COPI vesicle complex. We confirmed an endogenous interaction between RhoGAP6 and δ-COP in human platelets using GST-CD2AP which binds an N-terminal RhoGAP6 SH3 binding motif. Next, we confirmed that the MHD of δ-COP and the di-tryptophan motifs of RhoGAP6 mediate the interaction between both proteins. Each of the three di-tryptophan motifs appeared necessary for stable δ-COP binding. Proteomic analysis of other potential RhoGAP6 di-tryptophan motif binding partners indicated that the RhoGAP6/δ-COP interaction connects RhoGAP6 to the whole COPI complex. 14-3-3 was also established as a RhoGAP6 binding partner and its binding site was mapped to serine 37. We provide evidence of potential cross-regulation between 14-3-3 and δ-COP binding, however, neither δ-COP nor 14-3-3 binding to RhoGAP6 impacted RhoA activity. Instead, analysis of protein transport through the secretory pathway demonstrated that RhoGAP6/δ-COP binding increased protein transport to the plasma membrane, as did a catalytically inactive mutant of RhoGAP6. Overall, we have identified a novel interaction between RhoGAP6 and δ-COP which is mediated by conserved C-terminal di-tryptophan motifs, and which might control protein transport in platelets.

Gustavson syndrome is caused by an in-frame deletion in RBMX associated with potentially disturbed SH3 domain interactions

RNA binding motif protein X‐linked (RBMX) encodes the heterogeneous nuclear ribonucleoprotein G (hnRNP G) that regulates splicing, sister chromatid cohesion and genome stability. RBMX knock down experiments in various model organisms highlight the gene’s importance for brain development. Deletion of the RGG/RG motif in hnRNP G has previously been associated with Shashi syndrome, however involvement of other hnRNP G domains in intellectual disability remain unknown. In the current study, we present the underlying genetic and molecular cause of Gustavson syndrome. Gustavson syndrome was first reported in 1993 in a large Swedish five-generation family presented with profound X-linked intellectual disability and an early death. Extensive genomic analyses of the family revealed hemizygosity for a novel in-frame deletion in RBMX in affected individuals (NM_002139.4; c.484_486del, p.(Pro162del)). Carrier females were asymptomatic and presented with skewed X-chromosome inactivation, indicating silencing of the pathogenic allele. Affected individuals presented minor phenotypic overlap with Shashi syndrome, indicating a different disease-causing mechanism. Investigation of the variant effect in a neuronal cell line (SH-SY5Y) revealed differentially expressed genes enriched for transcription factors involved in RNA polymerase II transcription. Prediction tools and a fluorescence polarization assay imply a novel SH3-binding motif of hnRNP G, and potentially a reduced affinity to SH3 domains caused by the deletion. In conclusion, we present a novel in-frame deletion in RBMX segregating with Gustavson syndrome, leading to disturbed RNA polymerase II transcription, and potentially reduced SH3 binding. The results indicate that disruption of different protein domains affects the severity of RBMX-associated intellectual disabilities.

Using Linear Motif Database Resources to Identify SH2 Domain Binders.

The SH2-binding phosphotyrosine class of short linear motifs (SLiMs) are key conditional regulatory elements, particularly in signaling protein complexes beneath the cell's plasma membrane. In addition to transmitting cellular signaling information, they can also play roles in cellular hijack by invasive pathogens. Researchers can take advantage of bioinformatics tools and resources to predict the motifs at conserved phosphotyrosine residues in regions of intrinsically disordered protein. A candidate SH2-binding motif can be established and assigned to one or more of the SH2 domain subgroups. It is, however, not so straightforward to predict which SH2 domains are capable of binding the given candidate. This is largely due to the cooperative nature of the binding amino acids which enables poorer binding residues to be tolerated when the other residues are optimal. High-throughput peptide arrays are powerful tools used to derive SH2 domain-binding specificity, but they are unable to capture these cooperative effects and also suffer from other shortcomings. Tissue and cell type expression can help to restrict the list of available interactors: for example, some well-studied SH2 domain proteins are only present in the immune cell lineages. In this article, we provide a table of motif patterns and four bioinformatics strategies that introduce a range of tools that can be used in motif hunting in cellular and pathogen proteins. Experimental followup is essential to determine which SH2 domain/motif-containing proteins are the actual functional partners.

SH3- and actin-binding domains connect ADNP and SHANK3, revealing a fundamental shared mechanism underlying autism.

De novo heterozygous mutations in activity-dependent neuroprotective protein (ADNP) cause autistic ADNP syndrome. ADNP mutations impair microtubule (MT) function, essential for synaptic activity. The ADNP MT-associating fragment NAPVSIPQ (called NAP) contains an MT end-binding protein interacting domain, SxIP (mimicking the active-peptide, SKIP). We hypothesized that not all ADNP mutations are similarly deleterious and that the NAPV portion of NAPVSIPQ is biologically active. Using the eukaryotic linear motif (ELM) resource, we identified a Src homology 3 (SH3) domain-ligand association site in NAP responsible for controlling signaling pathways regulating the cytoskeleton, namely NAPVSIP. Altogether, we mapped multiple SH3-binding sites in ADNP. Comparisons of the effects of ADNP mutations p.Glu830synfs*83, p.Lys408Valfs*31, p.Ser404* on MT dynamics and Tau interactions (live-cell fluorescence-microscopy) suggested spared toxic function in p.Lys408Valfs*31, with a regained SH3-binding motif due to the frameshift insertion. Site-directed-mutagenesis, abolishing the p.Lys408Valfs*31 SH3-binding motif, produced MT toxicity. NAP normalized MT activities in the face of all ADNP mutations, although, SKIP, missing the SH3-binding motif, showed reduced efficacy in terms of MT-Tau interactions, as compared with NAP. Lastly, SH3 and multiple ankyrin repeat domains protein 3 (SHANK3), a major autism gene product, interact with the cytoskeleton through an actin-binding motif to modify behavior. Similarly, ELM analysis identified an actin-binding site on ADNP, suggesting direct SH3 and indirect SHANK3/ADNP associations. Actin co-immunoprecipitations from mouse brain extracts showed NAP-mediated normalization of Shank3-Adnp-actin interactions. Furthermore, NAP treatment ameliorated aberrant behavior in mice homozygous for the Shank3 ASD-linked InsG3680 mutation, revealing a fundamental shared mechanism between ADNP and SHANK3.

Ral GEF with the PH Domain and SH3 Binding Motif 1 Regulated by Splicing Factor Junction Plakoglobin and Pyrimidine Metabolism Are Prognostic in Uterine Carcinosarcoma.

Uterine carcinosarcoma (UCS) is a highly invasive malignant tumor that originated from the uterine epithelium. Many studies suggested that the abnormal changes of alternative splicing (AS) of pre-mRNA are related to the occurrence and metastasis of the tumor. This study investigates the mechanism of alternative splicing events (ASEs) in the tumorigenesis and metastasis of UCS. RNA-seq of UCS samples and alternative splicing event (ASE) data of UCS samples were downloaded from The Cancer Genome Atlas (TCGA) and TCGASpliceSeq databases, several times. Firstly, we performed the Cox regression analysis to identify the overall survival-related alternative splicing events (OSRASEs). Secondly, a multivariate model was applied to approach the prognostic values of the risk score. Afterwards, a coexpressed network between splicing factors (SFs) and OSRASEs was constructed. In order to explore the relationship between the potential prognostic signaling pathways and OSRASEs, we fabricated a network between these pathways and OSRASEs. Finally, validations from multidimension platforms were used to explain the results unambiguously. 1,040 OSRASEs were identified by Cox regression. Then, 6 OSRASEs were incorporated in a multivariable model by Lasso regression. The area under the curve (AUC) of the receiver operator characteristic (ROC) curve was 0.957. The risk score rendered from the multivariate model was corroborated to be an independent prognostic factor (P < 0.001). In the network of SFs and ASEs, junction plakoglobin (JUP) noteworthily regulated RALGPS1-87608-AT (P < 0.001, R = 0.455). Additionally, RALGPS1-87608-AT (P = 0.006) showed a prominent relationship with distant metastasis. KEGG pathways related to prognosis of UCS were selected by gene set variation analysis (GSVA). The pyrimidine metabolism (P < 0.001, R = −0.470) was the key pathway coexpressed with RALGPS1. We considered that aberrant JUP significantly regulated RALGPS1-87608-AT and the pyrimidine metabolism pathway might play a significant part in the metastasis and prognosis of UCS.

Itk Promotes the Integration of TCR and CD28 Costimulation through Its Direct Substrates SLP-76 and Gads.

The costimulatory receptor CD28 synergizes with the TCR to promote IL-2 production, cell survival, and proliferation; yet the obligatory interdependence of TCR and CD28 signaling is not well understood. Upon TCR stimulation, Gads, a Grb2-family adaptor, bridges the interaction of two additional adaptors, LAT and SLP-76, to form a TCR-induced effector signaling complex. SLP-76 binds the Tec-family tyrosine kinase, Itk, which phosphorylates SLP-76 Y173 and PLC-γ1 Y783. In this study, we identified TCR-inducible, Itk-mediated phosphorylation of Gads Y45 in a human T cell line and in mouse primary T cells. Y45 is found within the N-terminal SH3 domain of Gads, an evolutionarily conserved domain with no known signaling function. Gads Y45 phosphorylation depended on the interaction of Gads with SLP-76 and on the dimerization-dependent binding of Gads to phospho-LAT. We provide evidence that Itk acts through SLP-76 and Gads to promote the TCR/CD28-induced activation of the RE/AP transcriptional element from the IL-2 promoter. Two Itk-related features of SLP-76, Y173 and a proline-rich Itk SH3 binding motif on SLP-76, were dispensable for activation of NFAT but selectively required for the TCR/CD28-induced increase in cytoplasmic and nuclear c-Rel and consequent RE/AP activation. We provide evidence that unphosphorylated, monomeric Gads mediates an RE/AP-directed inhibitory activity that is mitigated upon Gads dimerization and Y45 phosphorylation. This study illuminates a new, to our knowledge, regulatory module, in which TCR-induced, Itk-mediated phosphorylation sites on SLP-76 and Gads control the transcriptional response to TCR/CD28 costimulation, thus enforcing the obligatory interdependence of the TCR and CD28 signaling pathways.

Aging modulated by the Drosophila insulin receptor through distinct structure-defined mechanisms.

Mutations of the Drosophila melanogaster insulin/IGF signaling system slow aging, while also affecting growth and reproduction. To understand this pleiotropy, we produced an allelic series of single codon substitutions in the Drosophila insulin receptor, InR. We generated InR substitutions using homologous recombination and related each to emerging models of receptor tyrosine kinase structure and function. Three mutations when combined as trans-heterozygotes extended lifespan while retarding growth and fecundity. These genotypes reduced insulin-stimulated Akt phosphorylation, suggesting they impede kinase catalytic domain function. Among these genotypes, longevity was negatively correlated with egg production, consistent with life-history trade-off theory. In contrast, one mutation (InR353) was located in the kinase insert domain, a poorly characterized element found in all receptor tyrosine kinases. Remarkably, wild-type heterozygotes with InR353 robustly extended lifespan without affecting growth or reproduction and retained capacity to fully phosphorylate Akt. The Drosophila insulin receptor kinase insert domain contains a previously unrecognized SH2 binding motif. We propose the kinase insert domain interacts with SH2-associated adapter proteins to affect aging through mechanisms that retain insulin sensitivity and are independent of reproduction.

IRSp53 is a novel interactor of SHIP2: A role of the actin binding protein Mena in their cellular localization in breast cancer cells

A tight control of the machineries regulating membrane bending and actin dynamics is very important for the generation of membrane protrusions, which are crucial for cell migration and invasion. Protein/protein and protein/phosphoinositides complexes assemble and disassemble to coordinate these mechanisms, the scaffold properties of the involved proteins playing a prominent role in this organization. The PI 5-phosphatase SHIP2 is a critical enzyme modulating PI(3,4,5)P3, PI(4,5)P2 and PI(3,4)P2 content in the cell. The scaffold properties of SHIP2 contribute to the specific targeting or retention of the protein in particular subcellular domains. Here, we identified IRSp53 as a new binding interactor of SHIP2 proline-rich domain. Both proteins are costained in HEK293T cells protrusions, upon transfection. We showed that the SH3-binding polyproline motif recognized by IRSp53 in SHIP2 is different from the regions targeted by other PRR binding partners i.e., CIN85, ITSN or even Mena a common interactor of both SHIP2 and IRSp53. We presented evidence that IRSp53 phosphorylation on S366 did not influence its interaction with SHIP2 and that Mena is not necessary for the association of SHIP2 with IRSp53 in MDA-MB-231 cells. The absence of Mena in MDA-MB-231 cells decreased the intracellular content in F-actin and modified the subcellular localization of SHIP2 and IRSp53 by increasing their relative content at the plasma membrane. Together our data suggest that SHIP2, through interaction with the cell protrusion regulators IRSp53 and Mena, participate to the formation of multi-protein complexes. This ensures the appropriate modulations of PIs which is important for regulation of membrane dynamics.

ELM-the eukaryotic linear motif resource in 2020.

The eukaryotic linear motif (ELM) resource is a repository of manually curated experimentally validated short linear motifs (SLiMs). Since the initial release almost 20 years ago, ELM has become an indispensable resource for the molecular biology community for investigating functional regions in many proteins. In this update, we have added 21 novel motif classes, made major revisions to 12 motif classes and added >400 new instances mostly focused on DNA damage, the cytoskeleton, SH2-binding phosphotyrosine motifs and motif mimicry by pathogenic bacterial effector proteins. The current release of the ELM database contains 289 motif classes and 3523 individual protein motif instances manually curated from 3467 scientific publications. ELM is available at: http://elm.eu.org.

Sequence feature and expression profile of a tumor suppressor QM protein gene from hard clam Meretrix meretrix

The present study describes the molecular characterization and transcriptional features of a tumor suppressor QM protein gene, MmQM, in Meretrix meretrix. The full-length cDNA (819 bp) of MmQM consists of a 657 bp opening reading frame (ORF) encoding a 218 amino acid protein with a calculated molecular mass of 25.3 kDa and theoretical isoelectric point of 10.2. A ribosomal protein L10 signature, an SH3-binding motif, an antibiotic binding site, an amidation site and two protein kinase C phosphorylation sites were revealed from the MmQM sequence. Phylogenetic analysis showed that MmQM is clustered with previously identified mollusk QM proteins. MmQM mRNA transcripts were detectable in all of the examined tissues in a constitutive manner, and were significantly different from each other. After bacterial stimulation, the mRNA transcripts of MmQM in the hepatopancreas significantly increased. Hence, we conclude that MmQM could respond to pathogenic infections and it might play an important role in the innate immunity against microorganisms in the clam M. meretrix.

Wnt5a Induces ROR1 Dependent Tyrosine Phosphorylation of DOCK2, and Enhanced Activation of ERK to Promote Proliferation of CLL Cells

ROR1 (receptor tyrosine kinase-like orphan receptor 1) is a conserved, oncoembryonic surface antigen expressed in chronic lymphocytic leukemia (CLL) B cells, but not on most normal post-partem tissues. Patients with high-level leukemia-cell expression of ROR1 have been found to have a shorter median time from diagnosis to therapy and shorter median survival than patients with CLL cells that express low-to-negligible amounts of ROR1 (Cui B. et al., Blood, 128:2931, 2016). In prior studies we found that Wnt5a could induce ROR1 to recruit DOCK2 at proline 808 and activate Rac1/2, leading to enhanced leukemia-cell proliferation (Hasan M.K. et al., Blood, 132:170, 2018). Moreover, Wnt5a-enhanced proliferation could be inhibited by cirmtuzumab, a first-in-class humanized mAb specific for a functional epitope of the ROR1; this mAb is undergoing phase II clinical testing in patients with CLL (Choi MY, et al, Cell Stem Cell22:951, 2018). However, the molecular mechanism(s) of ROR1/DOCK2 signaling in the activation of Rac1/2, or downstream signaling molecules such as ERK, or AKT remains elusive. Here, we report that Wnt5a can induce tyrosine phosphorylation of DOCK2 in serum-starved primary CLL cells. The capacity of Wnt5a to induce DOCK2 phosphorylation could be inhibited by cirmtuzumab, but not by ibrutinib, even when used at concentrations that exceeded those required to inhibit B-cell receptor signaling. Phosphorylation of DOCK2 was associated with activation of ERK1/2. Furthermore, siRNA mediated knockdown of DOCK2 could inhibit Wnt5a-induced activation of ERK1/2, as did treatment of the CLL cells with cirmtuzumab. We corroborated these findings in CLL-cell line MEC1, which expresses endogenous Wnt5a but lacks expression of ROR1. We stably transfected ROR1 into MEC1 cells to generate MEC1-ROR1 cells. We found higher-levels phosphorylated DOCK2 and activation of ERK1/2 in MEC1-ROR1 cells than in MEC1 cells lacking ROR1. Treatment of MEC1-ROR1 cells with neutralizing antibodies to Wnt5a or cirmtuzumab suppresed DOCK2 phosphorylation and activation of ERK1/2. Moreover, siRNA mediated knockdown of DOCK2 reduced the level of activation of ERK1/2 in MEC1-ROR1 cells. We examined whether the proline-rich-domain (PRD) of ROR1, and SH3-binding motifs with the PRD that were necessary for these effects. We found that PRD and more specifically proline at 808 position of ROR1 was required to enhance phosphorylation of DOCK2 and activation of ERK1/2 over that seen in MEC1 cells lacking ROR1. We also found that inhibition of Rac using a small molecule inhibitor of Rac1/2 could suppress the capacity of Wnt5a to induce activation of ERK, suggesting that Wnt5a induced activation of Rac was required for activation of ERK. Finally, Wnt5a could not enhance the proliferation of CLL cells when treated with an ERK inhibitor or citmuzumab. Collectively, our results support that Wnt5a/ROR1/DOCK2 signaling axis contributes enhanced activation of ERK to promote proliferation of CLL cells independently of BTK. DisclosuresNo relevant conflicts of interest to declare.

EGF Regulates the Interaction of Tks4 with Src through Its SH2 and SH3 Domains.

The nonreceptor tyrosine kinase Src is a central component of the epidermal growth factor (EGF) signaling pathway. Our group recently showed that the Frank-ter Haar syndrome protein Tks4 (tyrosine kinase substrate with four Src homology 3 domains) is also involved in EGF signaling. Here we demonstrate that Tks4 and Src bind directly to each other and elucidate the details of the molecular mechanism of this complex formation. Results of GST pull-down and fluorescence polarization assays show that both a proline-rich SH3 binding motif (PSRPLPDAP, residues 466-474) and an adjacent phosphotyrosine-containing SH2 binding motif (pYEEI, residues 508-511) in Tks4 are responsible for Src binding. These motifs interact with the SH3 and SH2 domains of Src, respectively, leading to a synergistic enhancement of binding strength and a highly stable, "bidentate"-type of interaction. In agreement with these results, we found that the association of Src with Tks4 is permanent and the complex lasts at least 3 h in living cells. We conclude that the interaction of Tks4 with Src may result in the long term stabilization of the kinase in its active conformation, leading to prolonged Src activity following EGF stimulation.

ICOS Signaling Controls Induction and Maintenance of Collagen-Induced Arthritis.

ICOS is a key costimulatory receptor facilitating differentiation and function of follicular helper T cells and inflammatory T cells. Rheumatoid arthritis patients were shown to have elevated levels of ICOS+ T cells in the synovial fluid, suggesting a potential role of ICOS-mediated T cell costimulation in autoimmune joint inflammation. In this study, using ICOS knockout and knockin mouse models, we found that ICOS signaling is required for the induction and maintenance of collagen-induced arthritis (CIA), a murine model of rheumatoid arthritis. For the initiation of CIA, the Tyr181-based SH2-binding motif of ICOS that is known to activate PI3K was critical for Ab production and expansion of inflammatory T cells. Furthermore, we found that Tyr181-dependent ICOS signaling is important for maintenance of CIA in an Ab-independent manner. Importantly, we found that a small molecule inhibitor of glycolysis, 3-bromopyruvate, ameliorates established CIA, suggesting an overlap between ICOS signaling, PI3K signaling, and glucose metabolism. Thus, we identified ICOS as a key costimulatory pathway that controls induction and maintenance of CIA and provide evidence that T cell glycolytic pathways can be potential therapeutic targets for rheumatoid arthritis.

Abstract P1-01-05: Wnt5a induces ROR1 to associate with cortactin, which undergoes tyrosine phosphorylation, and enhances migration of breast cancer cells

Abstract Background: ROR1is an evolutionarily conserved, oncoembryonic surface-antigen expressed in breast cancer. Previously, we found that ROR1 can serve as a receptor for Wnt5a, which can induce non-canonical Wnt signaling that enhances cancer-cell migration. Recently we found that Wnt5a could induce ROR1 to complex with a cytoskeletal protein designated HS1, which recruited ARHGEF1, enhanced activation of RhoA, and promoted leukemia-cell migration. These effects of Wnt5a on CLL cells could be inhibited by cirmtuzumab, a humanized, high-affinity monoclonal antibody (mAb) specific for ROR1. As expression of HS1 is limited to hematopoietic cells we examined breast cancer cell lines and primary breast cancer cells for expression of cortactin. Cortactin (also called EMS1), is a cytoskeletal protein, which is homologous in structure and function to HS1 and is broadly expressed in human cancers, including breast cancer. Methods: We performed immunoprecipitation studies using high-affinity mAb to ROR1 or cortactin and immunoblot analysis to examine for the association of ROR1 with cortactin and tyrosine phosphorylation of cortactin at Y421. We also assessed the expression levels of cortactin, ROR1, and ARHGEF1, in the MCF7 breast cancer cell line and in breast-cancer patient-derived xenografts (PDX). We also generated MCF7-ROR1 cells, which were stably transfected to express ROR1 or various mutant forms of ROR1 generated to study the structure-function requirements for effective ROR1-cortactin interactions. Results: We found that ROR1 associates with cortactin in freshly-isolated breast cancer PDX tumors or in PDX cells cultured with exogenous Wnt5a. We corroborated these results in breast cancer cell line MCF7-ROR1 cells, which were transfected to express ROR1 and found to migrate more effectively than the parental MCF7 lacking expression of ROR1. Wnt5a also induced cortactin tyrosine phosphorylation at Y421, recruitment of ARHGEF1, and activation of RhoA, which we found associated with enhanced breast-cancer cell migation. The capacity of Wnt5a to induce such changes could be blocked by treatment of the cells with cirmtuzumab. We generated truncated forms of ROR1 without a proline-rich domain (PRD) and found PRD was necessary for this association or Wnt5a-induced cortactin phosphorylation and enhanced cancer-cell migration. Accordingly, we 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 motifs. In contrast to wild-type ROR1, or other ROR1P→A mutants, ROR1P(841)A had impaired capacity to recruit cortactin and ARHGEF1 to ROR1 in response to Wnt5a. Moreover, Wnt5a could not induce cells expressing ROR1P(841)A to phosphorylate cortactin or activate ARHGEF1, and was unable to enhance the motility of the MCF7 cells transfected with this mutant form of ROR1. Conclusions: Collectively, these studies indicate the capacity of cortactin to complex with ROR1 plays an important role in ROR1-dependent Wnt5a-enhanced breast cancer cell migration. These studies also demonstrate that cirmtuzumab can inhibit the formation of cortactin-ROR1 complexes, cortactin phosphorylation, and cancer-cell migration, and metastasis. Citation Format: Hasan MK, Tripple V, Widhopf II GF, Yu J, Zhang S, Parker BA, Kipps TJ. Wnt5a induces ROR1 to associate with cortactin, which undergoes tyrosine phosphorylation, and enhances migration of breast cancer cells [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-01-05.