Undergoes Tyrosine Phosphorylation Research Articles

Dual role of valosin-containing protein (VCP/p97) in mouse sperm during capacitation.

Valosin-containing protein (VCP; aka p97), a member of the AAA (ATPases Associated with various cellular Activities) family, has been associated with a wide range of cellular functions. While previous evidence has shown its presence in mammalian sperm, our study unveils its function in mouse sperm. Notably, we found that mouse VCP does not undergo tyrosine phosphorylation during capacitation and exhibits distinct localization patterns. In the sperm head, it resides within the equatorial segment and, following acrosomal exocytosis, it is released and cleaved. In the flagellum, VCP is observed in the principal and midpiece. Furthermore, our research highlights a unique role for VCP in the cAMP/PKA pathway during capacitation. Pharmacological inhibition of sperm VCP led to reduced intracellular cAMP levels that resulted in decreased phosphorylation in PKA substrates and tyrosine residues and diminished fertilization competence. Our results show that in mouse sperm, VCP plays a pivotal role in regulating cAMP production, probably by the modulation of soluble adenylyl cyclase activity.

Morphological Changes Induced by TKS4 Deficiency Can Be Reversed by EZH2 Inhibition in Colorectal Carcinoma Cells.

The scaffold protein tyrosine kinase substrate 4 (TKS4) undergoes tyrosine phosphorylation by the epidermal growth factor receptor (EGFR) pathway via Src kinase. The TKS4 deficiency in humans is responsible for the manifestation of a genetic disorder known as Frank-Ter Haar syndrome (FTHS). Based on our earlier investigation, the absence of TKS4 triggers migration, invasion, and epithelial-mesenchymal transition (EMT)-like phenomena while concurrently suppressing cell proliferation in HCT116 colorectal carcinoma cells. This indicates that TKS4 may play a unique role in the progression of cancer. In this study, we demonstrated that the enhancer of zeste homolog 2 (EZH2) and the histone methyltransferase of polycomb repressive complex 2 (PRC2) are involved in the migration, invasion, and EMT-like changes in TKS4-deficient cells (KO). EZH2 is responsible for the maintenance of the trimethylated lysine 27 on histone H3 (H3K27me3). We performed transcriptome sequencing, chromatin immunoprecipitation, protein and RNA quantitative studies, cell mobility, invasion, and proliferation studies combined with/without the EZH2 activity inhibitor 3-deazanoplanocine (DZNep). We detected an elevation of global H3K27me3 levels in the TKS4 KO cells, which could be reduced with treatment with DZNep, an EZH2 inhibitor. Inhibition of EZH2 activity reversed the phenotypic effects of the knockout of TKS4, reducing the migration speed and wound healing capacity of the cells as well as decreasing the invasion capacity, while the decrease in cell proliferation became stronger. In addition, inhibition of EZH2 activity also reversed most epithelial and mesenchymal markers. We investigated the wider impact of TKS4 deletion on the gene expression profile of colorectal cancer cells using transcriptome sequencing of wild-type and TKS4 knockout cells, particularly before and after treatment with DZNep. Additionally, we observed changes in the expression of several protein-coding genes and long non-coding RNAs that showed a recovery in expression levels following EZH2 inhibition. Our results indicate that the removal of TKS4 causes a notable disruption in the gene expression pattern, leading to the disruption of several signal transduction pathways. Inhibiting the activity of EZH2 can restore most of these transcriptomics and phenotypic effects in colorectal carcinoma cells.

Blockade of the immunosuppressive KIR2DL5/PVR pathway elicits potent human NK cell-mediated antitumor immunity.

Cancer immunotherapy targeting the TIGIT/PVR pathway is currently facing challenges. KIR2DL5, a member of the human killer cell, immunoglobulin-like receptor (KIR) family, has recently been identified as another binding partner for PVR. The biology and therapeutic potential of the KIR2DL5/PVR pathway are largely unknown. Here we report that KIR2DL5 was predominantly expressed on human NK cells with mature phenotype and cytolytic function and that it bound to PVR without competition with the other 3 known PVR receptors. The interaction between KIR2DL5 on NK cells and PVR on target cells induced inhibitory synapse formation, whereas new monoclonal antibodies blocking the KIR2DL5-PVR interaction robustly augmented the NK cytotoxicity against PVR+ human tumors. Mechanistically, both intracellular ITIM and ITSM of KIR2DL5 underwent tyrosine phosphorylation after engagement, which was essential for KIR2DL5-mediated NK suppression by recruiting SHP-1 and/or SHP-2. Subsequently, ITIM/SHP-1/SHP-2 and ITSM/SHP-1 downregulated the downstream Vav1/ERK1/2/p90RSK/NF-κB signaling. KIR2DL5+ immune cells infiltrated in various types of PVR+ human cancers. Markedly, the KIR2DL5 blockade reduced tumor growth and improved overall survival across multiple NK cell-based humanized tumor models. Thus, our results revealed functional mechanisms of KIR2DL5-mediated NK cell immune evasion, demonstrated blockade of the KIR2DL5/PVR axis as a therapy for human cancers, and provided an underlying mechanism for the clinical failure of anti-TIGIT therapies.

Lawsonia intracellularis LI0666 is a new EPIYA effector exported by the Yersinia enterocolitica type III secretion system

Lawsonia intracellularis is the causative agent of proliferative enteropathy. While it harbors genes encoding the entire apparatus required for the type III secretion system (T3SS) and the expression of some of these components has been detected during experimental infection, the identification of L. intracellularis T3SS substrates (effector proteins) has been hampered. The Yersinia T3SS and yeast growth inhibition assays are two important heterologous systems used for the characterization of effector proteins. Bacterial EPIYA effectors are a distinct class of bacterial effectors defined by the presence of EPIYA or the EPIYA-related motif. When delivered into host cells via a T3SS or type IV secretion system, these effectors undergo tyrosine phosphorylation of the EPIYA motif, which enables them to manipulate host cell signaling by promiscuously interacting with multiple SH2 domain-containing proteins. A previous study showed that L. intracellularis LI0666 contains two EPIYA motifs and speculated that this protein could be a T3SS effector. In this study, we show that LI0666 is secreted by Yersinia in a T3SS-dependent manner and inhibits yeast growth. LI0666 is phosphorylated at tyrosine residues in porcine intestinal epithelial cells and in human epithelial cells. Like the archetypal EPIYA effector CagA, the EPIYA-containing region is not required for LI0666 association with yeast and mammalian cell membranes. Our results indicate that LI0666 is an authentic bacterial EPIYA effector. Identification of the tyrosine kinases that are responsible for LI0666 phosphorylation and the SH2 domain-containing host proteins that LI0666 interacts with will help to explore the molecular mechanisms of LI0666 in disease development.

Wnt5a Induces ROR1 to Bind and Activate Cortactin, Which Functions in Planar Cell Polarity and Migration of Chronic Lymphocytic Leukemia Cells

Studies have revealed a dynamic trafficking of chronic lymphocytic leukemia cells (CLL) between the blood and lymphoid compartments. Targeted therapies that impair chemokine-receptor signaling, such as ibrutinib, disrupt the capacity of leukemia cells to re-enter lymphoid compartments, precluding CLL cells from receiving growth/survival signals from the accessory cells within the lymphoid-tissue microenvironment, thereby causing attrition in the leukemia-cell population over time in a patient undergoing such targeted therapy. Studying the signaling pathways and intracellular proteins that influence/govern leukemia-cell migration may identify additional agents that can further disrupt CLL-cell trafficking and potentially enhance the therapeutic activity of drugs such as ibrutinib. Factoring in leukemia-cell migration may be the cytoskeletal protein cortactin (also called EMS1), which possesses a SH3 domain and a F-actin-binding domain, allowing it link cell-surface receptor-complexes to the actin cytoskeleton. Upon tyrosine phosphorylation, cortactin is activated, thereby facilitating RhoGTPase activation and polymerization and rearrangement of the actin cytoskeleton, especially the actin cortex around the cellular periphery, which is required for planar cell polarity, lamellipodia formation, invadopodia formation, cell migration, and endocytosis. We find cortactin abundantly is expressed in CLL B cells. Moreover, in freshly isolated CLL cells, we find that cortactin associates with ROR1, an evolutionarily conserved, oncoembryonic surface-protein expressed on many cancers, including CLL. Culture of CLL cells in serum-free medium results in the dissociation/inactivation of the ROR1-cortactin complex unless the leukemia cells are stimulated with the ligand for ROR1, namely Wnt5a, which, relative to the plasma of age-matched healthy adults, is present at high levels in the plasma of patients with CLL, including patients undergoing therapy with ibrutinib. Moreover, we find that Wnt5a induces ROR1 to complex with cortactin, which then undergoes tyrosine phosphorylation at Y421, recruit ARHGEF1, and activate RhoA, thereby enhancing/enabling leukemia-cell migration. These effects of Wnt5a on CLL cells could not be inhibited by ibrutinib, but could be blocked by cirmtuzumab, a humanized, high-affinity monoclonal antibody specific for ROR1 that is undergoing clinical evaluation in patients with CLL. We corroborated these findings using the CLL cell line MEC1, which also expresses cortactin, but does not express ROR1. MEC1 cells can be transduced to express ROR1 or various mutant forms of ROR1, allowing us to examine the structure-function relationships required for ROR1-cortactin complex formation. We confirmed that cortactin complexes with ROR1 and undergoes activation in response to Wnt5a in MEC1-ROR1 cells. We generated truncated forms of ROR1 and found the cytoplasmic proline-rich domain (PRD) of ROR1 was required for ROR1 to complex with cortactin 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, 841, or 850 in potential SH3-binding sites. In contrast to wild-type ROR1, or other ROR1P==>A mutants, ROR1P(841)A had impaired capacity to recruit cortactin to ROR1 in response to Wnt5a. Moreover, Wnt5a could not induce cells expressing ROR1P(841)A to phosphorylate cortactin, and was not able to enhance MEC1-ROR1-cell F-actin polymerization. Collectively, these studies reveal that cortactin plays an important role in Wnt5a-enhanced CLL cell migration/F-actin polymerization via a ROR1-dependent signaling pathway that cannot be inhibited by ibrutinib or other inhibitors of BTK. DisclosuresKipps:AbbVie: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria; Oncternal: Research Funding; Gilead: Consultancy, Honoraria, Speakers Bureau; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding.

Helicobacter pylori CagA oncoprotein interacts with SHIP2 to increase its delivery into gastric epithelial cells.

Chronic infection with Helicobacter pylori cagA‐positive strains is causally associated with the development of gastric diseases, most notably gastric cancer. The cagA‐encoded CagA protein, which is injected into gastric epithelial cells by bacterial type IV secretion, undergoes tyrosine phosphorylation at the Glu‐Pro‐Ile‐Tyr‐Ala (EPIYA) segments (EPIYA‐A, EPIYA‐B, EPIYA‐C, and EPIYA‐D), which are present in various numbers and combinations in its C‐terminal polymorphic region, thereby enabling CagA to promiscuously interact with SH2 domain‐containing host cell proteins, including the prooncogenic SH2 domain‐containing protein tyrosine phosphatase 2 (SHP2). Perturbation of host protein functions by aberrant complex formation with CagA has been considered to contribute to the development of gastric cancer. Here we show that SHIP2, an SH2 domain‐containing phosphatidylinositol 5′‐phosphatase, is a hitherto undiscovered CagA‐binding host protein. Similar to SHP2, SHIP2 binds to the Western CagA‐specific EPIYA‐C segment or East Asian CagA‐specific EPIYA‐D segment through the SH2 domain in a tyrosine phosphorylation‐dependent manner. In contrast to the case of SHP2, however, SHIP2 binds more strongly to EPIYA‐C than to EPIYA‐D. Interaction with CagA tethers SHIP2 to the plasma membrane, where it mediates production of phosphatidylinositol 3,4‐diphosphate [PI(3,4)P2]. The CagA‐SHIP2 interaction also potentiates the morphogenetic activity of CagA, which is caused by CagA‐deregulated SHP2. This study indicates that initially delivered CagA interacts with SHIP2 and thereby strengthens H. pylori‐host cell attachment by altering membrane phosphatidylinositol compositions, which potentiates subsequent delivery of CagA that binds to and thereby deregulates the prooncogenic phosphatase SHP2.

Mouse t-complex protein 11 is important for progressive motility in sperm†.

The t-complex is defined as naturally occurring variants of the proximal third of mouse chromosome 17 and has been studied by mouse geneticists for decades. This region contains many genes involved in processes from embryogenesis to sperm function. One such gene, t-complex protein 11 (Tcp11), was identified as a testis-specific gene whose protein is present in elongating spermatids. Later work on Tcp11 localized TCP11 to the sperm surface and acrosome cap and implicated TCP11 as important for sperm capacitation through the cyclic AMP/Protein Kinase A pathway. Here, we show that TCP11 is cytoplasmically localized to elongating spermatids and absent from sperm. In the absence of Tcp11, male mice have severely reduced fertility due to a significant decrease in progressively motile sperm; however, Tcp11-null sperm continues to undergo tyrosine phosphorylation, a hallmark of capacitation. Interestingly, null sperm displays reduced PKA activity, consistent with previous reports. Our work demonstrates that TCP11 functions in elongated spermatids to confer proper motility in mature sperm.

RIP2 promotes FcγR-mediated reactive oxygen species production

Receptor-interacting protein 2 (RIP2) is a kinase that mediates signaling downstream of the bacterial peptidoglycan sensors NOD1 and NOD2. Genetic loss or pharmaceutical inhibition of RIP2 has been shown to be beneficial in multiple inflammatory disease models with the effects largely attributed to reducing proinflammatory signaling downstream of peptidoglycan recognition. However, given the widespread expression of this kinase and its reported interactions with numerous other proteins, it is possible that RIP2 may also function in roles outside of peptidoglycan sensing. In this work, we show that RIP2 undergoes tyrosine phosphorylation and activation in response to engagement of the Fc γ receptor (FcγR). Using bone marrow-derived macrophages from WT and RIP2-KO mice, we show that loss of RIP2 leads to deficient FcγR signaling and reactive oxygen species (ROS) production upon FcγR cross-linking without affecting cytokine secretion, phagocytosis, or nitrate/nitrite production. The FcγR-induced ROS response was still dependent on NOD2, as macrophages deficient in this receptor showed similar defects. Mechanistically, we found that different members of the Src family kinases (SFKs) can promote RIP2 tyrosine phosphorylation and activation. Altogether, our findings suggest that RIP2 is functionally important in pathways outside of bacterial peptidoglycan sensing and that involvement in such pathways may depend on the actions of SFKs. These findings will have important implications for future therapies designed to target this kinase.

Wnt5a causes ROR1 to complex and activate cortactin to enhance migration of chronic lymphocytic leukemia cells

Chronic lymphocytic leukemia cells (CLL) migrate between the blood and lymphoid tissues in response to chemokines. Such migration requires structured cytoskeletal-actin polymerization, which may involve the protein cortactin. We discovered that treatment of CLL cells with Wnt5a causes Receptor tyosin kinase-like orphan receptor 1 (ROR1) to bind cortactin, which undergoes tyrosine phosphorylation at Y421, recruits ARHGEF1, and activates RhoA, thereby enhancing leukemia-cell migration; such effects could be inhibited by cirmtuzumab, a humanized mAb specific for ROR1. We transfected the CLL-cell-line MEC1 with either full-length ROR1 or various mutant forms of ROR1 to examine the structural features required for binding cortactin. We found that the proline-rich domain (PRD) was necessary for ROR1 to recruit cortactin. We generated MEC1 cells that each expressed a mutant form of ROR1 with a single amino-acid substitution of alanine (A) for proline (P) in potential SH3-binding sites in the ROR1-PRD at positions 784, 808, 826, 841, or 850. In contrast to wild-type ROR1, or other ROR1P=>A mutants, ROR1P(841)A failed to complex with cortactin or ARHGEF1 in response to Wnt5a. Moreover, Wnt5a could not induce MEC1-ROR1P(841)A to phosphorylate cortactin or enhance CLL-cell F-actin polymerization. Taken together, these studies show that cortactin plays an important role in ROR1-dependent Wnt5a-enhanced CLL-cell migration.

YAP Tyrosine Phosphorylation and Nuclear Localization in Cholangiocarcinoma Cells Are Regulated by LCK and Independent of LATS Activity.

The Hippo pathway effector, Yes-associated protein (YAP), is a transcriptional coactivator implicated in cholangiocarcinoma (CCA) pathogenesis. YAP is known to be regulated by a serine/threonine kinase relay module (MST1/2-LATS1/2) culminating in phosphorylation of YAP at Serine 127 and cytoplasmic sequestration. However, YAP also undergoes tyrosine phosphorylation, and the role of tyrosine phosphorylation in YAP regulation remains unclear. Herein, YAP regulation by tyrosine phosphorylation was examined in human and mouse CCA cells, as well as patient-derived xenograft (PDX) models. YAP was phosphorylated on tyrosine 357 (Y357) in CCA cell lines and PDX models. SRC family kinase (SFK) inhibition with dasatinib resulted in loss of YAPY357 phosphorylation, promoted its translocation from the nucleus to the cytoplasm, and reduced YAP target gene expression, including cell lines expressing a LATS1/2-resistant YAP mutant in which all serine residues were mutated to alanine. Consistent with these observations, precluding YAPY357 phosphorylation by site-directed mutagenesis (YAPY357F) excluded YAP from the nucleus. Targeted siRNA experiments identified LCK as the SFK that most potently mediated YAPY357 phosphorylation. Likewise, inducible CRISPR/Cas9-targeted LCK deletion decreased YAPY357 phosphorylation and its nuclear localization. The importance of LCK in CCA biology was demonstrated by clinical observations suggesting LCK expression levels were associated with early tumor recurrence following resection of CCA. Finally, dasatinib displayed therapeutic efficacy in PDX models. Mol Cancer Res; 16(10); 1556-67. ©2018 AACR.

Positive regulatory role of c-Src-mediated TRIM25 tyrosine phosphorylation on RIG-I ubiquitination and RIG-I-mediated antiviral signaling pathway

Retinoic acid-inducible gene I (RIG-I) detects viral RNAs and induces antiviral responses. During viral RNA recognition by RIG-I, tripartite motif protein 25 (TRIM25) plays a critical regulatory role by inducing K63-linked RIG-I polyubiquitination. Previous proteomics analysis revealed several phosphorylation sites on TRIM25, including tyrosine 278 (Y278), yet the roles of these modifications remain elusive. Here, we demonstrated that TRIM25 interacted with c-Src and underwent tyrosine phosphorylation by c-Src kinase upon viral infection and the phosphorylation is required for the complete activation of RIG-I signaling. Analysis using a c-Src inhibitor and TRIM25 mutant, in which tyrosine 278 is substituted by phenylalanine (Y278F), suggested that the phosphorylation positively regulates K63-linked polyubiquitination of RIG-I and subsequent antiviral signaling. The TRIM25 Y278F mutant displayed decreased E3-ubiquitin ligase activity in vitro, suggesting that this phosphorylation event affects the E3-ligase activity of TRIM25. Thus, we provide a molecular mechanism of c-Src-mediated positive regulation of RIG-I signaling.

Transcriptional Co-activator Functions of YAP and TAZ Are Inversely Regulated by Tyrosine Phosphorylation Status of Parafibromin

SummaryYAP and TAZ, the Hippo signal-regulated transcriptional co-activators, play crucial roles in morphogenesis and organogenesis. Here we report that the YAP/TAZ activities are stimulated upon complex formation with Parafibromin, which undergoes tyrosine phosphorylation and dephosphorylation by kinases such as PTK6 and phosphatases such as SHP2, respectively. Furthermore, TAZ and the Wnt effector β-catenin interact cooperatively with tyrosine-dephosphorylated Parafibromin, which synergistically stimulates the co-activator functions of TAZ and β-catenin. On the other hand, YAP is selectively activated through binding with tyrosine-phosphorylated Parafibromin, which does not interact with β-catenin and thus cannot co-activate YAP and β-catenin. These findings indicate that Parafibromin inversely regulates the activities of YAP and TAZ depending on its tyrosine phosphorylation status. They also suggest that YAP and TAZ exert their redundant and non-redundant biological actions through mutually exclusive interaction with Parafibromin, which is regulated by a balance of kinase and phosphatase activities toward Parafibromin.

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.

\u03b23 phosphorylation of platelet \u03b1IIb\u03b23 is crucial for stability of arterial thrombus and microparticle formation in vivo

BackgroundIt is well accepted that functional activity of platelet integrin αIIbβ3 is crucial for hemostasis and thrombosis. The β3 subunit of the complex undergoes tyrosine phosphorylation shown to be critical for outside-in integrin signaling and platelet clot retraction ex vivo. However, the role of this important signaling event in other aspects of prothrombotic platelet function is unknown.MethodHere, we assess the role of β3 tyrosine phosphorylation in platelet function regulation with a knock-in mouse strain, where two β3 cytoplasmic tyrosines are mutated to phenylalanine (DiYF). We employed platelet transfusion technique and intravital microscopy for observing the cellular events involved in specific steps of thrombus growth to investigate in detail the role of β3 tyrosine phosphorylation in arterial thrombosis in vivo.ResultsUpon injury, DiYF mice exhibited delayed arterial occlusion and unstable thrombus formation. The mean thrombus volume in DiYF mice formed on collagen was only 50% of that in WT. This effect was attributed to DiYF platelets but not to other blood cells and endothelium, which also carry these mutations. Transfusion of isolated DiYF but not WT platelets into irradiated WT mice resulted in reversal of the thrombotic phenotype and significantly prolonged blood vessel occlusion times. DiYF platelets exhibited reduced adhesion to collagen under in vitro shear conditions compared to WT platelets. Decreased platelet microparticle release after activation, both in vitro and in vivo, were observed in DiYF mice compared to WT mice.Conclusionβ3 tyrosine phosphorylation of platelet αIIbβ3 regulates both platelet pro-thrombotic activity and the formation of a stable platelet thrombus, as well as arterial microparticle release.

Subversion of host kinases: a key network in cellular signaling hijacked by Helicobacter pylori CagA.

Helicobacter pylori is a paradigm of persistent pathogens and major risk factor for developing severe diseases including adenocarcinoma in the human stomach. An important bacterial factor linked to gastric disease progression is the cag pathogenicity island-encoded type-IV secretion system (T4SS) effector protein CagA. Translocated CagA undergoes tyrosine phosphorylation at EPIYA-motifs and then activates or inactivates multiple host signaling proteins in a phosphorylation-dependent and phosphorylation-independent fashion. In this way, intracellular CagA acts as a 'masterkey' or 'picklock', which evolved during evolution to hijack key host cell signal transduction functions. Crucial targets of CagA represent a variety of serine/threonine and tyrosine kinases, which control major checkpoints of eukaryotic signaling. Here we review the signal transmission by translocated CagA on multiple receptor kinases (c-Met and EGFR) and non-receptor kinases (Src, Abl, Csk, aPKC, Par1, PI3K, Akt, FAK, GSK-3, JAK, PAK1, PAK2 and MAP kinases), manipulating a selection of fundamental processes in the human gastric epithelium such as cell adhesion, polarity, proliferation, motility, receptor endocytosis, cytoskeletal rearrangements, apoptosis, inflammation and cell cycle progression. This enormous complexity generates a highly remarkable and puzzling scenario during H. pylori infection. The contribution of these signaling pathways to bacterial survival, persistence and gastric pathogenesis is discussed.

Tyrosine Phosphorylation of NLRP3 by Lyn Suppresses NLRP3 Inflammasome Activation

Abstract The NLRP3 inflammasome is a multi-protein complex that triggers the activation of inflammatory caspase-1 and the maturation of IL-1β and IL-18 in response to microbes and danger signals in the cytosol of host cells. However, how the NLRP3 inflammasome is regulated is not well defined. Here, we showed that NLRP3 undergoes tyrosine phosphorylation in macrophages upon stimulation with ATP, a NLRP3 inflammsome stimulus, and the tyrosine phosphorylation of NLRP3 correlates with its ubiquitination. Pretreating the macrophages with a specific Src kinase inhibitor inhibits NLRP3 tyrosine phosphorylation and ubiquitination, which leads to enhanced production of IL-1β. Further we found that Src family PTK Lyn phosphorylates NLRP3 at Tyr918 which facilitates its ubiquitination and proteasome-mediated degradation. Consistent with these data, NLRP3 tyrosine phosphorylation and ubiquitination is abrogated in macrophages lacking Lyn, which correlates with heightened-activation of NLRP3 inflammasome. Therefore, our data demonstrates that the Lyn-mediated tyrosine phosphorylation of NLRP3 is prerequisite for its ubiquitination, thus dampening the NLRP3 inflammasome activity.

Type IV Secretion and Signal Transduction of Helicobacter pylori CagA through Interactions with Host Cell Receptors.

Helicobacter pylori is a highly successful human bacterium, which is exceptionally equipped to persistently inhabit the human stomach. Colonization by this pathogen is associated with gastric disorders ranging from chronic gastritis and peptic ulcers to cancer. Highly virulent H. pylori strains express the well-established adhesins BabA/B, SabA, AlpA/B, OipA, and HopQ, and a type IV secretion system (T4SS) encoded by the cag pathogenicity island (PAI). The adhesins ascertain intimate bacterial contact to gastric epithelial cells, while the T4SS represents an extracellular pilus-like structure for the translocation of the effector protein CagA. Numerous T4SS components including CagI, CagL, CagY, and CagA have been shown to target the integrin-β1 receptor followed by translocation of CagA across the host cell membrane. The interaction of CagA with membrane-anchored phosphatidylserine and CagA-containing outer membrane vesicles may also play a role in the delivery process. Translocated CagA undergoes tyrosine phosphorylation in C-terminal EPIYA-repeat motifs by oncogenic Src and Abl kinases. CagA then interacts with an array of host signaling proteins followed by their activation or inactivation in phosphorylation-dependent and phosphorylation-independent fashions. We now count about 25 host cell binding partners of intracellular CagA, which represent the highest quantity of all currently known virulence-associated effector proteins in the microbial world. Here we review the research progress in characterizing interactions of CagA with multiple host cell receptors in the gastric epithelium, including integrin-β1, EGFR, c-Met, CD44, E-cadherin, and gp130. The contribution of these interactions to H. pylori colonization, signal transduction, and gastric pathogenesis is discussed.

Milk fat globule epithelial growth factor VIII (MFG-E8) sustains survival of cancer cells by prompting tumor angiogenesis and suppressing host immunities ⁎

Abstract Milk fat globule epithelial growth factor VIII (MFG-E8) is a novel adhesion protein mainly produced by macrophages and dendritic cells; it is expressed in most of the human tissues and functions to prompt cancer progression and survival. MFG-E8 contains a signal sequence for secretion, two epidermal growth factor (EGF)-like domains at the NH2 terminus and two discoidin domains with blood-clotting factor V/factor VIII (C1 and C2) at the COOH terminus. The second EGF domain contains an arginine-glycine-aspartic (RGD) integrin-binding motif that engages αvβ5 integrins to facilitate cell adhesion and induce integrinmediated signal transduction. Integrin αvβ3 associates with VEGF receptor 2, engagement of integrins can promote angiogenesis, which plays key roles in growth, proliferation, and survival of cancer cells. VEGF stimulates the expression of αvβ3 and αvβ5 integrins on angiogenic vasculature, thereby potentiating effects of VEGF receptor engagement. Mice expressing a mutant form of αvβ3 integrin are unable to undergo tyrosine phosphorylation, confirming the important role that this integrin plays in pathological angiogenesis and providing important mechanistic insights. The C-terminus discoidin-like domains promote binding to membrane phospholipids, functioning close to VEGF like angiogenesis. MFG-E8 is an opsonin for apoptotic cells, and it acts as a bridging protein between apoptotic cells and phagocytes. It also influences cell immunities by altering CD4+ and/or CD8+ cells. Antibody or small peptide works with MFG-E8 at different functional sites or interacts with EGF-like domains and/or discoidin-like domains may play an important role in anti-angiogenesis or immune restoration. Altering the structures and/or functions of MFG-E8 and/or its domains is promising for development of novel anti-cancer strategies. List of abbreviations: milk fat globule epithelial growth factor VIII (MFG-E8); vascular endothelial growth factors (VEGFs); fibroblast growth factor (FGF); tripeptide Arg-Gly-Asp (RGD); granulocyte/monocyte colony-stimulating factor (GM-CSF); cyclin-dependent kinase inhibitor 1 (P21WAF1/CIP1); B-cell lymphoma 2/Bcl-2 associated X protein (Bcl-2/Bax); platelet-derived growth factor receptor β (PDGFRβ); tumor cells proliferation rate index (Ki-67); toll-like receptor (TLR)

Structure and function of Helicobacter pylori CagA, the first-identified bacterial protein involved in human cancer.

Chronic infection with Helicobacter pylori cagA-positive strains is the strongest risk factor of gastric cancer. The cagA gene-encoded CagA protein is delivered into gastric epithelial cells via bacterial type IV secretion, where it undergoes tyrosine phosphorylation at the Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs. Delivered CagA then acts as a non-physiological scaffold/hub protein by interacting with multiple host signaling molecules, most notably the pro-oncogenic phosphatase SHP2 and the polarity-regulating kinase PAR1/MARK, in both tyrosine phosphorylation-dependent and -independent manners. CagA-mediated manipulation of intracellular signaling promotes neoplastic transformation of gastric epithelial cells. Transgenic expression of CagA in experimental animals has confirmed the oncogenic potential of the bacterial protein. Structural polymorphism of CagA influences its scaffold function, which may underlie the geographic difference in the incidence of gastric cancer. Since CagA is no longer required for the maintenance of established gastric cancer cells, studying the role of CagA during neoplastic transformation will provide an excellent opportunity to understand molecular processes underlying “Hit-and-Run” carcinogenesis.

Wnt5a Induces ROR1 to Complex with HS1, Which Undergoes Tyrosine Phosphorylation and Contributes to Planar-Cell-Polarity Migration in Chronic Lymphocytic Leukemia

ROR1 (Receptor tyrosine kinase-like orphan receptor 1) is an evolutionarily conserved, oncoembryonic antigen that is expressed on CLL cells, but not on normal postpartum tissues. In prior studies we found that ROR1 was a receptor for Wnt5a, which could activate RhoGTPases and enhance chemokine-directed leukemia-cell migration. Moreover, Wnt5a-enhanced migration could be inhibited by cirmtuzumab, a first-in-class humanized mAb specific for a functional epitope of the ROR1 extracellular domain; this mAb is undergoing phase I clinical testing in patients with CLL. However, it is not known whether ROR1 contributes to cell migration only through its recruitment/activation of guanine exchange factors (GEFs) or whether it also can complex with proteins that directly interact with the actin cytoskeleton. We performed mass spectrometry-based proteomics to interrogate immune-precipitates of Wnt5a-activated ROR1 from CLL cells and identified HS1 (or HCLS1, hematopoietic cell-specific Lyn substrate 1), a conserved cytoplasmic protein that can interact with the actin cytoskeleton. Moreover, tyrosine phosphorylation of HS1 has been found in prior studies to be associated with enhanced leukemia-cell migration and adverse prognosis in CLL, although the stimuli/mechanism(s) contributing to HS1 activation were unknown. We validated that Wnt5a induced the association of ROR1 with HS1 in primary CLL cells via co-immunoprecipitation studies and immunoblot analyses. Moreover, we found that Wnt5a could induce tyrosine phosphorylation of HS1 in CLL cells previously deprived of Wnt5a. On the other hand, treatment of CLL cells with the anti-ROR1 mAb cirmtuzumab inhibited the capacity of Wnt5a to induce the association of ROR1 with HS1 or tyrosine phosphorylation of HS1. We speculated that the Src-homology 3 (SH3) domain of HS1 allowed it to dock onto the cytoplasmic proline-rich domain (PRD) of ROR1. Consistent with this notion we found that a mutant-form of ROR1 lacking the PRD was unable to complex with HS1 or facilitate F-actin polymerization in response to Wnt5a. Accordingly, we used site-directed mutagenesis to introduce single amino acid substitutions of proline (P) to alanine (A) in the P-X-X-P- motifs of the ROR1 PRD at positions 784, 808, 826, 841, or 850. Each mutant form of ROR1, except for P841A-ROR1, could complex with HS1, permit tyrosine phosphorylation of HS1, and allow for enhanced leukemia-cell motility. Collectively, these data indicate that Wnt5a can induce HS1 to dock at P841 in the PRD of ROR1 and undergo tyrosine phosphorylation, thereby enhancing F-actin polymerization and planar-cell-polarity leukemia-cell migration. Moreover, we found that cirmtuzumab can inhibit activation of HS1 and F-actin polymerization in response to Wnt5a, potentially accounting in part for its capacity to inhibit Wnt5a-enhanced leukemia-cell migration. DisclosuresKipps:Gilead: Consultancy, Honoraria, Speakers Bureau; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria.