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The cellular mechanisms mediating norepinephrine (NE) functions in brain to result in behaviors are unknown. We identified the L-type Ca2+ channel (LTCC) CaV1.2 as a principal target for Gq-coupled α1-adrenergic receptors (ARs). α1AR signaling increased LTCC activity in hippocampal neurons. This regulation required protein kinase C (PKC)-mediated activation of the tyrosine kinases Pyk2 and, downstream, Src. Pyk2 and Src were associated with CaV1.2. In model neuroendocrine PC12 cells, stimulation of PKC induced tyrosine phosphorylation of CaV1.2, a modification abrogated by inhibition of Pyk2 and Src. Upregulation of LTCC activity by α1AR and formation of a signaling complex with PKC, Pyk2, and Src suggests that CaV1.2 is a central conduit for signaling by NE. Indeed, a form of hippocampal long-term potentiation (LTP) in young mice requires both the LTCC and α1AR stimulation. Inhibition of Pyk2 and Src blocked this LTP, indicating that enhancement of CaV1.2 activity via α1AR-Pyk2-Src signaling regulates synaptic strength.

Dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) is an evolutionarily conserved protein kinase belonging to the CMGC kinase family, which is closely related to Down syndrome (DS) and Alzheimer's disease (AD). In recent years, not only the treatment of diabetes, but also the treatment of cancer gradually focuses on targeting DYRK1A. Therefore, a series of DYRK1A inhibitors have been developed to treat relevant diseases and clarify their treatment mechanism furtherly. DYRK1A inhibitors are mainly divided into natural products and synthetic compounds. Among them, harmine is an excellent DYRK1A inhibitor. Therefore, the synthetic DYRK1A inhibitors are mainly based on harmine, which greatly enriches the structure and quantity of DYRK1A inhibitors. The interaction between the inhibitors and the DYRK1A protein has a guiding significance in predicting the activity of the inhibitors, and plays an irreplaceable role in the design of the compounds. This paper mainly reviews DYRK1A inhibitors found in recent years and their structure activity relationship, looking forward to providing a theoretical basis for the development of DYRK1A inhibitors.

Hepatocellular carcinoma (HCC), a heterogeneous cancer with high mortality, is resistant to single targeted therapy; thus, combination therapy based on synthetic lethality is a promising therapeutic strategy for HCC. Poly (adenosine diphosphate [ADP]-ribose) polymerase 1 (PARP1) is the most recognized target for synthetic lethality; however, the therapeutic effect of PARP1 inhibition on HCC is disappointing. Therefore, exploring new synthetic lethal partners for the efficient manipulation of HCC is urgently required. In this study, we identified Src and PARP1 as novel synthetic lethal partners, and the combination therapy produced significant anti-tumor effects without causing obvious side effects. Mechanistically, Src interacted with PARP1 and phosphorylated PARP1 at the Y992 residue, which further mediated resistance to PARP1 inhibition. Overall, this study revealed that Src-mediated PARP1 phosphorylation induced HCC resistance to PARP1 inhibitors and indicated a therapeutic window of the Y992 phosphorylation of PARP1 for HCC patients. Moreover, synthetic lethal therapy by co-targeting PARP1 and Src have the potential to broaden the strategies for HCC and might benefit HCC patients with high Src activation and resistance to PARP1 inhibitors alone.

Ras genes are frequently mutated in many cancer types; however, there are currently no conclusively effective anticancer drugs against Ras-induced cancer. Therefore, the downstream effectors of Ras signaling need to be identified for the development of promising novel therapeutic approaches. We previously reported that oncogenic Ras induced the expression of NF-HEV/IL-33, a member of the interleukin-1 family, and showed that intracellular IL-33 was required for oncogenic Ras-induced cellular transformation. In the present study, we demonstrated that the c-Mer proto-oncogene tyrosine kinase (MerTK), a receptor tyrosine kinase, played essential roles in oncogenic Ras/IL-33 signaling. The expression of MerTK was enhanced in transformed NIH-3T3 cells by the expression of oncogenic Ras, H-Ras (G12V), in an IL-33-dependent manner. In human colorectal cancer tissues, MerTK expression also correlated with IL-33 expression. The knockdown of IL-33 or MerTK effectively attenuated the migration of NIH-3T3 cells transformed by H-Ras (G12V) and A549, LoVo, and HCT116 cells harboring an oncogenic K-Ras mutation. Furthermore, the suppression of Ras-induced cell migration by the knockdown of IL-33 was rescued by the enforced expression of MerTK. The present results also revealed that MerTK was effectively phosphorylated in NIH-3T3 cells transformed by Ras (G12V). Ras signaling was essential for the tyrosine phosphorylation of MerTK, and the kinase activity of MerTK was indispensable for accelerating cell migration. Collectively, the present results reveal a novel role for MerTK in cancer malignancy, which may be utilized to develop novel therapeutic strategies that target Ras-transformed cells.

Background C3G is a guanine nucleotide exchange factor (GEF) that activates Rap1 to promote cell adhesion. Resting C3G is autoinhibited and the GEF activity is released by stimuli that signal through tyrosine kinases. C3G is activated by tyrosine phosphorylation and interaction with Crk adaptor proteins, whose expression is elevated in multiple human cancers. However, the molecular details of C3G activation and the interplay between phosphorylation and Crk interaction are poorly understood. Methods We combined biochemical, biophysical, and cell biology approaches to elucidate the mechanisms of C3G activation. Binding of Crk adaptor proteins to four proline-rich motifs (P1 to P4) in C3G was characterized in vitro using isothermal titration calorimetry and sedimentation velocity, and in Jurkat and HEK293T cells by affinity pull-down assays. The nucleotide exchange activity of C3G over Rap1 was measured using nucleotide-dissociation kinetic assays. Jurkat cells were also used to analyze C3G translocation to the plasma membrane and the C3G-dependent activation of Rap1 upon ligation of T cell receptors. Results CrkL interacts through its SH3N domain with sites P1 and P2 of inactive C3G in vitro and in Jurkat and HEK293T cells, and these sites are necessary to recruit C3G to the plasma membrane. However, direct stimulation of the GEF activity requires binding of Crk proteins to the P3 and P4 sites. P3 is occluded in resting C3G and is essential for activation, while P4 contributes secondarily towards complete stimulation. Tyrosine phosphorylation of C3G alone causes marginal activation. Instead, phosphorylation primes C3G lowering the concentration of Crk proteins required for activation and increasing the maximum activity. Unexpectedly, optimal activation also requires the interaction of CrkL-SH2 domain with phosphorylated C3G. Conclusion Our study revealed that phosphorylation of C3G by Src and Crk-binding form a two-factor mechanism that ensures tight control of C3G activation. Additionally, the simultaneous SH2 and SH3N interaction of CrkL with C3G, required for the activation, reveals a novel adaptor-independent function of Crk proteins relevant to understanding their role in physiological signaling and their deregulation in diseases.

A previous study has shown that Cetrimonium bromide has a slight cytotoxicity to head and neck cancer cells due to the presence of the cationic quaternary amine group in this molecule that affects the function of mitochondria.The same study also found that the cytotoxicity of Cetrimonium bromide increased when combined with gamma radiation and this could be used to treat head and neck cancer in a mice in vivo tumor model.In the current study, an in vitro photochemical reaction between Cetrimonium chloride and tyrosine was investigated in order to understand the molecular mechanism of the in vivo observations and data. I found that Cetrimonium chloride could react with tyrosine upon simulated solar irradiation to produce an imine Schiff base which turned into cyano-tyrosine and a melanin polymer.It is possible that in the in vivo study mentioned earlier, the gamma radiation and Cetrimonium bromide together destroyed the tyrosine residues in some cellular proteins chemically, and as a result, tyrosine phosphorylation was inhibited in the cancer cells which slowed the growth of the tumor.

STAT3 plays a prominent role in proliferation and survival of tumor cells. Thus, STAT3 has been considered to be a prime target for development of anti-cancer therapeutics. The electrophilic cyclopentenone prostaglandin,15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) has been well recognized for its capability to modulate intracellular signaling pathways involved in cancer cell growth and progression. We previously reported that 15d-PGJ2 had potent cytotoxicity against harvey-ras transformed human mammary epithelial cells through direct interaction with STAT3. In this study, we have attempted to verify the inhibitory effects of 15d-PGJ2 on STAT3 signaling in human breast tumor cells. The triple negative breast cancer cell lines, MDA-MB-231 and MDA-MB-468 displaying constitutive phosphorylation of STAT3 on the tyrosine 705 (Tyr705) residue, underwent apoptosis upon inhibition of STAT3 by 15d-PGJ2. In contrast, estrogen receptor positive MCF-7 breast cancer cells that do not exhibit elevated STAT3 phosphorylation were much less susceptible to 15d-PGJ2-induced apoptosis as assessed by PARP cleavage. Furthermore, 15d-PGJ2 inhibited interleukin-6-induced tyrosine phosphorylation of STAT3 in LNCaP cells. According to molecular docking studies, 15d-PGJ2 may preferentially bind to the cysteine 259 residue (Cys259) present in the coiled-coil domain of STAT3. Site-directed mutagenesis of STAT3 identified Cys259 to be the critical amino acid for the 15d-PGJ2-induced apoptosis as well as epithelial-to-mesenchymal transition. Taken together, these findings suggest STAT3 inactivation through direct chemical modification of its Cys259 as a potential therapeutic approach for treatment of triple negative breast cancer treatment.

Chronic respiratory diseases (CRDs), including pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), lung cancer, and asthma, are significant global health problems due to their prevalence and rising incidence. The roles of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) in controlling tyrosine phosphorylation of targeting proteins modulate multiple physiological cellular responses and contribute to the pathogenesis of CRDs. Src homology-2 domain-containing PTP2 (SHP2) plays a pivotal role in modulating downstream growth factor receptor signaling and cytoplasmic PTKs, including MAPK/ERK, PI3K/AKT, and JAK/STAT pathways, to regulate cell survival and proliferation. In addition, SHP2 mutation and activation are commonly implicated in tumorigenesis. However, little is known about SHP2 in chronic pulmonary inflammation and fibrosis. This review discusses the potential involvement of SHP2 deregulation in chronic pulmonary inflammation and fibrosis, as well as the therapeutic effects of targeting SHP2 in CRDs.

The membrane protein SIRPα is a cold stress-responsive signaling molecule in neurons. Cold stress directly induces tyrosine phosphorylation of SIRPα in its cytoplasmic region, and phosphorylated SIRPα is involved in regulating experience-dependent behavioral changes in mice. Here, we examined the mechanism of cold stress-induced SIRPα phosphorylation in vitro and in vivo. The levels of activated Src family protein tyrosine kinases (SFKs), which phosphorylate SIRPα, were not increased by lowering the temperature in cultured neurons. Although the SFK inhibitor dasatinib markedly reduced SIRPα phosphorylation, low temperature induced an increase in SIRPα phosphorylation even in the presence of dasatinib, suggesting that SFK activation is not required for low temperature-induced SIRPα phosphorylation. However, in the presence of pervanadate, a potent inhibitor of protein tyrosine phosphatases (PTPases), SIRPα phosphorylation was significantly reduced by lowering the temperature, suggesting that either the inactivation of PTPase(s) that dephosphorylate SIRPα or increased protection of phosphorylated SIRPα from the PTPase activity is important for low temperature-induced SIRPα phosphorylation. Inactivation of PTPase Shp2 by the allosteric Shp2 inhibitor SHP099, but not by the competitive inhibitor NSC-87877, reduced SIRPα phosphorylation in cultured neurons. Shp2 knockout also reduced SIRPα phosphorylation in the mouse brain. Our data suggest that Shp2, but not SFKs, positively regulates cold stress-induced SIRPα phosphorylation in a PTPase activity-independent manner.

SHP2 is a protein tyrosine phosphatase (PTP) that can regulate the tyrosine phosphorylation level. Overexpression of SHP2 will promote the development of cancer diseases, so SHP2 has become one of the popular targets for the treatment of cancer. Studies have reported that both SHP099 and SHP844 are inhibitors of SHP2 and bind to different allosteric sites 1 and 2, respectively. Studies have shown that combining SHP099 with SHP844 will enhance pharmacological pathway inhibition in cells. This study uses molecular dynamic simulations to explore the dual allosteric targeted inhibition mechanism. The result shows that the residues THR108-TRP112 (allosteric site 1) move to LEU236-GLN245 (αB-αC link loop in PTP domain) , the residues of GLN79-GLN87 (allosteric site 2) get close to LEU262-GLN269 (αA-αB link loop in PTP domain) and HIS458-ARG465 (P-loop) come near to ARG501-THR507 (Q-loop) in SHP2-SHP099-SHP844 system, which makes the "inactive conformation" more stable and prevents the substrate from entering the catalytic site. Meanwhile, residue GLU110 (allosteric site 1), ARG265 (allosteric site 2), and ARG501 (Q-loop) are speculated to be the key residues that causing the SHP2 protein in auto-inhibition conformation. It is hoped that this study will provide clues for the development of the dual allosteric targeted inhibition of SHP2.

Clostridium butyricum (C. butyricum) is a probiotic that could promote animal growth and protect gut health. So far, current studies mainly keep up with the basic biological functions of C. butyricum, missing the effective strategy to further improve its protective efficiency. A recent report about C. butyricum alleviating intestinal injury through epidermal growth factor receptor (EGFR) inspired us to bridge this gap by porcine epidermal growth factor (EGF) overexpression. Lacking a secretory overexpression system, we constructed the recombinant strains overexpressing pEGF in C. butyricum for the first time and obtained 4 recombinant strains for highly efficient secretion of pEGF (BC/pPD1, BC/pSPP, BC/pGHF, and BC/pDBD). Compared to the wild-type strain, we confirmed that the expression level ranges of the intestinal development-related genes (Claudin-1, GLUT-2, SUC, GLP2R, and EGFR) and anti-inflammation-related gene (IL-10) in IPECs were upregulated under recombinant strain stimulation, and the growth of Staphylococcus aureus and Salmonella typhimurium was significantly inhibited as well. Furthermore, a particular inhibitor (stattic) was used to block STAT3 tyrosine phosphorylation, resulting in the downregulation on antibacterial effect of recombinant strains. This study demonstrated that the secretory overexpression of pEGF in C. butyricum could upregulate the expression level of EGFR, consequently improving the intestinal protective functions of C. butyricum partly following STAT3 signal activation in IPECs and making it a positive loop. These findings on the overexpression strains pointed out a new direction for further development and utilization of C. butyricum. KEY POINTS: • By 12 signal peptide screening in silico, 4 pEGF overexpression strains of C. butyricum/pMTL82151-pEGF for highly efficient secretion of pEGF were generated for the first time. • The secretory overexpression of pEGF promoted the intestinal development, antimicrobial action, and anti-inflammatory function of C. butyricum. • The overexpressed pEGF upregulated the expression level of EGFR and further magnified the gut protective function of recombinant strains which in turn partly depended on STAT3 signal pathway in IPECs.

Grb2-associated-binding protein-2 (Gab2) is a member of the Gab/DOS family and functions as an adapter protein downstream of several growth factor signaling pathways. Gab2 is considered an Alzheimer's disease susceptibility gene. However, the role of Gab2 in the brain is still largely unknown. Herein, we report that Gab2 is involved in the postnatal development of microglia in mice. The Gab2 expression in the brain was detected at postnatal day 1 (P1) and increased until P14 but decreased thereafter. The tyrosine phosphorylation of Gab2 (pGab2) was also detected at P1 and increased until P14. Next, we focused on microglial development in Gab2 knockout and heterozygous mice. Although differences were not detected in the cytoplasmic area of Iba1-labeled microglia between Gab2(±) and Gab2(-/-) mice, the analysis of CD68 and cathepsin D (indicators of microglial lysosomal activation) immunolabeling within Iba1+ cells revealed significant underdevelopment of microglial lysosomes in Gab2(-/-) mice at P60. In addition to the developmental abnormality of microglia in Gab2(-/-) mice, lipopolysaccharide-induced lysosomal activation was selectively suppressed in Gab2(-/-) mice compared to that in Gab2(±) mice. Our findings suggest that Gab2 is involved not only in postnatal development but also in lysosomal activation of microglia, therefore Gab2 dysfunction in microglia might potentially contribute to the development of neurodegenerative diseases.

Mammalian sperm have to undergo capacitation to fertilize the egg. At the molecular level, capacitation involves cAMP synthesis, protein kinase A activation, and downstream increase in tyrosine phosphorylation. In addition, during capacitation, mammalian sperm actively generate reactive oxygen species (ROS). It has been proposed that ROS modulate phosphorylation pathways; however, the crosstalk between these signaling processes is not well-understood. In the present study, we used loss- and gain-of-function approaches to evaluate the interconnection between ROS and phosphorylation. We showed that BSA and HCO3 - , but not Ca2+ , in the capacitation media are required for ROS production. The synergic effect of these compounds was neither mediated by HCO3 - stimulation of cAMP synthesis nor by BSA-induced cholesterol efflux. The capacitation-induced ROS generation was blocked in the presence of superoxide dismutase (SOD), catalase, and apocynin. However, none of these compounds affected cAMP-dependent or tyrosine phosphorylation. On the other hand, the addition of NADPH to the media induced ROS generation in sperm incubated in the absence of BSA and HCO3 - without upregulating cAMP-dependent or tyrosine phosphorylation signaling. Most interestingly, catalase, but not SOD, blocked in vitro fertilization suggesting a role for H2 O2 in this process.

Fetal alcohol syndrome (FAS) is characterized by disrupted fetal brain development and postnatal cognitive impairment. The targets of alcohol are diverse, and it is not clear whether there are common underlying molecular mechanisms producing these disruptions. Prior work established that acute ethanol exposure causes a transient increase in tyrosine phosphorylation of multiple proteins in cultured embryonic cortical cells. In this study, we show that a similar tyrosine phosphorylation transient occurs in the fetal brain after maternal dosing with ethanol. Using phospho-specific antibodies and immunohistochemistry, we mapped regions of highest tyrosine phosphorylation in the fetal cerebral cortex and found that areas of dendritic and axonal growth showed elevated tyrosine phosphorylation 10 min after maternal ethanol exposure. These were also areas of Src expression and Src family kinase (SFK) activation loop phosphorylation (pY416) expression. Importantly, maternal pretreatment with the SFK inhibitor dasatinib completely prevents both the pY416 increase and the tyrosine phosphorylation response. The phosphorylation response was observed in the perisomatic region and neurites of immature migrating and differentiating primary neurons. Importantly, the initial phosphotyrosine transient (~ 30 min) targets both Src and Dab1, two critical elements in Reelin signaling, a pathway required for normal cortical development. This initial phosphorylation response is followed by sustained reduction in Ser3 phosphorylation of n-cofilin, a critical actin severing protein and an identified downstream effector of Reelin signaling. This biochemical disruption is associated with sustained reduction of F-actin content and disrupted Golgi apparatus morphology in developing cortical neurons. The finding outlines a model in which the initial activation of SFKs by ethanol has the potential to disrupt multiple developmentally important signaling systems for several hours after maternal exposure. Supplementary Information The online version contains supplementary material available at 10.1007/s12035-021-02467-x.

Protein-tyrosine phosphorylation is one of the posttranslational modifications and plays critical roles in regulating a wide variety of cellular processes, such as cell proliferation, differentiation, adhesion, migration, survival, and apoptosis. Protein-tyrosine phosphorylation is reversibly regulated by protein-tyrosine kinases and protein-tyrosine phosphatases. Strong inhibition of protein-tyrosine phosphatase activities is required to undoubtedly detect tyrosine phosphorylation. Our extremely careful usage of Na3VO4, a potent protein-tyrosine phosphatase inhibitor, has revealed not only the different intracellular trafficking pathways of Src-family tyrosine kinase members but also novel tyrosine phosphorylation signals in the nucleus and on mitotic spindle fibers and lysosomes. Furthermore, despite that the first identified oncogene product v-Src is generally believed to induce transformation through continuous stimulation of proliferation signaling by its strong tyrosine kinase activity, v-Src-driven transformation was found to be caused not by continuous proliferation signaling but by v-Src tyrosine kinase activity-dependent stochastic genome alterations. Here, I summarize our findings regarding novel tyrosine phosphorylation signaling in a spatiotemporal sense and highlight the significance of the roles of tyrosine phosphorylation in transcriptional regulation inside the nucleus and chromosome dynamics.

Congenital myasthenia (CM) is a devastating neuromuscular disease, and mutations in DOK7, an adaptor protein that is crucial for forming and maintaining neuromuscular synapses, are a major cause of CM1,2. The most common disease-causing mutation (DOK71124_1127 dup) truncates DOK7 and leads to the loss of two tyrosine residues that are phosphorylated and recruit CRK proteins, which are important for anchoring acetylcholine receptors at synapses. Here we describe a mouse model of this common form of CM (Dok7CM mice) and a mouse with point mutations in the two tyrosine residues (Dok72YF). We show that Dok7CM mice had severe deficits in neuromuscular synapse formation that caused neonatal lethality. Unexpectedly, these deficits were due to a severe deficiency in phosphorylation and activation of muscle-specific kinase (MUSK) rather than a deficiency in DOK7 tyrosine phosphorylation. We developed agonist antibodies against MUSK and show that these antibodies restored neuromuscular synapse formation and prevented neonatal lethality and late-onset disease in Dok7CM mice. These findings identify an unexpected cause for disease and a potential therapy for both DOK7 CM and other forms of CM caused by mutations in AGRIN, LRP4 or MUSK, and illustrate the potential of targeted therapy to rescue congenital lethality.

SH2 domain-containing inositol 5-phosphatase 2 (SHIP2) plays an essential role in regulating phosphatidylinositol level in human cell, and is recruited to many phosphotyrosine (pY)-dependent signal transduction pathways by the SH2 domain. In immunity signaling, immunoreceptor FcγRIIB binds to SHIP2-SH2 via its Y292-phosphorylated immunoreceptor tyrosine-based inhibitory motif (ITIM) and transmits inhibitory signal, which regulates B cell and neuronal cell activity and is associated with immune diseases and Alzheimer's disease. To date, the interaction between SHIP2 and FcγRIIB has not been analyzed from a structural point of view. Here, the binding of SHIP2-SH2 with Y292-phosphorylated FcγRIIB-ITIM was analyzed using NMR spectroscopy. The results demonstrated that SHIP2-SH2 mainly utilizes two regions including a pY-binding pocket and a specificity pocket formed by βD, βE, and EF-loop, to bind with FcγRIIB-ITIM in high affinity. In addition to the two regions, the BG-loop of SHIP2-SH2 functions as an auxiliary interface enhancing affinity. By comparing the binding of SHIP2-SH2 with ligands from FcγRIIB and c-MET, a hepatocyte growth factor receptor associated with tumorigenesis, significant differences in interface and affinity were found, suggesting that SHIP2-SH2 applies diverse patterns for binding to different ligand proteins. Moreover, S49, S51, and R70 of SHIP2 were identified to mediate the binding of both FcγRIIB and c-MET, while R28 and Q107 were found to only participate in the binding of c-MET and FcγRIIB respectively. Taken together, this study reveals the diverse mechanisms of SHIP2-SH2 for recognizing different ligands, and provides important clues for selectively manipulating various signaling pathways and specific drug design.

The sperm-derived oocyte activating factor, phospholipase C zeta (PLC ζ), is the only PLC isoform reported in cattle. The objectives were to (1) localize PLC ζ in fresh and capacitated bovine sperm and (2) investigate the activation of PLC ζ during bull sperm capacitation and contributions of PLC activity to this process. We confirmed interaction of testis-specific isoform of Na/K-ATPase (ATP1A4) with PLC ζ (immunolocalization and immunoprecipitation) and tyrosine phosphorylation (immunoprecipitation) of PLC ζ (a post-translational protein modification commonly involved in activation of PLC in somatic cells) during capacitation. Furthermore, incubation of sperm under capacitating conditions upregulated PLC-mediated hyperactivated motility, tyrosine phosphoprotein content, acrosome reaction, and F-actin formation (flow cytometry), implying that PLC activity is enhanced during capacitation and contributing to these capacitation processes. In conclusion, we inferred that PLC ζ is activated during capacitation by tyrosine phosphorylation through a mechanism involving ATP1A4, contributing to capacitation-associated biochemical events.

The cytoplasmic region of the γ chain of the high-affinity receptor for IgE (FcεRI) contains a consensus sequence termed the immunoreceptor tyrosine-based activation motif (ITAM). Phosphorylation of the two tyrosine residues (N-terminal Y47 and C-terminal Y58) in the ITAM sequence is crucial for the recruitment and activation of Syk, a cytoplasmic tyrosine kinase with central signaling roles in mast cells. Using a reconstitution system in which individual tyrosine-to-phenylalanine substituted γ chains were expressed in γ-chain-deficient mast cells, we previously reported differential dephosphorylation of these tyrosines. Herein, we developed monoclonal antibodies highly specific to the phosphorylated Y47 and Y58 residues, which enables monitoring their phosphorylation under more physiological conditions. Using these antibodies, preferential dephosphorylation of Y58 following FcεRI stimulation was confirmed. Furthermore, Y58 is potentially more susceptible to phosphorylation than is Y47. Consistent with this, an in vitro kinase assay using these phospho-specific antibodies demonstrated that the Src family kinase Lyn, which is primarily responsible for ITAM phosphorylation, phosphorylates Y58 more efficiently than Y47. These results indicate that Y58 is more susceptible to dephosphorylation and phosphorylation than is Y47. Because a phosphate group on Y58 is more important for Syk binding than is a phosphate group on Y47, the preferential phosphorylation and dephosphorylation of Y58 may contribute to the fine tuning of Syk activity by promoting rapid recruitment and reducing excessive activation.

ABL, a human tyrosine protein kinase, and its substrate are co-expressed in Escherichia coli. Tyrosine phosphorylation of the substrate in E. coli was detected using Phos-tag SDS-PAGE. The bacterial co-expression system was used as a field for the kinase reaction to evaluate the enzymatic activity of five types of ABL kinase domain mutants. Relative to wild-type ABL, kinase activity was comparable in the H396P mutant, reduced in both Y253F and E255K mutants and undetectable in T315I and M351T mutants. These comparative results demonstrated that the phosphorylation states of the mutants correlated with their activity. The bacterial co-expression system permits rapid production of tyrosine kinase variants and provides a simple approach for examining their structure–activity relationships.

Even though the search for methods improving cryopreservation of canine spermatozoa led to an improvement of post-thaw quality, fertilizing results after insemination with frozen–thawed semen are still not satisfying. In this study, we focused on modification of spermatozoa membrane fluidity and investigated whether kinematic parameters as assessed by computer-assisted semen analyzer (CASA) can be improved. The primary aim of our study was to investigate whether the use of cholesterol-loaded cyclodextrins (CLC; 0.5 mg, 1 mg, 2 mg) and 2-Hydroxypropyl-ß-cyclodextrin (HBCD; 1 mg) positively influence capacitation status as examined by tyrosinphosphorylation, cholesterol efflux and zona binding assay (ZBA) of spermatozoa. The use of 0.5 mg of CLC increased the percentage of motile, progressive and rapid spermatozoa compared to the control. Addition of HBCD decreased motility and progressive motility of spermatozoa and the population with rapid movement in comparison to the control. The percentage of live spermatozoa without efflux of cholesterol compared to the control was increased when extender with 0.5 mg of CLC was used. There was no change in capacitation status. The zona binding ability of spermatozoa was significantly lower in the group with 0.5 mg of CLC than in the control. In conclusion, these results suggest that improvement of kinematic parameters does not necessarily coincide with better zona pellucida binding ability of spermatozoa.

Glucose-6-phosphate dehydrogenase (G6PD) is the first and rate-limiting enzyme in pentose phosphate pathway (PPP), excessive activation of which has been considered to be involved in tumorigenesis. Here, we show that tyrosine kinase c-Src interacts with and phosphorylates G6PD at Tyr 112. This phosphorylation enhances catalytic activity of G6PD by dramatically decreasing its Km value and increasing its Kcat value for substrate glucose-6-phosphate. Activated G6PD therefore augments the PPP flux for NADPH and ribose-5-phosphate production which is required for detoxification of intracellular reactive oxygen species (ROS) and biosynthesis of cancer cells, and eventually contributes to tumorigenesis. Consistently, c-Src activation is closely correlated with tyrosine phosphorylation and activity of G6PD in clinical colorectal cancer samples. We thus uncover another aspect of c-Src in promoting cell proliferation and tumorigenesis, deepening our understanding of c-Src as a proto-oncogene.

Background A gain-of-function mutation in germline ABL1 causes a syndrome including congenital heart defects. However, the molecular mechanisms of this syndrome remain unknown. In this study, we found a novel ABL1 mutation in a Japanese family with ventricular septal defect, finger contracture, skin abnormalities and failure to thrive, and the molecular mechanisms of these phenotypes were investigated. Methods and results Whole-exome sequencing on several family members revealed a novel mutation (c.1522A > C, p.I508L) in the tyrosine kinase domain of ABL1, and complete co-segregation with clinical presentations was confirmed in all members. Wild-type and mutant ABL1 were transfected into human embryonic kidney 293 cells for functional analysis. Western blotting confirmed that tyrosine phosphorylation in STAT5, a substrate of ABL1, was enhanced, and the novel mutation was proved to be a gain-of-function mutation. Since this novel mutation in ABL1 enhances tyrosine kinase activity, phosphorylated proteome analysis was used to elucidate the molecular pathology. The proteome analysis showed that phosphorylation in proteins such as UFD1, AXIN1, ATRX, which may be involved in the phenotypes, was enhanced in the mutant group. Conclusions The onset of congenital heart defects associated with this syndrome appears to involve a mechanism caused by UFD1 common to 22q.11.2 deletion syndrome. On the other hand, AXIN1 and ATRX may be important in elucidating the mechanisms of other phenotypes, such as finger contracture and failure to thrive. Verification of these hypotheses would lead to further understanding of the pathophysiology and the development of treatment methods.

C-Src kinase is localized in several subcellular compartments, including mitochondria where it is involved in the regulation of organelle functions and overall metabolism. Surprisingly, the characterization of the intramitochondrial Src interactome has never been fully determined. Using in vitro proximity-dependent biotin identification (BioID) coupled to mass spectrometry, we identified 51 candidate proteins that may interact directly or indirectly with c-Src within the mitochondrial matrix. Pathway analysis suggests that these proteins are involved in a large array of mitochondrial functions such as protein folding and import, mitochondrial organization and transport, oxidative phosphorylation, tricarboxylic acid cycle and metabolism of amino and fatty acids. Among these proteins, we identified 24 tyrosine phosphorylation sites in 17 mitochondrial proteins (AKAP1, VDAC1, VDAC2, VDAC3, LonP1, Hsp90, SLP2, PHB2, MIC60, UBA1, EF-Tu, LRPPRC, ACO2, OAT, ACAT1, ETFβ and ATP5β) as potential substrates for intramitochondrial Src using in silico prediction of tyrosine phospho-sites. Interaction of c-Src with SLP2 and ATP5β was confirmed using coimmunoprecipitation. This study suggests that the intramitochondrial Src could target several proteins and regulate different mitochondrial functions.

Phospholipase D2 (PLD2) has been implicated in the tyrosine kinase-mediated signaling pathways, but the regulation events are yet to be identified. Herein, we demonstrate that pleckstrin homology (PH) domain of PLD2 (PLD2-PH) exerts an antitumorigenic effect via the suppression of PLD2 and focal adhesion kinase (FAK). The kinase domain of FAK interacts with PLD2-PH and induces tyrosine phosphorylation and activation of PLD2. Furthermore, PLD2 increased tyrosine phosphorylation of FAK. However, ectopic expression of the PLD2-PH competes for binding to FAK and reduces the interaction between PLD2 and FAK, thereby suppressing FAK-induced PLD activation and tyrosine phosphorylation of FAK. The PLD2-PH suppressed the migration and invasion of glioblastoma cells, as well as tumor formation in a xenograft mouse model. This study uncovers a novel role of PLD2-PH as a negative regulator of PLD2 and FAK. [BMB Reports 2021; 54(2): 112-117].

Reversible phosphorylation has emerged as an important mechanism for regulating 26S proteasome function in health and disease. Over 100 phospho-tyrosine (pTyr) sites of the human proteasome have been detected, and yet their function and regulation remain poorly understood. Here we show that the 19S subunit Rpt2 is phosphorylated at Tyr439, a strictly conserved residue within the C-terminal HbYX motif of Rpt2 that is essential for 26S proteasome assembly. Unexpectedly, we found that Y439 phosphorylation depends on Rpt2 membrane localization mediated by its N-myristoylation. Multiple receptor tyrosine kinases (RTKs) can trigger Rpt2-Y439 phosphorylation by activating Src, a N-myristoylated tyrosine kinase. Src directly phosphorylates Rpt2-Y439 in vitro and negatively regulates 26S proteasome integrity and activity at cellular membranes, which can be reversed by the membrane-associated isoform of protein tyrosine phosphatase non-receptor type 2 (PTPN2). In H1975 lung cancer cells with activated Src, blocking Rpt2-Y439 phosphorylation by the Y439F mutation conferred partial resistance to the Src inhibitor saracatinib both in vitro and in a mouse xenograft tumor model, and caused significant changes of cellular responses to saracatinib at the proteome level. Our study has defined a novel mechanism involved in the spatial regulation of proteasome function and provided new insights into tyrosine kinase inhibitor-based anti-cancer therapies.

Platinum-based complexes are one of the most successful chemotherapeutic agents having a significant ground in cancer chemotherapy despite their side effects. During the past few decades, Ru(II) complexes have been emerging as efficient alternatives owing to their promising activities against platinum-resistant cancer. The pathway of action, lipophilicity, and cytotoxicity of a Pt or Ru complex may be tuned by varying the attached ligands, the coordination mode, and the leaving group. In this work, we report a family of Pt(II) and Ru(II) complexes (1-5) of three N,O and N,N donor-based trimethoxyanilines containing Schiff bases with the general formula [PtII(L)(DMSO)Cl], [RuII(L)(p-cymene)Cl], [RuII(L)(p-cymene)Cl]+, and [PtII(L)Cl2]. All of the complexes are characterized by different analytical techniques. 1H NMR and electrospray ionization mass spectrometry (ESI-MS) data suggest that the N,O-coordinated Pt(II) complexes undergo slower aquation compared to the Ru(II) analogues. The change of the coordination mode to N,N causes the Ru complexes to be more inert to aquation. The N,O-coordinating complexes show superiority over N,N-coordinating complexes by displaying excellent in vitro antiproliferative activity against different aggressive cancer cells, viz., triple-negative human metastatic breast adenocarcinoma MDA-MB-231, human pancreatic carcinoma MIA PaCa-2, and hepatocellular carcinoma Hep G2. In vitro cytotoxicity studies suggest that Pt(II) complexes are more effective than their corresponding Ru(II) analogues, and the most cytotoxic complex 3 is 10-15 times more toxic than the clinical drugs cisplatin and oxaliplatin against MDA-MB-231 cells. Cellular studies show that all of the N,O-coordinated complexes (1-3) initiate disruption of the microtubule network in MDA-MB-231 cells in a dose-dependent manner within 6 h of incubation and finally lead to the arrest of the cell cycle in the G2/M phase and render apoptotic cell death. The disruption of the microtubule network affects the agility of the cytoskeleton rendering inhibition of tyrosine phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2), a key step in angiogenesis. Complexes 1 and 2 inhibit VEGFR2 phosphorylation in a dose-dependent fashion. Among the Pt(II) and Ru(II) complexes, the former displays higher cytotoxicity, a stronger effect on the cytoskeleton, better VEGFR2 inhibition, and strong interaction with the model nucleobase 9-ethylguanine (9-EtG).

Glioblastoma multiforme (GBM) is an aggressive malignant brain tumour that is resistant to existing therapeutics. Identifying signalling pathways deregulated in GBM that can be targeted therapeutically is critical to improve the present dismal prognosis for GBM patients. In this report, we have identified that the BRG1 (Brahma‐Related Gene‐1) catalytic subunit of the SWI/SNF chromatin remodelling complex promotes the malignant phenotype of GBM cells. We found that BRG1 is ubiquitously expressed in tumour tissue from GBM patients, and high BRG1 expression levels are localized to specific brain tumour regions. Knockout (KO) of BRG1 by CRISPR‐Cas9 gene editing had minimal effects on GBM cell proliferation, but significantly inhibited GBM cell migration and invasion. BRG1‐KO also sensitized GBM cells to the anti‐proliferative effects of the anti‐cancer agent temozolomide (TMZ), which is used to treat GBM patients in the clinic, and selectively altered STAT3 tyrosine phosphorylation and gene expression. These results demonstrate that BRG‐1 promotes invasion and migration, and decreases chemotherapy sensitivity, indicating that it functions in an oncogenic manner in GBM cells. Taken together, our findings suggest that targeting BRG1 in GBM may have therapeutic benefit in the treatment of this deadly form of brain cancer.

Epithelial junctions ensure cell–cell adhesion and establish permeability barriers between cells. At the corners of epithelia, the tricellular junction (TCJ) is formed by three adjacent epithelial cells and generates a functional barrier. In Drosophila, a key TCJ protein is Gliotactin (Gli) where loss of Gli disrupts barrier formation and function. Conversely, overexpressed Gli spreads away from the TCJ and triggers apoptosis, delamination, and cell migration. Thus, Gli protein levels are tightly regulated and by two mechanisms, at the protein levels by tyrosine phosphorylation and endocytosis and at the mRNA level through microRNA-184. Regulation of Gli mRNA is mediated through a Gli–BMP–miR184 feedback loop. Excessive Gli triggers BMP signaling pathway through the activation of Tkv type-I BMP receptor and Mad. Elevated level of pMad induces micrRNA-184 expression which in turn targets the Gli 3′UTR and mRNA degradation. Gli activation of Tkv is not through its ligand Dpp but rather through the inhibition of Dad, an inhibitory-Smad. Here, we show that ectopic expression of Gli interferes with Tkv–Dad association by sequestering Dad away from Tkv. The reduced inhibitory effect of Dad on Tkv results in the increased Tkv–pMad signaling activity, and this effect is continuous through larval and pupal wing formation.

Collapsin response mediator proteins (CRMPs) have been identified as mediating proteins of repulsive axon guidance cue Semaphorin-3A (Sema3A). Phosphorylation of CRMPs plays a crucial role in the Sema3A signaling cascade. It has been shown that Fyn phosphorylates CRMP1 at Tyrosine 504 residue (Tyr504); however, the physiological role of this phosphorylation has not been examined. We found that CRMP1 was the most strongly phosphorylated by Fyn among the five members of CRMPs. We confirmed Tyr504 phosphorylation of CRMP1 by Fyn. Immunocytochemistry of mouse dorsal root ganglion (DRG) neurons showed that phosphotyrosine signal in the growth cones was transiently increased in the growth cones upon Sema3A stimulation. Tyr504-phosphorylated CRMP1 also tended to increase after Sema3A simulation. Ectopic expression of a single amino acid mutant of CRMP1 replacing Tyr504 with phenylalanine (CRMP1-Tyr504Phe) suppressed Sema3A-induced growth cone collapse response in chick DRG neurons. CRMP1-Tyr504Phe expression in mouse hippocampal neurons also suppressed Sema3A but not Sema3F-induced growth cone collapse response. Immunohistochemistry showed that Tyr504-phosphorylated CRMP1 was present in the cell bodies and in the dendritic processes of mouse cortical neurons. CRMP1-Tyr504Phe suppressed Sema3A-induced dendritic growth of primary cultured mouse cortical neurons as well as the dendritic development of cortical pyramidal neurons in vivo. Fyn± ; Crmp1± double heterozygous mutant mice exhibited poor development of cortical layer V basal dendrites, which was the similar phenotype observed in Sema3a-/- , Fyn-/- , and Crmp1-/- mice. These findings demonstrate that Tyr504 phosphorylation of CRMP1 by Fyn is an essential step of Sema3A-regulated dendritic development of cortical pyramidal neurons. (247 words).

Alpha-1-syntrophin (SNTA1) is emerging as a novel modulator of the actin cytoskeleton. SNTA1 binds to F-actin and regulates intracellular localization and activity of various actin organizing signaling molecules. Aberration in syntrophin signaling has been closely linked with deregulated growth connected to tumor development/metastasis and its abnormal over expression has been observed in breast cancer. In the present work the effect of jasplakinolide, an actin-binding cyclodepsipeptide, on the SNTA1 protein activity and SNTA1 mediated downstream cellular events was studied in MDA-MB-231 breast cancer cell line. SNTA1 protein levels and phosphorylation status were determined in MDA-MB-231 cells post jasplakinolide exposure using western blotting and immunoprecipitation techniques respectively. MDA-MB-231 cells were transfected with WT SNTA1 and DM SNTA1 (Y215/229 phospho mutant) and simultaneously treated with jasplakinolide. The effect of jasplakinolide and SNTA1 protein on cell migration was determined using the boyden chamber assay. Jasplakinolide treatment decreases proliferation of MDA-MB-231 cells in both dose and time dependent manner. Results suggest that subtoxic doses of jasplakinolide induce morphological changes in MDA-MB-231 cells from flat spindle shape adherent cells to round weakly adherent forms. Mechanistically, jasplakinolide treatment was found to decrease SNTA1 protein levels and its tyrosine phosphorylation status. Moreover, migratory potential of jasplakinolide treated cells was significantly inhibited in comparison to control cells. Our results demonstrate that jasplakinolide inhibits cell migration by impairing SNTA1 functioning in breast cancer cells

Post-translational modifications (PTMs) can affect the normal function and pathology of α-synuclein (αS), an amyloid-fibril-forming protein linked to Parkinson's disease. Phosphorylation of αS Tyr39 has recently been found to display a dose-dependent effect on fibril formation kinetics and to alter the morphology of the fibrils. Existing methods to access site-specifically phosphorylated αS for biochemical studies include total or semi-synthesis by native chemical ligation (NCL) as well as chemoenzymatic methods to phosphorylate peptides, followed by NCL. Here, we investigated a streamlined method to produce large quantities of phosphorylated αS by co-expressing a kinase with a protein fragment in Escherichia coli. We also introduced the use of methyl thioglycolate (MTG) to enable one-pot NCL and desulfurization. We compare our optimized methods to previous reports and show that we can achieve the highest yields of site-specifically phosphorylated protein through chemoenzymatic methods using MTG, and that our strategy is uniquely well suited to producing 15 N-labeled, phosphorylated protein for NMR studies.

Background Rhus toxicodendron (R. tox) has been used as a homeopathic remedy for the treatment of inflammatory conditions. Previously, we reported that R. tox modulated inflammation in the mouse chondrocyte and pre-osteoblastic MC3T3-e1 cell line. During the inflammatory process, cells adhere to the extracellular matrix (ECM) and then migrate to the inflammation site. We examine here the process of cell adhesion in MC3T3-e1 cells after their stimulation with homeopathic R. tox.Methods For the cell–substrate adhesion assay, the cultured MC3T3-e1 cells were trypsinized, starved for 1 h in serum-free media, and plated onto culture plates coated with fibronectin (FN), 30c R. tox or gelatin, respectively. The cells were allowed to adhere for 20 min incubation and unattached cells were washed out. Adherent cells were measured using the water-soluble tetrazolium salt-8 assay. The intracellular signals after stimulation of R. tox were examined by analyzing the tyrosine phosphorylation of focal adhesion kinase (FAK), Src kinase, and Paxillin using immunoblot assay. Formation of focal adhesion (FA, an integrin-containing multi-protein structure that forms between intracellular actin bundles and the ECM) was analyzed by immunocytochemistry using NIH ImageJ software.Results Cell adhesion increased after stimulation with R. tox (FN, 20.50%; R. tox, 44.80%; and gelatin, 17.11% vs. uncoated cells [control]). Tyrosine phosphorylation of FAK, Paxillin, and Src increased compared with that of gelatin when stimulated with R. tox. Additionally, R. tox-stimulated cells formed many FAs (number of FAs per cell, 35.82 ± 7.68) compared with gelatin-stimulated cells (number of FAs per cell, 19.80 ± 7.18) and exhibited extensive formation of actin stress fibers anchored by FAs formed at the cell periphery.Conclusion Homeopathic R. tox promotes the formation of cell adhesions in vitro.

The NF-κB family of transcriptional activators is responsible for the expression of numerous genes that control key functions such as cell development and survival. Subunit p50 has been studied extensively and is known to include 13 tyrosines, but the extent and pattern of tyrosine phosphorylation that accompanies p50 function has not been defined in the literature, especially at the level of selectivity of gene expression. In this study, phosphorylated tyrosine (pTyr) was site-selectively incorporated into the p50 subunit using an E. coli in vitro expression system containing a modified ribosome. In human T cells, the NF-κBs containing a pTyr at position 60 or 82 of p50 strongly increased the expression of CD40, which is a potential target for cancer or viral immunotherapy. Promoter DNA binding was studied for CD40 promoters, and verified two pTyr residues in NF-κB p50/p65 heterodimers that facilitated this process, and that support the possible importance of phosphorylation stoichiometry. This study defines a new approach for studying tyrosine residues whose phosphorylation alters protein binding to DNA promoters, and contributes to the facility of DNA expression.

Spermatozoa capacitation is a time-dependent physiological process essential for acquiring the fertilizing capacity. In this context, reorganization of spermatozoa surface sugars and tyrosine phosphorylation are some of the most important biochemical changes involved in capacitation. However, the relationship between these 2 biomarkers remains poorly defined. By cytofluorescence we simultaneously characterized the head concanavalin A (ConA)-binding sites and the flagellar tyrosine phosphorylation before capacitation, during different capacitation times (1 and 4 h), and after acrosome reaction induction in human spermatozoa. The results showed a strong connection between ConA-label patterns and tyrosine phosphorylation according to the spermatozoa capacitation time and acrosomal status. Specifically, the spermatozoa subpopulation with phosphotyrosine presented proper sugar location (label in acrosomal and postacrosomal region) just after 1 h of capacitation, while cells without phosphotyrosine needed 4 h to do it. Moreover, after induction of spermatozoa acrosome reaction, phosphorylation was significantly correlated (p < 0.05) with the relocation of ConA-binding residues to the equatorial region, regardless of capacitation time. Overall, these observations provide novel insights regarding spermatozoa subpopulations based on essential physiological events like capacitation and acrosome reaction, which could have potential implications in the improvement of spermatozoa selection techniques. This research was funded by the Human Fertility Cathedra of the University of Alicante and the VIGROB-186.

Multimodal gadolinium fluoride nanoparticles belong to potential contrast agents useful for bimodal optical fluorescence and magnetic resonance imaging. However, the metallic nature of the nanoparticles, similarly to some paramagnetic iron oxides, might induce allergic and anaphylactic reactions in patients after administration. A reduction of these adverse side effects is a priority for the safe application of the nanoparticles. Herein, we prepared paramagnetic poly(4-styrenesulfonic acid-co-maleic acid) (PSSMA)-stabilized GdF3 nanoparticles with surface modified by Atto 488-labeled poly(styrene-grad-2-dimethylaminoethyl acrylate)-block-poly(2-dimethylaminoethyl acrylate) (PSDA-A488) with reactive amino groups for introduction of an additional imaging (luminescence) modality and possible targeting of anticancer drugs. The saturation magnetization of GdF3@PSSMA particles according to SQUID magnetometry reached 157 Am2 kg-1 at 2 K and magnetic field of 7 T. GdF3@PSSMA-PSDA-A488 nanoparticles were well tolerated by human cervical adenocarcinoma (HeLa), mouse bone marrow-derived mast cells (BMMC), and rat basophilic mast cells (RBL-2H3); the particles also affected cell morphology and protein tyrosine phosphorylation in mast cells. Moreover, the nanoparticles interfered with the activation of mast cells by multivalent antigens and inhibited calcium mobilization and cell degranulation. These findings show that the new multimodal GdF3-based nanoparticles possess properties useful for various imaging methods and might minimize mast cell degranulation incurred after future nanoparticle diagnostic administration.

MCP-1–induced monocyte chemotaxis is a crucial event in inflammation and atherogenesis. Identifying the important signal transduction pathways that control monocyte chemotaxis can unravel potential targets for preventive therapies in inflammatory disease conditions. Previous studies have shown that the focal adhesion kinase Pyk2 plays a critical role in monocyte motility. In this study, we investigated the MCP-1–mediated activation of Pyk2 (particularly by the phosphorylation of Tyr402) in primary human peripheral blood monocytes. We showed that MCP-1 induces Src phosphorylation in a similar time frame and that the MCP-1–induced Pyk2 tyrosine phosphorylation is controlled by the Src family kinase. We also report, in this study, that PKCβ, an isoform of PKC, is required for both Src and Pyk2 activation/phosphorylation in response to MCP-1 stimulation. We identified Lyn as the specific Src kinase isoform that is activated by MCP-1 and acts upstream of Pyk2 in primary monocytes. Furthermore, Lyn is found to be indispensable for monocyte migration in response to MCP-1 stimulation. Moreover, our coimmunoprecipitation studies in monocytes revealed that PKCβ, Pyk2, and Lyn exist constitutively in a molecular complex. To our knowledge, our study has uncovered a novel PKCβ–Lyn–Pyk2 signaling cascade in primary monocytes that regulates MCP-1–induced monocyte adhesion and migration.

The discovery and the use of insulin to treat patients in January 1922 marked an important milestone in our quest for finding out the cause and cure for diabetes—a disease haunting humanity since ancient times. The pace of discovery accelerated since the discovery of the islets of langerhans. This review focuses on the advances since 1922 that have helped us understand the nature control secretion function and action of insulin. From its physical characterization in the mid-1920s to determination of its molecular structure, precursor and measurement in the 1950s through the discovery of its receptor tyrosine phosphorylation and gene in the eighties these advances have borrowed from and contributed richly to advances in protein genetics and molecular biology. Paralleling advances in purification modification and delivery of insulin, these advances form the basis of our understanding and the therapy of diabetes.

Glycoprotein (GP)VI and integrin αIIbβ3 are key signaling receptors in collagen-dependent platelet aggregation and in arterial thrombus formation under shear. The multiple downstream signaling pathways are still poorly understood. Here, we focused on disclosing the integrin-dependent roles of focal adhesion kinase (protein tyrosine kinase 2, PTK2), the shear-dependent collagen receptor GPR56 (ADGRG1 gene), and calcium and integrin-binding protein 1 (CIB1). We designed and synthetized peptides that interfered with integrin αIIb binding (pCIB and pCIBm) or mimicked the activation of GPR56 (pGRP). The results show that the combination of pGRP with PTK2 inhibition or of pGRP with pCIB > pCIBm in additive ways suppressed collagen- and GPVI-dependent platelet activation, thrombus buildup, and contraction. Microscopic thrombus formation was assessed by eight parameters (with script descriptions enclosed). The suppressive rather than activating effects of pGRP were confined to blood flow at a high shear rate. Blockage of PTK2 or interference of CIB1 no more than slightly affected thrombus formation at a low shear rate. Peptides did not influence GPVI-induced aggregation and Ca2+ signaling in the absence of shear. Together, these data reveal a shear-dependent signaling axis of PTK2, integrin αIIbβ3, and CIB1 in collagen- and GPVI-dependent thrombus formation, which is modulated by GPR56 and exclusively at high shear. This work thereby supports the role of PTK2 in integrin αIIbβ3 activation and signaling.

Heterozygous gain-of-function (GOF) mutations in the interferon-driven transcription factor STAT1 (signal transducer and activator of transcription 1) cause chronic mucocutaneous candidiasis (CMC). In this study, we characterized the molecular basis of a CMC-associated missense mutation by introducing a threonine-to-alanine exchange in the STAT1 DNA-binding domain at position 387. This substitution had previously been described in a CMC patient with suppurative eyelid infection and cutaneous abscesses, which are both unusual symptoms in this immunodeficiency. The STAT1-T387A mutant generated was compared to the wild-type protein and, in addition, to the missense mutant in the neighbouring position 386. Our results showed that the T387A mutant displayed distinct properties different from the wild-type molecule, namely elevated levels of tyrosine phosphorylation in conjunction with increased DNA-binding activity, hyperactive transcriptional regulation, and prolonged nuclear accumulation. The elevated tyrosine phosphorylation of the T387A mutant did not result in an increased mRNA production above the level of the wild-type molecule for all transcripts tested, indicating that the transcriptional activity of this mutant is largely gene-dependent. Nonetheless, these data demonstrate that the pathogenic T387A mutation associated with an atypical CMC symptomatology is biochemically similar to other well-characterized GOF mutants, while the H386A mutant was indistinguishable from the wild-type molecule. Our findings are in line with the assumption that the phenotype of this dominant STAT1 GOF mutation probably results from a disturbed shift in the equilibrium between the parallel and antiparallel dimer conformation, which is required for physiological gene activation.

Background Radioresistance poses a major challenge in nasopharyngeal carcinoma (NPC) treatment. Protein tyrosine phosphorylation has emerged as a key device in the control of resistance to therapy in cancer cells. Methods Using tandem mass tag (TMT) labeling and phospho-antibody affinity enrichment followed by high-resolution LC-MS/MS analysis, quantitative tyrosine phosphorylome analysis was performed in CNE2 (parental) and its radioresistant subline CNE2-IR. Results Altogether, 233 tyrosine phosphorylation sites in 179 protein groups were identified, among which 179 sites in 140 proteins were quantified. Among the quantified proteins, 38 tyrosine phosphorylation proteins are up-regulated and 18 tyrosine phosphorylation proteins are down-regulated in CNE2-IR vs CNE2. Increased tyrosine phosphorylation in multiple receptor/protein tyrosine kinases (EPHA2, EGFR, IGF1R, ABL1 and LYN) was identified in CNE2-IR vs CNE2 cells. Intensive bioinformatic analyses revealed robust activation of multiple biological processes/pathways including E-cadherin stabilization, cell-cell adhesion, and cell junction organization in radioresistant CNE2-IR cells. Specifically, we observed that the CNE2 cells incubated with EphrinA1-Fc exhibited higher EPHA2 Y772 phosphorylation and lower E-cadherin expression, as compared with PBS control. Furthermore, an ATP-competitive EPHA2 RTK inhibitor (ALW-II-41-27, ALW) reduced EPHA2 Y772 phosphorylation and increased the expression of E-cadherin in CNE2-IR cells. Colony formation analysis showed that EFNA1 (EFNA1 is the ligand of EPHA2) treatment in CNE2 significantly promoted colony formation after 6Gy irradiation; while incubation with EPHA2 inhibitor ALW-II-41-27 in CNE2-IR cells impaired colony formation after irradiation, as compared with solvent control (DMSO). Conclusion In conclusion, phosphoproteomic approach allowed us to link tyrosine kinases signaling with radioresistance in NPC. Further studies are necessary to delineate the molecular function of EPHA2/E-cadherin signaling in radioresistant NPC and to explore rational combination therapy and its underlying mechanism.

IQGAP1 is a key scaffold protein that regulates numerous cellular processes and signaling pathways. Analogous to many other cellular proteins, IQGAP1 undergoes post-translational modifications, including phosphorylation. Nevertheless, very little is known about the specific sites of phosphorylation or the effects on IQGAP1 function. Here, using several approaches, including MS, site-directed mutagenesis, siRNA-mediated gene silencing, and chemical inhibitors, we identified the specific tyrosine residues that are phosphorylated on IQGAP1 and evaluated the effect on function. Tyr-172, Tyr-654, Tyr-855, and Tyr-1510 were phosphorylated on IQGAP1 when phosphotyrosine phosphatase activity was inhibited in cells. IQGAP1 was phosphorylated exclusively on Tyr-1510 under conditions with enhanced MET or c-Src signaling, including in human lung cancer cell lines. This phosphorylation was significantly reduced by chemical inhibitors of MET or c-Src or by siRNA-mediated knockdown of MET. To investigate the biological sequelae of phosphorylation, we generated a nonphosphorylatable IQGAP1 construct by replacing Tyr-1510 with alanine. The ability of hepatocyte growth factor, the ligand for MET, to promote AKT activation and cell migration was significantly greater when IQGAP1-null cells were reconstituted with IQGAP1 Y1510A than when cells were reconstituted with WT IQGAP1. Collectively, our data suggest that phosphorylation of Tyr-1510 of IQGAP1 alters cell function. Because increased MET signaling is implicated in the development and progression of several types of carcinoma, IQGAP1 may be a potential therapeutic target in selected malignancies.

Nucleophosmin‐anaplastic lymphoma kinase (NPM‐ALK) inactivates tumor suppressor p53 by negatively regulating the activation of the Akt‐mTORC1 pathway through an interaction with EBP2 in the nucleolus in anaplastic large‐cell lymphoma cells., The oncogenic fusion protein nucleophosmin‐anaplastic lymphoma kinase (NPM‐ALK), found in anaplastic large‐cell lymphoma (ALCL), localizes to the cytosol, nucleoplasm, and nucleolus. However, the relationship between its localization and transforming activity remains unclear. We herein demonstrated that NPM‐ALK localized to the nucleolus by binding to nucleophosmin 1 (NPM1), a nucleolar protein that exhibits shuttling activity between the nucleolus and cytoplasm, in a manner that was dependent on its kinase activity. In the nucleolus, NPM‐ALK interacted with Epstein–Barr virus nuclear antigen 1‐binding protein 2 (EBP2), which is involved in rRNA biosynthesis. Moreover, enforced expression of NPM‐ALK induced tyrosine phosphorylation of EBP2. Knockdown of EBP2 promoted the activation of the tumor suppressor p53, leading to G0/G1‐phase cell cycle arrest in Ba/F3 cells transformed by NPM‐ALK and ALCL patient‐derived Ki‐JK cells, but not ALCL patient‐derived SUDH‐L1 cells harboring p53 gene mutation. In Ba/F3 cells transformed by NPM‐ALK and Ki‐JK cells, p53 activation induced by knockdown of EBP2 was significantly inhibited by Akt inhibitor GDC‐0068, mTORC1 inhibitor rapamycin, and knockdown of Raptor, an essential component of mTORC1. These results suggest that the knockdown of EBP2 triggered p53 activation through the Akt‐mTORC1 pathway in NPM‐ALK‐positive cells. Collectively, the present results revealed the critical repressive mechanism of p53 activity by EBP2 and provide a novel therapeutic strategy for the treatment of ALCL.

It has been reported that chemokine CX3 CL1 can regulate various tumours by binding to its unique receptor CX3 CR1. However, the effect of CX3 CL1-CX3 CR1 on the lung adenocarcinoma and lung squamous cell carcinoma is still unclear. Here, we showed that CX3 CL1 can further invasion and migration of lung adenocarcinoma A549 and lung squamous cell carcinoma H520. In addition, Western blot and immunofluorescence test indicated CX3 CL1 up-regulated the phosphorylation level of cortactin, which is a marker of cell pseudopodium. Meanwhile, the phosphorylation levels of c-Src and c-Abl, which are closely related to the regulation of cortactin phosphorylation, are elevated. Nevertheless, the src/abl inhibitor bosutinib and mutations of cortactin phosphorylation site could inhibit the promotion effect of CX3 CL1 on invasion and migration of A549 and H520. Moreover, these results of MTT, Hoechst staining and Western blot suggested that CX3 CL1 had no effect on the proliferation and apoptosis of A549 and H520 in vitro. The effects of CX3 CL1 were also verified by the subcutaneous tumour formation in nude mice, which showed that it could promote proliferation and invasion of A549 in vivo. In summary, our results indicated that CX3 CL1 furthered invasion and migration in lung cancer cells partly via activating cortactin, and CX3 CL1 may be a potential molecule in regulating the migration and invasion of lung cancer.

Zinc (Zn2+) is an essential micronutrient and the second most abundant trace metal in the human body. The important role that Zn2+ plays in hemostasis is exemplified by platelet-related bleeding phenotypes coinciding with dietary Zn2+ deficiency. These phenotypes are rectified upon Zn2+ supplementation. Labile (unbound) Zn2+ is present in the plasma at micromolar levels, but is also detected in atherosclerotic plaques, and released from platelet α granules. Therefore, it is likely that localized Zn2+ concentrations are higher at sites of thrombosis and hemostasis. Exogenous Zn2+ is a regulator of the hemostatic responses, with roles during coagulation and platelet activation. Extracellular Zn2+ gains access to the platelet cytosol and induces full platelet activation at high concentrations, and potentiates platelets to activation by conventional agonists at lower concentrations. Zn2+-induced platelet activation is dependent on PKC and integrin αIIbβ3, and is associated with tyrosine phosphorylation of platelet proteins. Agonist evoked platelet activation results in intracellular Zn2+ ([Zn2+]i) fluctuations that are sensitive to the platelet redox state. Increases in [Zn2+]i correlate with activation responses, including shape change, granule release, αIIbβ3 activation and phosphatidyl-serine exposure, consistent with a role as a second messenger. This review provides insight into the numerous demonstrated and potential roles for Zn2+ in platelet function during thrombosis and hemostasis, highlighting its increasing acceptance as an intracellular and extracellular platelet regulatory agent.

Interleukin-17 receptor D (IL-17RD), also known as similar expression to Fgf genes (SEF), is proposed to act as a signaling hub that negatively regulates mitogenic signaling pathways, like the ERK1/2 MAP kinase pathway, and innate immune signaling. The expression of IL-17RD is downregulated in certain solid tumors, which has led to the hypothesis that it may exert tumor suppressor functions. However, the role of IL-17RD in tumor biology remains to be studied in vivo. Here, we show that genetic disruption of Il17rd leads to the increased formation of spontaneous tumors in multiple tissues of aging mice. Loss of IL-17RD also promotes tumor development in a model of colitis-associated colorectal cancer, associated with an exacerbated inflammatory response. Colon tumors from IL-17RD-deficient mice are characterized by a strong enrichment in inflammation-related gene signatures, elevated expression of pro-inflammatory tumorigenic cytokines, such as IL-17A and IL-6, and increased STAT3 tyrosine phosphorylation. We further show that RNAi depletion of IL-17RD enhances Toll-like receptor and IL-17A signaling in colon adenocarcinoma cells. No change in the proliferation of normal or tumor intestinal epithelial cells was observed upon genetic inactivation of IL-17RD. Our findings establish IL-17RD as a tumor suppressor in mice and suggest that the protein exerts its function mainly by limiting the extent and duration of inflammation.

Since tyrosine phosphorylation appears to play important functions in photoreceptor cells, we searched here for retinal nonreceptor tyrosine kinases of the Src family. We demonstrated that Src family tyrosine kinases were present in the cytosolic fraction of extracted bovine retinas. A Src family tyrosine kinase with an apparent molecular mass of about 62 kDa was purified to homogeneity from the soluble fraction of dark-adapted bovine retinas after three consecutive purification steps: ω-aminooctyl-agarose hydrophobic chromatography, Cibacron blue 3GA-agarose pseudo-affinity chromatography, and α-casein-agarose affinity chromatography. The purified protein was subjected to N-terminal amino acid sequencing and the sequence Gly-Ile-Ile-Lys-Ser-Glu-Glu was obtained, which displayed homology with the first seven residues of the Src family tyrosine kinase c-Yes from Bos taurus (Gly-Cys-Ile-Lys-Ser-Lys-Glu). Although the cytosolic fraction from dark-adapted retinas contained tyrosine kinases of the Src family capable of phosphorylating the α-subunit of transducin, which is the heterotrimeric G protein involved in phototransduction, the purified tyrosine kinase was not capable of using transducin as a substrate. The cellular role of this retinal Src family member remains to be found.

Recently, it was reported that hamster sperm hyperactivation is regulated by extracellular Na+. Two types of catalytic Na+/K+-ATPase (NKA) α subunits (α1 and α4) are present in spermatozoa. In this work, the contribution of these NKA subunits to the regulation of hamster sperm hyperactivation was investigated using the specific inhibitor ouabain. When 10−6 M ouabain was added to the modified Tyrode’s albumin lactate pyruvate medium (mTALP) medium, hyperactivation was significantly inhibited, whereas 10−5-10−4 M ouabain was needed to significantly reduce the amount of motile spermatozoa. When a more detailed analysis of flagellar movement was performed, 10−6 M ouabain suppressed the hyperactivation-associated change in the patterns of flagellar motion without affecting the sliding velocity of microtubules. Since 10−6 M ouabain specifically inhibits the α4 subunit while 10−5-10−4 M ouabain inhibits both the α1 and α4 subunits, these results suggest the α1 subunit is necessary for the maintenance of motility while the α4 subunit is necessary for the hyperactivation-associated change in flagellar movement. Ouabain did not inhibit tyrosine phosphorylation, and activation of tyrosine phosphorylation-dependent signaling had no effect on the inhibition of hyperactivation by ouabain. The immediate recovery of hyperactivation was observed when ouabain was washed out after a 3-h incubation. whereas the administration of ouabain after the onset of hyperactivation significantly inhibited hyperactivation. These results suggest ouabain inhibited hyperactivation in a manner that was independent of time-requiring phosphorylation-mediated signaling.