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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.

Increased lipogenesis has been linked to an increased cancer risk and poor prognosis; however, the underlying mechanisms remain obscure. Here we show that phosphatidic acid phosphatase (PAP) lipin-1, which generates diglyceride precursors necessary for the synthesis of glycerolipids, interacts with and is a direct substrate of the Src proto-oncogenic tyrosine kinase. Obesity-associated microenvironmental factors and other Src-activating growth factors, including the epidermal growth factor, activate Src and promote Src-mediated lipin-1 phosphorylation on Tyr398, Tyr413 and Tyr795 residues. The tyrosine phosphorylation of lipin-1 markedly increases its PAP activity, accelerating the synthesis of glycerophospholipids and triglyceride. Alteration of the three tyrosine residues to phenylalanine (3YF-lipin-1) disables lipin-1 from mediating Src-enhanced glycerolipid synthesis, cell proliferation and xenograft growth. Re-expression of 3YF-lipin-1 in PyVT;Lpin1−/− mice fails to promote progression and metastasis of mammary tumours. Human breast tumours exhibit increased p-Tyr-lipin-1 levels compared to the adjacent tissues. Importantly, statistical analyses show that levels of p-Tyr-lipin-1 correlate with tumour sizes, lymph node metastasis, time to recurrence and survival of the patients. These results illustrate a direct lipogenesis-promoting role of the pro-oncogenic Src, providing a mechanistic link between obesity-associated mitogenic signaling and breast cancer malignancy., Altered lipid metabolism has been associated with tumour malignancy, but underlying mechanisms are not clear. Here the authors show that proto-oncogene Src interacts and phosphorylates metabolic enzyme phosphatidic acid phosphatase LPIN1 (lipin-1) to promote growth and metastasis in breast cancer.

Inflammasomes are essential for inflammation and pathogen elimination in response to microbial infection and endogenous danger signals. However, the mechanism of inflammasome activation by endogenous danger signals mediated posttranslational modification and the connection between inflammasomes and inflammatory diseases remains elusive. In this study, we found that ADP was highly released from injured colonic tissue as a danger signal during inflammatory bowel disease. Consequently, extracellular ADP activated the NLRP3 inflammasome through P2Y1 receptor-mediated calcium signaling, which led to the maturation and secretion of IL-1β and further aggravation of experimental colitis. Genetic ablation or pharmacological blockade of the P2Y1 receptor significantly ameliorated DSS-induced colitis and endotoxic shock through reducing NLRP3 inflammasome activation. Moreover, ERK5-mediated tyrosine phosphorylation of ASC was essential for activation of the NLRP3 inflammasome. Thus, our study provides a novel theoretical basis for posttranslational modification of ASC in NLRP3 inflammasome activation and revealed that ADP/P2Y1 is a potential drug target for inflammatory bowel disease.

Antibodies that antagonize extracellular receptor–ligand interactions are used as therapeutic agents for many diseases to inhibit signalling by cell-surface receptors1. However, this approach does not directly prevent intracellular signalling, such as through tonic or sustained signalling after ligand engagement. Here we present an alternative approach for attenuating cell-surface receptor signalling, termed receptor inhibition by phosphatase recruitment (RIPR). This approach compels cis-ligation of cell-surface receptors containing ITAM, ITIM or ITSM tyrosine phosphorylation motifs to the promiscuous cell-surface phosphatase CD452,3, which results in the direct intracellular dephosphorylation of tyrosine residues on the receptor target. As an example, we found that tonic signalling by the programmed cell death-1 receptor (PD-1) results in residual suppression of T cell activation, but is not inhibited by ligand-antagonist antibodies. We engineered a PD-1 molecule, which we denote RIPR-PD1, that induces cross-linking of PD-1 to CD45 and inhibits both tonic and ligand-activated signalling. RIPR-PD1 demonstrated enhanced inhibition of checkpoint blockade compared with ligand blocking by anti-PD1 antibodies, and increased therapeutic efficacy over anti-PD1 in mouse tumour models. We also show that the RIPR strategy extends to other immune-receptor targets that contain activating or inhibitory ITIM, ITSM or ITAM motifs; for example, inhibition of the macrophage SIRPα ‘don’t eat me’ signal with a SIRPα–CD45 RIPR molecule potentiates antibody-dependent cellular phagocytosis beyond that of SIRPα blockade alone. RIPR represents a general strategy for direct attenuation of signalling by kinase-activated cell-surface receptors. A approach termed ‘receptor inhibition by phosphatase recruitment’ is described for attenuating both tonic and ligand-activated cell-surface receptor signalling.

Rab GTPases are master regulators of membrane trafficking in eukaryotic cells. Phosphorylation of Rab GTPases was characterized in the 1990s and there have been intermittent reports of its relevance to Rab functions. Phosphorylation as a regulatory mechanism has gained prominence through the identification of Rabs as physiological substrates of leucine‐rich repeat kinase 2 (LRRK2). LRRK2 is a Ser/Thr kinase that is associated with inherited and sporadic forms of Parkinson disease. In recent years, numerous kinases and their associated signaling pathways have been identified that lead to phosphorylation of Rabs. These emerging studies suggest that serine/threonine and tyrosine phosphorylation of Rabs may be a widespread and under‐appreciated mechanism for controlling their membrane trafficking functions. Here we survey current knowledge of Rab phosphorylation and discuss models for how this post‐translational mechanism exerts control of membrane trafficking., Phosphorylation of Rab GTPases by serine/threonine and tyrosine kinases is emerging as a critical regulatory mechanism in membrane trafficking. This post‐translational modification can tune the strength of binding between Rabs and their interacting proteins. Phosphorylation can also influence the lifetime of active Rabs on subcellular compartments. We survey the current literature and discuss the various cellular processes connected with Rab phosphorylation.

Epstein-Barr virus (EBV) infects human B cells and reprograms them to allow virus replication and persistence. One key viral factor in this process is latent membrane protein 2A (LMP2A), which has been described as a B cell receptor (BCR) mimic promoting malignant transformation. However, how LMP2A signaling contributes to tumorigenesis remains elusive. By comparing LMP2A and BCR signaling in primary human B cells using phosphoproteomics and transcriptome profiling, we identified molecular mechanisms through which LMP2A affects B cell biology. Consistent with the literature, we found that LMP2A mimics a subset of BCR signaling events, including tyrosine phosphorylation of the kinase SYK, the calcium initiation complex consisting of BLNK, BTK, and PLCγ2, and its downstream transcription factor NFAT. However, the majority of LMP2A-induced signaling events markedly differed from those induced by BCR stimulation. These included differential phosphorylation of kinases, phosphatases, adaptor proteins, transcription factors such as nuclear factor κB (NF-κB) and TCF3, as well as widespread changes in the transcriptional output of LMP2A-expressing B cells. LMP2A affected apoptosis and cell-cycle checkpoints by dysregulating the expression of apoptosis regulators such as BCl-xL and the tumor suppressor retinoblastoma-associated protein 1 (RB1). LMP2A cooperated with MYC and mutant cyclin D3, two oncogenic drivers of Burkitt lymphoma, to promote proliferation and survival of primary human B cells by counteracting MYC-induced apoptosis and by inhibiting RB1 function, thereby promoting cell-cycle progression. Our results indicate that LMP2A is not a pure BCR mimic but rather rewires intracellular signaling in EBV-infected B cells that optimizes cell survival and proliferation, setting the stage for oncogenic transformation.

Tyrosine phosphorylation (pTyr), much of which occurred on localized multiple sites, initiates cellular signaling, governs cellular functions, and its dysregulation is implicated in many diseases, especially cancers. pTyr-specific sensing is of great significance for understanding disease states and developing targeted anticancer drugs, however, it is very challenging due to the slight difference from serine (pSer) or threonine phosphorylation (pThr). Here we present polyethylenimine-g-phenylguanidine (PEI-PG)-modified nanochannels that can address the challenge. Rich guanidinium groups enabled PEI-PG to form multiple interactions with phosphorylated residues, especially pTyr residue, which triggered the conformational change of PEI-PG. By taking advantage of the "OFF-ON" change of the ion flux arising from the conformational shrinkage of the grafted PEI-PG, the nanochannels could distinguish phosphorylated peptide (PP) from nonmodified peptide, recognize PPs with pSer, pThr, or pTyr residue and PPs with different numbers of identical residues, and importantly could sense pTyr peptides in a biosample. Benefiting from the strong interaction between the guanidinium group and the pTyr side-chain, the specific sensing of pTyr peptide was achieved by performing a simple logic operation based on PEI-PG-modified nanochannels when Ca2+ was introduced as an interferent. The excellent pTyr sensing capacity makes the nanochannels available for real-time monitoring of the pTyr process by c-Abl kinase on a peptide substrate, even under complicated conditions, and the proof-of-concept study of monitoring the kinase activity demonstrates its potential in kinase inhibitor screening.

Mammalian ejaculated spermatozoa must undergo a series of changes in the female reproductive tract, collectively called capacitation, in order to fertilize the oocyte. We reported that fibronectin (Fn), a glycoprotein from the extracellular matrix, and anandamide (AEA), one of the major members of the endocannabinoid family, are present in the bovine oviductal fluid and regulate bull sperm function. Also, AEA induces bovine sperm capacitation, through CB1 and TRPV1 receptors. In this work, we investigated if Fn induces bovine sperm capacitation thought the activation of the endocannabinoid system in this process. We incubated sperm with Fn (100 μg/ml) and/or capsazepine, a TRPV1 antagonist (0.1 μM) and some events related to sperm capacitation such as LPC-induced acrosome reaction, sperm-release from the oviduct, induction of PKA phosphorylated substrates (pPKAs) and protein tyrosine phosphorylation (pY) and nitric oxide (NO) production were assessed. Also, we studied the activity of fatty acid amide hydrolase (FAAH), the enzyme that degrades AEA. We found that Fn, via α5β1 integrin, induced capacitation-associated events. Also, Fn stimulated signaling pathways associated to capacitation as cAMP/PKA and NO/NO synthase. Moreover, Fn decreased the FAAH activity and this correlated with sperm capacitation. Capsazepine reversed fibronectin-induced capacitation, and pPKAs and NO levels. The incubation of spermatozoa with R-methanandamide (1.4 nM), a stable analogue of AEA, increased cAMP and pPKAs levels. The presence of H89 (50 μM) or KT5720 (100 nM) (PKA inhibitors) prevented AEA-induced capacitation. In addition, R-methanandamide and capsaicin (0.01 μM), a TRPV1 agonist, increased NO production via the PKA pathway. These results indicate that Fn, through α5β1, supports capacitation in bovine spermatozoa. This effect is dependent on the activation of TRPV1 through cAMP/PKA and NO signaling pathways. We propose that Fn could be considered as a new agent that promotes sperm capacitation in bull sperm. Our findings contribute to better understand the significance of Fn signaling in the capacitating events that lead to successful fertilization and embryo development in mammals including humans.

Canine parvovirus (CPV) has been used in cancer control as a drug delivery vehicle or anti-tumor reagent due to its multiple natural advantages. However, potential host cell cycle arrest induced by virus infection may impose a big challenge to CPV associated cancer control as it could prevent host cancer cells from undergoing cell lysis and foster them regain viability once the virotherapy was ceased. To explore CPV-induced cell cycle arrest and the underlying mechanism toward improved virotherapeutic design, we focus on epidermal growth factor receptor (EGFR), a cellular receptor interacting with TfR that mediates CPV-host interactions, and alterations on its tyrosine phosphorylation sites in response to CPV infection. We found that CPV could trigger host G1/S cell cycle arrest via the EGFR (Y1086)/p27 and EGFR (Y1068)/STAT3/cyclin D1 axes, and EGFR inhibitor could not reverse this process. Our results contribute to our understandings on the mechanism of CPV-induced host cellular response and can be used in the onco-therapeutic design utilizing CPV by preventing host cancer cells from entering cell cycle arrest.

Diffuse large B‐cell lymphoma (DLBCL) is characterized by extensive genetic heterogeneity, and this results in unpredictable responses to the current treatment, R‐CHOP, which consists of a cancer drug combination supplemented with the humanized CD20‐targeting monoclonal antibody rituximab. Despite improvements in the patient response rate through rituximab addition to the treatment plan, up to 40% of DLBCL patients end in a relapsed or refractory state due to inherent or acquired resistance to the regimen. Here, we employ a lentiviral genome‐wide clustered regularly interspaced short palindromic repeats library screening approach to identify genes involved in facilitating the rituximab response in cancerous B cells. Along with the CD20‐encoding MS4A1 gene, we identify genes related to B‐cell receptor (BCR) signaling as mediators of the intracellular signaling response to rituximab. More specifically, the B‐cell linker protein (BLNK) and Bruton's tyrosine kinase (BTK) genes stand out as pivotal genes in facilitating direct rituximab‐induced apoptosis through mechanisms that occur alongside complement‐dependent cytotoxicity (CDC). Our findings demonstrate that rituximab triggers BCR signaling in a BLNK‐ and BTK‐dependent manner and support the existing notion that intertwined CD20 and BCR signaling pathways in germinal center B‐cell‐like‐subtype DLBCL lead to programmed cell death., Genome‐wide clustered regularly interspaced short palindromic repeats‐based screening identify B‐cell receptor (BCR) signaling as a core mediator of rituximab sensitivity in OCI‐Ly‐7 cells, specifically through actions of the B‐cell linker protein and Bruton's tyrosine kinase proteins. This supports the notion of intertwined signaling pathways in germinal center B‐cell‐like‐subtype diffuse large B‐cell lymphoma involving stimulation of CD20 and BCR on the cellular path toward programmed cell death and reduction of CD20 levels.

Vanadium is a chemical element that enters the atmosphere via anthropogenic pollution. Exposure to vanadium affects cancer development and can result in toxic effects. Multiple studies have focused on vanadium's detrimental effect on male reproduction using conventional sperm analysis techniques. This study focused on vanadium's effect on spermatozoa following capacitation at the molecular level, in order to provide a more detailed assessment of vanadium's reproductive toxicity. We observed a decrease in germ cell density and a structural collapse of the testicular organ in seminiferous tubules during vanadium treatment. In addition, various sperm motion parameters were significantly decreased regardless of capacitation status, including sperm motility, rapid sperm motility, and progressive sperm motility. Curvilinear velocity, straight-line velocity, average path velocity, beat cross frequency, and mean amplitude of head lateral displacement were also decreased after capacitation. Capacitation status was altered after capacitation. Vanadium dramatically enhanced protein kinase A (PKA) activity and tyrosine phosphorylation. Taken together, our results suggest that vanadium is detrimental to male fertility by negatively influencing sperm motility, motion kinematics, and capacitation status via abnormal PKA activity and tyrosine phosphorylation before and after capacitation.

Mitochondrial morphology and function are crucial for tissue homeostasis, such as for skeletal development, but the cellular and molecular mechanisms remain unclear. Here, we provide evidence that regulator of G-protein signaling 12 (RGS12) is present in the mitochondria of primary chondrocytes and cartilage tissues. Deletion of RGS12 in type II collagen-positive cells led to a significant decrease in mitochondrial number, membrane potential, and oxidative phosphorylation function. Mechanistically, RGS12 promoted the function of ATP5A as an enhancer of tyrosine phosphorylation. Mice with RGS12 deficiency in the chondrocyte lineage showed serious body retardation, decreased bone mass, and chondrocyte apoptosis due to the defective activity of ATP synthase. To our knowledge, this is the first report that RGS12 is required for maintaining the function of mitochondria, which may allow it to orchestrate responses to cellular homeostasis.

BACKGROUND Cerebral complications in preeclampsia are leading causes of maternal mortality worldwide but pathophysiology is largely unknown and a challenge to study. Using an in vitro model of the human blood–brain barrier (BBB), we explored the role of vascular endothelial growth factor receptor 2 (VEGFR2) in preeclampsia. METHODS The human brain endothelial cell line (hCMEC/D3) cultured on Tranwells insert was exposed (12 hours) to plasma from women with preeclampsia (n = 28), normal pregnancy (n = 28), and nonpregnant (n = 16) controls. Transendothelial electrical resistance (TEER) and permeability to 70 kDa fluorescein isothiocyanate (FITC)-dextran were measured for the assessment of BBB integrity. We explored possible underlying mechanisms, with a focus on the expression of tight junction proteins and phosphorylation of 2 tyrosine residues of VEGFR2, associated with vascular permeability and migration (pY951) and cell proliferation (pY1175). Plasma concentrations of soluble FMS-like tyrosine kinase-1 (sFlt-1) and placental growth factor (PlGF) were also measured. RESULTS hCMEC/D3 exposed to plasma from women with preeclampsia exhibited reduced TEER and increased permeability to 70 kDa FITC-dextran. These cells upregulated the messenger ribonucleic acid (mRNA) levels of VEGFR2, and pY951-VEGFR2, but reduced pY1175-VEGFR2 (P < 0.05 in all cases). No difference in mRNA expression of tight junction protein was observed between groups. There was no correlation between angiogenic biomarkers and BBB permeability. CONCLUSIONS We present a promising in vitro model of the BBB in preeclampsia. Selective tyrosine phosphorylation of VEGFR2 may participate in the increased BBB permeability in preeclampsia irrespective of plasma concentrations of angiogenic biomarkers.

Activation of REarranged during Transfection (RET) proto-oncogene is responsible for various human cancers such as papillary and medullary thyroid carcinomas and non-small cell lung carcinomas. RET activation in these tumors is caused by point mutations or gene rearrangements, resulting in constitutive activation of RET tyrosine kinase. Physiologically, RET is activated by glial cell line-derived neurotrophic factor (GDNF) ligands that bind to coreceptor GDNF family receptor alphas (GFRαs), leading to RET dimerization. GDNF-GFRα1-RET signaling plays crucial roles in the development of the enteric nervous system, kidney and lower urinary tract as well as in spermatogenesis. Intracellular tyrosine phosphorylation in RET and recruitment of adaptor proteins to phosphotyrosines are essential for various biological functions. Significance of intracellular RET signaling pathways activated by GDNF is discussed and summarized in this review.

Purpose Ureteropelvic junction (UPJ) obstruction is the most common cause of congenital hydronephrosis in children. The pathophysiology of UPJ obstruction and the exact mechanism of pelviureteral peristalsis are poorly understood. Anoctamin-1 (ANO1), a Ca2 +-activated chloride channel, has been shown to play a key role in muscle wall contractions in the gastrointestinal tract. We designed this study to investigate the hypothesis that ANO1 is expressed in smooth muscle cells (SMCs) of the human UPJ and that tyrosine phosphorylation is altered in UPJ obstruction. Materials and methods Fresh frozen specimens of UPJ obstruction (n = 28) and control specimens from patients who underwent Wilms' tumor nephrectomy (n = 20) were prepared. Western blot (WB) was performed to evaluate levels of ANO1 protein expression and changes in tyrosine phosphorylation. In addition analysis of ANO1 and phalloidin using confocal-immunofluoresence-double staining and 3D reconstruction were carried out. Results Our WB results revealed increased tyrosine phosphorylation in UPJ obstruction samples compared to controls, and decreased ANO1 expression in UPJ obstruction. Confocal microscopy showed that ANO1 immunoreactivity was decreased in SMCs of UPJ obstruction compared to controls. Conclusions We provide evidence, for the first time, of the presence of ANO1 expression in the human UPJ. We speculate that altered tyrosine phosphorylation, observed in UPJ obstruction, may lead to a failure of transmission of peristaltic waves in UPJ obstruction by inhibiting Ca2 +-activated chloride channels in SMCs.

ERBB4 is a member of the epidermal growth factor receptor (EGFR)/ERBB subfamily of receptor tyrosine kinases that regulates cellular processes including proliferation, migration, and survival. ERBB4 signaling is involved in embryogenesis and homeostasis of healthy adult tissues, but also in human pathologies such as cancer, neurological disorders, and cardiovascular diseases. Here, an MS-based analysis revealed the Vav guanine nucleotide exchange factor 3 (VAV3), an activator of Rho family GTPases, as a critical ERBB4-interacting protein in breast cancer cells. We confirmed the ERBB4–VAV3 interaction by targeted MS and coimmunoprecipitation experiments and further defined it by demonstrating that kinase activity and Tyr-1022 and Tyr-1162 of ERBB4, as well as the intact phosphotyrosine-interacting SH2 domain of VAV3, are necessary for this interaction. We found that ERBB4 stimulates tyrosine phosphorylation of the VAV3 activation domain, known to be required for guanine nucleotide exchange factor (GEF) activity of VAV proteins. In addition to VAV3, the other members of the VAV family, VAV1 and VAV2, also coprecipitated with ERBB4. Analyses of the effects of overexpression of dominant-negative VAV3 constructs or shRNA-mediated down-regulation of VAV3 expression in breast cancer cells indicated that active VAV3 is involved in ERBB4-stimulated cell migration. These results define the VAV GEFs as effectors of ERBB4 activity in a signaling pathway relevant for cancer cell migration.

SUMMARY: Letrozole (Letro) is a drug commonly used for breast cancer treatment since it can decrease estrogen level. In experimental animal, the Letro has been used to induce the polycystic ovarian syndrome (PCOS) model. Tyrosine phosphorylation (TyrPho) is an essential process in various biological functions both normal and abnormal conditions especially reproduction. Although some side effects of Letro are reported, the alterations of TyrPho responsible for liver and kidney functions have never been demonstrated. In this study, the blood serum, liver, and kidney of control and PCOS rats induced with Letro (orally, 1 mg/ KgBW) for consecutive 21 days were used to determine the serum biochemical components and to investigate the TyrPho expression using western blot analysis. Histopathology of such tissues was observed by Masson’s trichrome staining. The results showed that Letro did not affect histological structures but significantly increased the serum levels of urea nitrogen, cholesterol, triglyceride, HDL, LDL, ALT, AST, and alkaline phosphatase. Additionally, the TyrPho protein expressions of 32 and 27 kDas in liver and of 55 and 43 kDas in kidney were increased while of a kidney 26 kDa was decreased as compared to those of control. In conclusion, this recent study indicated that the changes of TyrPho proteins in liver and kidney induced with Letro associated with their functions by alteration of serum biochemical levels.

Background Heat shock protein 90 (Hsp90) signaling pathways participate in protein phosphorylation during sperm capacitation. However, the underlying mechanism is largely unknown. Objective The aim of this study was to explore the interaction between Hsp90 and its co-chaperone protein, cell division cycle protein Cdc37 (Cdc37), in human spermatozoa. Materials and methods We examined the effects of H-89 (a protein kinase A [PKA] inhibitor) and Go6983 (a protein kinase C [PKC] inhibitor) on the phosphorylation of serine, threonine, and tyrosine residues in Hsp90; the effect of 17-allylamino-17-demethoxygeldanamycin (17-AAG, Hsp90 inhibitor) on Y416-Src phosphorylation; and the effects of 17-AAG and geldanamycin on threonine phosphorylation during human sperm capacitation. Results Hsp90 co-localized and interacted with Cdc37. During human sperm capacitation, Hsp90 phosphorylation at serine, threonine, and tyrosine residues was inhibited by H-89 and Go6983. In addition, phosphorylation of residue Y416 in the tyrosine kinase Src (its active site) was inhibited by 17-AAG, and the threonine phosphorylation levels of some proteins were decreased by 17-AAG and geldanamycin. Discussion and conclusion Taken together, our data showed that the interaction of Hsp90 with Cdc37 regulates total protein threonine phosphorylation and Src phosphorylation via its serine, threonine, and tyrosine phosphorylation, which are controlled by PKA and PKC during human sperm capacitation. The results of this study help understand the mechanism underlying Hsp90 regulation of sperm function.

Aims Receptor-type vascular endothelial protein tyrosine phosphatase (VE-PTP) dephosphorylates Tie-2 as well as CD31, VE-cadherin, and vascular endothelial growth factor receptor 2 (VEGFR2). The latter form a signal transduction complex that mediates the endothelial cell response to shear stress, including the activation of the endothelial nitric oxide (NO) synthase (eNOS). As VE-PTP expression is increased in diabetes, we investigated the consequences of VE-PTP inhibition (using AKB-9778) on blood pressure in diabetic patients and the role of VE-PTP in the regulation of eNOS activity and vascular reactivity. Methods and results In diabetic patients AKB-9778 significantly lowered systolic and diastolic blood pressure. This could be linked to elevated NO production, as AKB increased NO generation by cultured endothelial cells and elicited the NOS inhibitor-sensitive relaxation of endothelium-intact rings of mouse aorta. At the molecular level, VE-PTP inhibition increased the phosphorylation of eNOS on Tyr81 and Ser1177 (human sequence). The PIEZO1 activator Yoda1, which was used to mimic the response to shear stress, also increased eNOS Tyr81 phosphorylation, an effect that was enhanced by VE-PTP inhibition. Two kinases, i.e. abelson-tyrosine protein kinase (ABL)1 and Src were identified as eNOS Tyr81 kinases as their inhibition and down-regulation significantly reduced the basal and Yoda1-induced tyrosine phosphorylation and activity of eNOS. VE-PTP, on the other hand, formed a complex with eNOS in endothelial cells and directly dephosphorylated eNOS Tyr81 in vitro. Finally, phosphorylation of eNOS on Tyr80 (murine sequence) was found to be reduced in diabetic mice and diabetes-induced endothelial dysfunction (isolated aortic rings) was blunted by VE-PTP inhibition. Conclusions VE-PTP inhibition enhances eNOS activity to improve endothelial function and decrease blood pressure indirectly, through the activation of Tie-2 and the CD31/VE-cadherin/VEGFR2 complex, and directly by dephosphorylating eNOS Tyr81. VE-PTP inhibition, therefore, represents an attractive novel therapeutic option for diabetes-induced endothelial dysfunction and hypertension.

BackgroundEPS8 is a scaffolding protein that regulates proliferation, actin dynamics and receptor trafficking. Its expression is increased in cancer, enhancing mitogenesis, migration and tumorigenesis. Src phosphorylates EPS8 at four tyrosine residues, although the function is unknown. Here we investigated the pro-oncogenic role of EPS8 tyrosine phosphorylation at Src target sites in HNSCC.MethodsPlasmids expressing EPS8 Src-mediated phosphorylation site mutants (Y485F, Y525F, Y602F, Y774F and all four combined [FFFF]) were expressed in cells containing a normal endogenous level of EPS8. In addition, cells were treated with dasatinib to inhibit Src activity. EPS8 downstream targets were evaluated by western blotting. Wound closure, proliferation, immunofluorescence and tumorgenicity assays were used to investigate the impact of phenylalanine mutations on EPS8 biological functions.ResultsFOXM1, AURKA, and AURKB were decreased in cells expressing FFFF- and Y602F-EPS8 mutants, while cells harbouring the Y485F-, Y525F- and Y774F-EPS8 mutants showed no differences compared to controls. Consistent with this, dasatinib decreased the expression of EPS8 targets. Moreover, Y602F- and FFFF-EPS8 mutants reduced mitogenesis and motility. Strikingly though, FFFF- or Y602F-EPS8 mutants actually promoted tumorigenicity compared with control cells.ConclusionsPhosphorylation of EPS8 at Y602 is crucial for signalling to the cell cycle and may provide insight to explain reduced efficacy of dasatinib treatment.

Transient Receptor Potential (TRP) channels are a superfamily of polymodal, non-selective receptors, expressed in the nervous system and several other tissues, where they play many physiological or pathological roles. TRP channels are sensitive to a diverse range of stimuli, such as temperature, osmolarity, oxidative stress, external compounds and intracellular signaling molecules. The activity of TRP channels can be modulated by protein phosphorylation, including tyrosine phosphorylation. In this review, we present the studies carried out so far regarding the modulation of TRP channels by tyrosine phosphorylation.

Schizophrenia is a debilitating disorder affecting young adults displaying symptoms of cognitive impairment, anxiety, and early social isolation prior to episodes of auditory hallucinations. Cannabis use has been tied to schizophrenia-like symptoms, indicating that dysregulated endogenous cannabinoid signaling may be causally linked to schizophrenia. Previously, we reported that glutamatergic neuron-selective ablation of Lmo4, an endogenous inhibitor of the tyrosine phosphatase PTP1B, impairs endocannabinoid (eCB) production from the metabotropic glutamate receptor mGluR5. These Lmo4-deficient mice display anxiety-like behaviors that are alleviated by local shRNA knockdown or pharmacological inhibition of PTP1B that restores mGluR5-dependent eCB production in the amygdala. Here, we report that these Lmo4-deficient mice also display schizophrenia-like behaviors: impaired working memory assessed in the Y maze and defective sensory gating by prepulse inhibition of the acoustic startle response. Modulation of inhibitory inputs onto layer 2/3 pyramidal neurons of the prefrontal cortex relies on eCB signaling from the brain-derived neurotrophic factor receptor trkB, rather than mGluR5, and this mechanism was defective in Lmo4-deficient mice. Genetic ablation of PTP1B in the glutamatergic neurons lacking Lmo4 restored tyrosine phosphorylation of trkB, trkB-mediated eCB signaling, and ameliorated schizophrenia-like behaviors. Pharmacological inhibition of PTP1B with trodusquemine also restored trkB phosphorylation and improved schizophrenia-like behaviors by restoring eCB signaling, since the CB1 receptor antagonist 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide blocked this effect. Thus, activation of PTP1B in pyramidal neurons contributes to schizophrenia-like behaviors in Lmo4-deficient mice and genetic or pharmacological intervention targeting PTP1B ameliorates schizophrenia-related deficits.

Voltage-gated sodium channel (VGSC) β1 subunits are multifunctional proteins that modulate the biophysical properties and cell-surface localization of VGSC α subunits and participate in cell–cell and cell–matrix adhesion, all with important implications for intracellular signal transduction, cell migration, and differentiation. Human loss-of-function variants in SCN1B, the gene encoding the VGSC β1 subunits, are linked to severe diseases with high risk for sudden death, including epileptic encephalopathy and cardiac arrhythmia. We showed previously that β1 subunits are post-translationally modified by tyrosine phosphorylation. We also showed that β1 subunits undergo regulated intramembrane proteolysis via the activity of β-secretase 1 and γ-secretase, resulting in the generation of a soluble intracellular domain, β1-ICD, which modulates transcription. Here, we report that β1 subunits are phosphorylated by FYN kinase. Moreover, we show that β1 subunits are S-palmitoylated. Substitution of a single residue in β1, Cys-162, to alanine prevented palmitoylation, reduced the level of β1 polypeptides at the plasma membrane, and reduced the extent of β1-regulated intramembrane proteolysis, suggesting that the plasma membrane is the site of β1 proteolytic processing. Treatment with the clathrin-mediated endocytosis inhibitor, Dyngo-4a, re-stored the plasma membrane association of β1-p.C162A to WT levels. Despite these observations, palmitoylation-null β1-p.C162A modulated sodium current and sorted to detergent-resistant membrane fractions normally. This is the first demonstration of S-palmitoylation of a VGSC β subunit, establishing precedence for this post-translational modification as a regulatory mechanism in this protein family.

Axon guidance is a critical process in forming the connections between a neuron and its target. The growth cone steers the growing axon toward the appropriate direction by integrating extracellular guidance cues and initiating intracellular signal transduction pathways downstream of these cues. The growth cone generates these responses by remodeling its cytoskeletal components. Regulation of microtubule dynamics within the growth cone is important for making guidance decisions. TACC3, as a microtubule plus-end binding (EB) protein, modulates microtubule dynamics during axon outgrowth and guidance. We have previously shown that Xenopus laevis embryos depleted of TACC3 displayed spinal cord axon guidance defects, while TACC3-overexpressing spinal neurons showed increased resistance to Slit2-induced growth cone collapse. Tyrosine kinases play an important role in relaying guidance signals to downstream targets during pathfinding events via inducing tyrosine phosphorylation. Here, in order to investigate the mechanism behind TACC3-mediated axon guidance, we examined whether tyrosine residues that are present in TACC3 have any role in regulating TACC3's interaction with microtubules or during axon outgrowth and guidance behaviors. We find that the phosphorylatable tyrosines within the TACC domain are important for the microtubule plus-end tracking behavior of TACC3. Moreover, TACC domain phosphorylation impacts axon outgrowth dynamics such as growth length and growth persistency. Together, our results suggest that tyrosine phosphorylation of TACC3 affects TACC3's microtubule plus-end tracking behavior as well as its ability to mediate axon growth dynamics in cultured embryonic neural tube explants.

Osteocytes, cells ensconced within mineralized bone matrix, are the primary skeletal mechanosensors. Osteocytes sense mechanical cues by changes in fluid flow shear stress (FFSS) across their dendritic projections. Loading-induced reductions of osteocytic Sclerostin (encoded by Sost) expression stimulates new bone formation. However, the molecular steps linking mechanotransduction and Sost suppression remain unknown. Here, we report that class IIa histone deacetylases (HDAC4 and HDAC5) are required for loading-induced Sost suppression and bone formation. FFSS signaling drives class IIa HDAC nuclear translocation through a signaling pathway involving direct HDAC5 tyrosine 642 phosphorylation by focal adhesion kinase (FAK), a HDAC5 post-translational modification that controls its subcellular localization. Osteocyte cell adhesion supports FAK tyrosine phosphorylation, and FFSS triggers FAK dephosphorylation. Pharmacologic FAK catalytic inhibition reduces Sost mRNA expression in vitro and in vivo. These studies demonstrate a role for HDAC5 as a transducer of matrix-derived cues to regulate cell type-specific gene expression., Osteocytes are mechanoresponsive within skeletal tissue. Here, the authors show that class IIa histone deacetylases are phosphorylated by focal adhesion kinase, suggesting that HDAC5 may propagate mechanobiological cues to regulate cell type-specific gene expression.

In response to genotoxic stress, multiple kinase signaling cascades are activated, many of them directed towards the tumor suppressor p53, which coordinates the DNA damage response (DDR). Defects in DDR pathways lead to an accumulation of mutations that can promote tumorigenesis. Emerging evidence implicates multiple members of the NimA-related kinase (NEK) family (NEK1, NEK10, and NEK11) in the DDR. Here, we describe a function for NEK10 in the regulation of p53 transcriptional activity through tyrosine phosphorylation. NEK10 loss increases cellular proliferation by modulating the p53-dependent transcriptional output. NEK10 directly phosphorylates p53 on Y327, revealing NEK10’s unexpected substrate specificity. A p53 mutant at this site (Y327F) acts as a hypomorph, causing an attenuated p53-mediated transcriptional response. Consistently, NEK10-deficient cells display heightened sensitivity to DNA-damaging agents. Further, a combinatorial score of NEK10 and TP53-target gene expression is an independent predictor of a favorable outcome in breast cancers.

Extracellular ATP released from stimulated and/or damaged cells modulates physiological responses via stimulation of various purinoceptors. We previously showed that ATP potentiated the Ag-induced mast cell (MC) degranulation via purinoceptors pharmacologically similar to the ionotropic P2X4 receptor. In this study, we investigated the role of P2X4 receptor in MC degranulation induced by stimulation of IgE-FcεRI complex with Ag, using bone marrow–derived MCs (BMMCs) prepared from wild type and P2X4 receptor–deficient (P2rx4−/−) mice. ATP significantly increased Ag-induced degranulation in BMMCs prepared from wild type mice. This effect of ATP was reduced in BMMCs prepared from P2rx4−/− mice. The potentiating effect of ATP was restored by expressing P2X4 receptor in P2rx4−/− BMMCs. The P2X4 receptor–mediated effects were maintained even after differentiating into the connective tissue-type MCs. P2X4 receptor stimulation did not affect the Ag-induced Ca2+ response but enhanced Ag-induced early signals, such as tyrosine phosphorylation of Syk and phospholipase C-γ. Interestingly, these effects of ATP on Syk phosphorylation were not impaired by pretreatment with Cu2+, an inhibitor of the P2X4 receptor channel, or removal of external Ca2+, suggesting that a mechanisms other than Ca2+ influx through ion channel activity may be involved. In vivo experiments revealed that systemic and intradermal passive anaphylaxis responses were significantly alleviated in P2rx4−/− mice. Taken together, the present data suggest that the P2X4 receptor plays an essential role in ATP-induced upregulation of MC degranulation in response to Ag, and also contributes to the Ag-induced allergic response in vivo.

The Hippo pathway and its effector protein YAP (a transcriptional coactivator) have been identified as important in the biology of both hepatocellular carcinoma and cholangiocarcinoma. First identified as a tumor suppressor pathway in Drosophila, the understanding of the mammalian YAP signaling and its regulation continues to expand. In its “on” function, the canonical regulatory Hippo pathway, a well-described serine/threonine kinase module, regulates YAP function by restricting its subcellular localization to the cytoplasm. In contrast, when the Hippo pathway is “off,” YAP translocates to the nucleus and drives cotranscriptional activity. Given the role of Hippo/YAP signaling in hepatic malignancies, investigators have sought to target these molecules; however, standard approaches have not been successful based on the pathways’ negative regulatory role. More recently, additional regulatory mechanisms, such as tyrosine phosphorylation, of YAP have been described. These represent positive regulatory events that may be targetable. Additionally, several groups have identified potentiating feed-forward signaling for YAP in multiple contexts, suggesting other experimental therapeutic approaches to interrupt these signaling loops. Herein we explore the current data supporting alternative YAP regulatory pathways, review the described feed-forward signaling cascades that are YAP dependent, and explore targeting strategies that have been employed in preclinical models of hepatic malignancies.

Gastric cancer is associated with chronic inflammation (gastritis) triggered by persistent Helicobacter pylori (H. pylori) infection. Elevated tyrosine phosphorylation of the latent transcription factor STAT3 is a feature of gastric cancer, including H. pylori-infected tissues, and aligns with nuclear transcriptional activity. However, the transcriptional role of STAT3 serine phosphorylation, which promotes STAT3-driven mitochondrial activities, is unclear. Here, by coupling serine-phosphorylated (pS)-STAT3-deficient Stat3SA/SA mice with chronic H. felis infection, which mimics human H. pylori infection in mice, we reveal a key role for pS-STAT3 in promoting Helicobacter-induced gastric pathology. Immunohistochemical staining for infiltrating immune cells and expression analyses of inflammatory genes revealed that gastritis was markedly suppressed in infected Stat3SA/SA mice compared with wild-type mice. Stomach weight and gastric mucosal thickness were also reduced in infected Stat3SA/SA mice, which was associated with reduced proliferative potential of infected Stat3SA/SA gastric mucosa. The suppressed H. felis-induced gastric phenotype of Stat3SA/SA mice was phenocopied upon genetic ablation of signaling by the cytokine IL-11, which promotes gastric tumorigenesis via STAT3. pS-STAT3 dependency by Helicobacter coincided with transcriptional activity on STAT3-regulated genes, rather than mitochondrial and metabolic genes. In the gastric mucosa of mice and patients with gastritis, pS-STAT3 was constitutively expressed irrespective of Helicobacter infection. Collectively, these findings suggest an obligate requirement for IL-11 signaling via constitutive pS-STAT3 in Helicobacter-induced gastric carcinogenesis.