The luminal domain of p23 (Tmp21) plays a critical role in p23 cell surface trafficking

p23 (Tmp21 or p24δ), a member of the p24 family, is important for maintaining the integrity of the secretory pathway in mammals. It is a type I protein with a receptor-like luminal domain and a short cytoplasmic tail. This cytoplasmic tail carries an atypical endoplasmic reticulum (ER) retention KKXX motif that binds to coat protein I. The trafficking of p23 has been thought to be restricted to the early secretory pathway. However, recent findings as well as this study demonstrate that p23 is also found in the plasma membrane. By tagging different domains of p23 with green fluorescent protein, it is shown that it is the luminal domain that is primarily responsible for the appearance of p23 in the plasma membrane, despite the presence of a functional KKXX-ER retention and retrieval motif. When the KKXX motif is abolished, p23 shows an extremely increased trafficking to the plasma membrane. These experiments reveal the presence of two fractions of p23 with distinct trafficking destinations. One fraction cycles through the ER–Golgi pathway using its functional KKXX retrieval motif. The transient appearance of p23 in the plasma membrane is supported by the luminal domain. These results help to explain the functional presence of p23 in plasma membrane protein complexes and post-Golgi compartments.     

Tyrosine 319, a newly identified phosphorylation site of ZAP-70, plays a critical role in T cell antigen receptor signaling

Following T cell antigen receptor (TCR) engagement, the protein tyrosine kinase (PTK) ZAP-70 is rapidly phosphorylated on several tyrosine residues, presumably by two mechanisms: an autophosphorylation and a trans-phosphorylation by the Src-family PTK Lck. These events have been implicated in both positive and negative regulation of ZAP-70 activity and in coupling this PTK to downstream signaling pathways in T cells. We show here that Tyr315 and Tyr319 in the interdomain B of ZAP-70 are autophosphorylated in vitro and become phosphorylated in vivo upon TCR triggering. Moreover, by mutational analysis, we demonstrate that phosphorylation of Tyr319 is required for the positive regulation of ZAP-70 function. Indeed, overexpression in Jurkat cells and in a murine T cell hybridoma of a ZAP-70 mutant in which Tyr319 was replaced by phenylalanine (ZAP-70-Y319F) dramatically impaired anti-TCR-induced activation of the nuclear factor of activated T cells and interleukin-2 production, respectively. Surprisingly, an analogous mutation of Tyr315 had little or no effect. The inhibitory effect of ZAP-70-Y319F correlated with a substantial loss of its activation-induced tyrosine phosphorylation and up-regulation of catalytic activity, as well as with a decreased in vivo capacity to phosphorylate known ZAP-70 substrates, such as SLP-76 and LAT. Collectively, our data reveal the pivotal role of Tyr319 phosphorylation in the positive regulation of ZAP-70 and in TCR-mediated signaling.     

Phospholipase D1 plays a key role in TNF-alpha signaling

The primary characteristic features of any inflammatory or infectious lesions are immune cell infiltration, cellular proliferation, and the generation of proinflammatory mediators. TNF-alpha is a potent proinflammatory and immuno-regulatory cytokine. Decades of research have been focused on the physiological/pathophysiological events triggered by TNF-alpha. However, the signaling network initiated by TNF-alpha in human leukocytes is still poorly understood. In this study, we report that TNF-alpha activates phospholipase D1 (PLD1), in a dose-dependent manner, and PLD1 is required for the activation of sphingosine kinase and cytosolic calcium signals. PLD1 is also required for NFkappaB and ERK1/2 activation in human monocytic cells. Using antisense oligonucleotides to reduce specifically the expression of PLD isozymes showed PLD1, but not PLD2, to be coupled to TNF-alpha signaling and that PLD1 is required to mediate receptor activation of sphingosine kinase and calcium transients. In addition, the coupling of TNF-alpha to activation of the phosphorylation of ERK1/2 and the activation of NFkappaB were inhibited by pretreating cells with antisense to PLD1, but not to PLD2; thus, demonstrating a specific requirement for PLD1. Furthermore, use of antisense oligonucleotides to reduce expression of PLD1 or PLD2 demonstrated that PLD1 is required for TNF-alpha-induced production of several important cytokines, such as IL-1beta, IL-5, IL-6, and IL-13, in human monocytes. These studies demonstrate the critical role of PLD1 in the intracellular signaling cascades initiated by TNF-alpha and its functional role for coordinating the signals to inflammatory responses.     

PP2A-mediated dephosphorylation of p107 plays a critical role in chondrocyte cell cycle arrest by FGF

FGF signaling inhibits chondrocyte proliferation, a cell type-specific response that is the basis for several genetic skeletal disorders caused by activating FGFR mutations. This phenomenon requires the function of the p107 and p130 members of the Rb protein family, and p107 dephosphorylation is one of the earliest distinguishing events in FGF-induced growth arrest. To determine whether p107 dephoshorylation played a critical role in the chondrocyte response to FGF, we sought to counteract this process by overexpressing in RCS chondrocytes the cyclin D1/cdk4 kinase complex. CyclinD/cdk4-expressing RCS cells became resistant to FGF-induced p107 dephosphorylation and growth arrest, and maintained significantly high levels of cyclin E/cdk2 activity and of phosphorylated p130 at later times of FGF treatment. We explored the involvement of a phosphatase in p107 dephosphorylation. Expression of the SV40 small T-Ag, which inhibits the activity of the PP2A phosphatase, or knockdown of the expression of the PP2A catalytic subunit by RNA interference prevented p107 dephosphorylation and FGF-induced growth arrest of RCS cells. Furthermore, an association between p107 and PP2A was induced by FGF treatment. Our data show that p107 dephosphorylation is a key event in FGF-induced cell cycle arrest and indicate that in chondrocytes FGF activates the PP2A phosphatase to promote p107 dephosphorylation.     

The wnt receptor ryk plays a role in Mammalian planar cell polarity signaling

Wnts are essential for a wide range of developmental processes, including cell growth, division, and differentiation. Some of these processes signal via the planar cell polarity (PCP) pathway, which is a β-catenin-independent Wnt signaling pathway. Previous studies have shown that Ryk, a member of the receptor tyrosine kinase family, can bind to Wnts. Ryk is required for normal axon guidance and neuronal differentiation during development. Here, we demonstrate that mammalian Ryk interacts with the Wnt/PCP pathway. In vitro analysis showed that the Wnt inhibitory factor domain of Ryk was necessary for Wnt binding. Detailed analysis of two vertebrate model organisms showed Ryk phenotypes consistent with PCP signaling. In zebrafish, gene knockdown using morpholinos revealed a genetic interaction between Ryk and Wnt11 during the PCP pathway-regulated process of embryo convergent extension. Ryk-deficient mouse embryos displayed disrupted polarity of stereociliary hair cells in the cochlea, a characteristic of disturbed PCP signaling. This PCP defect was also observed in mouse embryos that were double heterozygotes for Ryk and Looptail (containing a mutation in the core Wnt/PCP pathway gene Vangl2) but not in either of the single heterozygotes, suggesting a genetic interaction between Ryk and Vangl2. Co-immunoprecipitation studies demonstrated that RYK and VANGL2 proteins form a complex, whereas RYK also activated RhoA, a downstream effector of PCP signaling. Overall, our data suggest an important role for Ryk in Wnt/planar cell polarity signaling during vertebrate development via the Vangl2 signaling pathway, as demonstrated in the mouse cochlea.     

Angiopoietin 2 signaling plays a critical role in neural crest cell migration

Background

Collective neural crest cell migration is critical to the form and function of the vertebrate face and neck, distributing bone, cartilage, and nerve cells into peripheral targets that are intimately linked with head vasculature. The vasculature and neural crest structures are ultimately linked, but when and how these patterns develop in the early embryo are not well understood.

Results

Using in vivo imaging and sophisticated cell behavior analyses, we show that quail cranial neural crest and endothelial cells share common migratory paths, sort out in a dynamic multistep process, and display multiple types of motion. To better understand the underlying molecular signals, we examined the role of angiopoietin 2 (Ang2), which we found expressed in migrating cranial neural crest cells. Overexpression of Ang2 causes neural crest cells to be more exploratory as displayed by invasion of off-target locations, the widening of migratory streams into prohibitive zones, and differences in cell motility type. The enhanced exploratory phenotype correlates with increased phosphorylated focal adhesion kinase activity in migrating neural crest cells. In contrast, loss of Ang2 function reduces neural crest cell exploration. In both gain and loss of function of Ang2, we found disruptions to the timing and interplay between cranial neural crest and endothelial cells.

Conclusions

Together, these data demonstrate a role for Ang2 in maintaining collective cranial neural crest cell migration and suggest interdependence with endothelial cell migration during vertebrate head patterning.

     

MyD88 plays a critical T cell-intrinsic role in supporting CD8 T cell expansion during acute lymphocytic choriomeningitis virus infection

During acute lymphocytic choriomeningitis virus (LCMV) infection, CD8 T cells rapidly expand and differentiate into effectors that are required for viral clearance. The accumulation of activated T cells is greatly reduced in mice lacking the adaptor molecule MyD88. Although MyD88 has generally been considered to indirectly regulate adaptive immune responses by controlling inflammatory cytokine production and Ag presentation in innate immune cells, in this study, we identify an unappreciated cell-intrinsic role for MyD88 in LCMV-specific CD8 T cells. Using reciprocal adoptive transfer models and bone marrow chimeras, we show that Myd88(-/-) CD8 T cells are defective in their clonal expansion in response to LCMV infection, independent of their environment. Furthermore, we show that while MyD88 is dispensable for initial activation and division of LCMV-specific CD8 T cells during the early stages of viral infection, MyD88-dependent signals are critical for supporting their survival and sustained accumulation.     

p21-activated kinase 1 plays a critical role in cellular activation by Nef

The activation of Nef-associated kinase (NAK) by Nef from human and simian immunodeficiency viruses is critical for efficient viral replication and pathogenesis. This induction occurs via the guanine nucleotide exchange factor Vav and the small GTPases Rac1 and Cdc42. In this study, we identified NAK as p21-activated kinase 1 (PAK1). PAK1 bound to Nef in vitro and in vivo. Moreover, the induction of cytoskeletal rearrangements such as the formation of trichopodia, the activation of Jun N-terminal kinase, and the increase of viral production were blocked by an inhibitory peptide that targets the kinase activity of PAK1 (PAK1 83-149). These results identify NAK as PAK1 and emphasize the central role its kinase activity plays in cytoskeletal rearrangements and cellular signaling by Nef.     

The Coxsackie and adenovirus receptor binds microtubules and plays a role in cell migration

The Coxsackie and adenovirus receptor (CAR), a cell adhesion molecule of the immunoglobulin superfamily, inhibits cell growth of a variety of tumors. The cytoplasmic domain of CAR has been implicated in decreased invasion and intracerebral growth of human U87 glioma cells. Using affinity binding, we identified tubulin as an interaction partner for the cytoplasmic domain of CAR. The interaction was specific; CAR and tubulin co-immunoprecipitated in cells expressing endogenous CAR and partially co-localized in situ. The binding of CAR to tubulin heterodimers and to microtubules was direct, with dissociation constants of approximately 1 mum for tubulin and approximately 32 nm for in vitro assembled microtubules. Whereas CAR-expressing U87 glioma cells had decreased migration in a chemotactic assay in Boyden chambers as compared with control cells, an effect that depended on the presence of the cytoplasmic domain of CAR, the difference was abrogated at low, non-cytotoxic doses of the taxane paclitaxel, a microtubule-stabilizing agent. These results indicate that CAR may affect cell migration through its interaction with microtubules.     

ZFAT plays critical roles in peripheral T cell homeostasis and its T cell receptor-mediated response

ZFAT, originally identified as a candidate susceptibility gene for autoimmune thyroid disease, has been reported to be involved in apoptosis, development and primitive hematopoiesis. Zfat is highly expressed in T- and B-cells in the lymphoid tissues, however, its physiological function in the immune system remains totally unknown. Here, we generated the T cell-specific Zfat-deficient mice and demonstrated that Zfat-deficiency leads to a remarkable reduction in the number of the peripheral T cells. Intriguingly, a reduced expression of IL-7Rα and the impaired responsiveness to IL-7 for the survival were observed in the Zfat-deficient T cells. Furthermore, a severe defect in proliferation and increased apoptosis in the Zfat-deficient T cells following T cell receptor (TCR) stimulation was observed with a reduced IL-2Rα expression as well as a reduced IL-2 production. Thus, our findings reveal that Zfat is a critical regulator in peripheral T cell homeostasis and its TCR-mediated response.     

Identification of TNF-alpha binding peptides from a D-amino acid hexapeptide library that specifically inhibit TNF-alpha binding to recombinant p55 receptor

Tumour necrosis factor alpha (TNF-alpha) is a pro-inflammatory cytokine with pleiotropic activity that binds to two transmembrane receptors. Its role in mediating the inflammatory response to injury or infection has been well documented and it has been shown to be a causative factor in rheumatoid arthritis, inflammatory bowel disease and septic shock. Using synthetic peptide libraries composed exclusively of D-amino acids, two distinct hexapeptide families that block the binding of TNF-alpha to its receptors were identified. In the deconvolution of the library, activity increased from submillimolar to the low micromolar range with the most active compound having an IC50 of 0.33 microM. With the aid of biotinylated constructs of these hexapeptides it was possible to demonstrate that their antagonistic effect is due to specific binding to TNF-alpha and not to its receptor.     

Toll-like receptor 2 plays a critical role in maintaining mucosal integrity during Citrobacter rodentium-induced colitis

Inflammatory bowel diseases and infectious gastroenteritis likely occur when the integrity of intestinal barriers is disrupted allowing luminal bacterial products to cross into the intestinal mucosa, stimulating immune cells and triggering inflammation. While specific Toll-like receptors (TLR) are involved in the generation of inflammatory responses against enteric bacteria, their contributions to the maintenance of intestinal mucosal integrity are less clear. These studies investigated the role of TLR2 in a model of murine colitis induced by the bacterial pathogen Citrobacter rodentium . C. rodentium supernatants specifically activated TLR2 in vitro while infected TLR2–/– mice suffered a lethal colitis coincident with colonic mucosal ulcerations, bleeding and increased cell death but not increased pathogen burden. TLR2–/– mice suffered impaired epithelial barrier function mediated via zonula occludens (ZO)-1 in naïve mice and claudin-3 in infected mice, suggesting this could underlie their susceptibility. TLR2 deficiency was also associated with impaired production of IL-6 by bone marrow-derived macrophages and infected colons cultured ex vivo . As IL-6 has antiapoptotic and epithelial repair capabilities, its reduced expression could contribute to the impaired mucosal integrity. These studies report for the first time that TLR2 plays a critical role in maintaining intestinal mucosal integrity during infection by a bacterial pathogen.     

TRAIL/DR5 plays a critical role in NK cell-mediated negative regulation of dendritic cell cross-priming of T cells

Dendritic cells (DCs) are critical in initiating immune responses by cross-priming of tumor Ags to T cells. Previous results showed that NK cells inhibited DC-mediated cross-presentation of tumor Ags both in vivo and in vitro. In this study, enhanced Ag presentation was observed in draining lymph nodes in TRAIL(-/-) and DR5(-/-) mice compared with that of wild-type mice. NK cells inhibit DC cross-priming of tumor Ags in vitro, but not direct presentation of endogenous Ags. NK cells lacking TRAIL, but not perforin, were not able to inhibit DC cross-priming of tumor Ags. DCs that lack expression of TRAIL receptor DR5 were less susceptible to NK cell-mediated inhibition of cross-priming, and cross-linking of DR5 receptor led to reduced generation of MHC class I-Ag peptide complexes, followed by attenuated cross-priming of CD8(+) T cells. In addition, key molecules involved in the TRAIL/DR5 pathway during DC/NK cell interactions were determined. In summary, these data indicate a novel alternative pathway for DC/NK cell interactions in antitumor immunity and may reflect homeostasis of both DCs and NK cells for regulation of CD8(+) T cell function in physiological conditions.     

Signaling for myocardial depression in hemorrhagic shock: roles of Toll-like receptor 4 and p55 TNF-alpha receptor

Hemorrhagic shock causes myocardial contractile depression. Although this myocardial disorder is associated with increased expression of tumor necrosis factor-alpha (TNF-alpha), the role of TNF-alpha as a myocardial depressant factor in hemorrhagic shock remains to be determined. Moreover, it is unclear which TNF-alpha receptor mediates the myocardial depressive effects of TNF-alpha. Toll-like receptor 4 (TLR4) regulates cellular expression of proinflammatory mediators following lipopolysaccharide stimulation and may be involved in the tissue inflammatory response to injury. The contribution of TLR4 signaling to tissue TNF-alpha response to hemorrhagic shock and TLR4's role in myocardial depression during hemorrhagic shock are presently unknown. We examined the relationship of TNF-alpha production to myocardial depression in a mouse model of nonresuscitated hemorrhagic shock, assessed the influence of TLR4 mutation, resulting in defective signaling, on TNF-alpha production and myocardial depression, and determined the roles of TNF-alpha and TNF-alpha receptors in myocardial depression using a gene knockout (KO) approach. Hemorrhagic shock resulted in increased plasma and myocardial TNF-alpha (4.9- and 4.5-fold, respectively) at 30 min and induced myocardial contractile depression at 4 h. TLR4 mutation abolished the TNF-alpha response and attenuated myocardial depression (left ventricular developed pressure of 43.0 +/- 6.2 mmHg in TLR4 mutant vs. 30.0 +/- 3.6 mmHg in wild type, P < 0.05). TNF-alpha KO also attenuated myocardial depression in hemorrhagic shock, and the p55 receptor KO, but not the p75 receptor KO, mimicked the effect of TNF-alpha KO. The results suggest that TLR4 plays a novel role in signaling to the TNF-alpha response during hemorrhagic shock and that TNF-alpha through the p55 receptor activates a pathway leading to myocardial depression. Thus TLR4 and the p55 TNF-alpha receptor represent therapeutic targets for preservation of cardiac mechanical function during hemorrhagic shock.     

The V protein of mumps virus plays a critical role in pathogenesis

Mumps virus (MuV) causes an acute infection in humans characterized by a wide array of symptoms ranging from relatively mild manifestations, such as parotitis, to more-severe complications, such as meningitis and encephalitis. Widespread mumps vaccination has reduced mumps incidence dramatically; however, outbreaks still occur in vaccinated populations. The V protein of MuV, when expressed in cell culture, blocks interferon (IFN) expression and signaling and interleukin-6 (IL-6) signaling. In this work, we generated a recombinant MuV incapable of expressing the V protein (rMuVΔV). The rescued MuV was derived from a clinical wild-type isolate from a recent outbreak in the United States (MuV(Iowa/US/06), G genotype). Analysis of the virus confirmed the roles of V protein in blocking IFN expression and signaling and IL-6 signaling. We also found that the rMuV(Iowa/US/06)ΔV virus induced high levels of IL-6 expression in vitro, suggesting that V plays a role in reducing IL-6 expression. In vivo, the rMuV(Iowa/US/06)ΔV virus was highly attenuated, indicating that the V protein plays an essential role in viral virulence.     

The AKT-mTOR pathway plays a critical role in the development of leiomyosarcomas

We analyzed the PI3K-AKT signaling cascade in a cohort of sarcomas and found a marked induction of insulin receptor substrate-2 (IRS2) and phosphorylated AKT and a concomitant upregulation of downstream effectors in most leiomyosarcomas. To determine the role of aberrant PI3K-AKT signaling in leiomyosarcoma pathogenesis, we genetically inactivated Pten in the smooth muscle cell lineage by cross-breeding Pten(loxP/loxP) mice with Tagln-cre mice. Mice carrying homozygous deletion of Pten alleles developed widespread smooth muscle cell hyperplasia and abdominal leiomyosarcomas, with a very rapid onset and elevated incidence (approximately 80%) compared to other animal models. Constitutive mTOR activation was restricted to the leiomyosarcomas, revealing the requirement for additional molecular events besides Pten loss. The rapamycin derivative everolimus substantially decelerated tumor growth on Tagln-cre/Pten(loxP/loxP) mice and prolonged their lifespan. Our data show a new and critical role for the AKT-mTOR pathway in smooth muscle transformation and leiomyosarcoma genesis, and support treatment of selected sarcomas by the targeting of this pathway with new compounds or combinations of these with conventional chemotherapy agents.     

CDK11p110 plays a critical role in the tumorigenicity of esophageal squamous cell carcinoma cells and is a potential drug target

Esophageal squamous cell carcinoma (ESCC) is a serious malignancy with limited options for targeted therapy. The exploration of novel targeted therapies for combating ESCC is urgently needed. Cyclin-dependent kinases (CDKs) play important roles in the progression of cancers; however, the function of CDK11^p110 (cyclin-dependent kinase 11^p110) in ESCC is still unknown. Here, we investigated the effects and molecular mechanisms of CDK11^p110 in the proliferation and growth of ESCC by examining the expression of CDK11^p110 in ESCC tissues and by detecting phenotypic changes in ESCC cells after CDK11^p110 knockdown or overexpression in vitro and in vivo. According to the tissue microarray analysis, compared with its expression level in normal tissues, the expression level of CDK11^p110 was significantly elevated in ESCC tissues; this result was in concordance with the data in TCGA (The Cancer Genome Atlas) datasets. In addition, RNAi-mediated CDK11^p110 silencing exerted a substantial inhibitory effect on the proliferation, clonogenicity and migration ability of ESCC cells. Further study indicated that CDK11^p110 knockdown arrested ESCC cells in the G2/M phase of the cell cycle and induced cell apoptosis. Moreover, stable shRNA-mediated CDK11^p110 knockdown inhibited tumor growth in an ESCC xenograft model, and overexpression of CDK11^p110 enhanced tumor growth. In addition, the Ki67 proliferation index was closely associated with the elevation or depletion of CDK11^p110 in vivo. In summary, this study provides evidence that CDK11^p110 play a critical role in the tumorigenicity of ESCC cells, which suggests that CDK11^p110 may be a promising therapeutic target in ESCC.

Abbreviations: CDKs: cyclin-dependent kinases; CDK11: Cyclin-dependent kinase 11; CDK11^p110: Cyclin-dependent kinase 11^p110, the larger isomer of cyclin-dependent kinase 11; ESCC: esophageal squamous cell carcinoma; FACS: fluorescence-activated cell sorting; FDA: the Food and Drug Administration; TCGA: The Cancer Genome Atlas; TMA: tissue microarray.