A Novel Conserved Phosphotyrosine Motif in the Drosophila Fibroblast Growth Factor Signaling Adaptor Dof with a Redundant Role in Signal Transmission

The fibroblast growth factor receptor (FGFR) signals through adaptors constitutively associated with the receptor. In Drosophila melanogaster, the FGFR-specific adaptor protein Downstream-of-FGFR (Dof) becomes phosphorylated upon receptor activation at several tyrosine residues, one of which recruits Corkscrew (Csw), the Drosophila homolog of SHP2, which provides a molecular link to mitogen-activated protein kinase (MAPK) activation. However, the Csw pathway is not the only link from Dof to MAPK. In this study, we identify a novel phosphotyrosine motif present in four copies in Dof and also found in other insect and vertebrate signaling molecules. We show that these motifs are phosphorylated and contribute to FGF signal transduction. They constitute one of three sets of phosphotyrosines that act redundantly in signal transmission: (i) a Csw binding site, (ii) four consensus Grb2 recognition sites, and (iii) four novel tyrosine motifs. We show that Src64B binds to Dof and that Src kinases contribute to FGFR-dependent MAPK activation. Phosphorylation of the novel tyrosine motifs is required for the interaction of Dof with Src64B. Thus, Src64B recruitment to Dof through the novel phosphosites can provide a new link to MAPK activation and other cellular responses. This may give a molecular explanation for the involvement of Src kinases in FGF-dependent developmental events.Fibroblast growth factor (FGF) receptors (FGFRs) are highly conserved molecules that belong to the family of receptor tyrosine kinases (RTKs). They form dimers which are activated by autophosphorylation upon ligand binding. Activated RTKs can directly recruit signaling molecules via their phosphorylated tyrosine residues. They can also phosphorylate other signaling molecules, and the phosphosites of those molecules can serve as additional interaction surfaces for downstream signal transducers (8).RTKs activate conserved intracellular signaling cascades, one of the most frequently activated being the Ras-mitogen-activated protein kinase (MAPK) pathway. Ras activation at the plasma membrane occurs via the recruitment of the phosphotyrosine binding adaptor molecule Grb2 in a complex with the Ras GTP exchange factor (RasGEF) Sos (26). The FGF signal transduction pathway also uses the Ras-MAPK cascade, but while most other RTKs access this cascade by using phosphotyrosines in their intracellular domains to recruit either Grb2 itself or signaling molecules such as Shc or SHP2, which can then recruit Grb2 upon phosphorylation, this is not the case for FGF receptors (8).Instead, FGF receptors use constitutively bound adaptor proteins, which are tyrosine phosphorylated upon receptor activation at several sites and provide binding surfaces for many signaling molecules, including the known activators of the Ras-MAPK pathway (8). Surprisingly, although the FGF receptor and its downstream signal transducers are conserved between vertebrates and insects, and in both cases an adaptor is required to connect them, the adaptor function is carried out by unrelated molecules in vertebrates and insects. In vertebrates, the adaptor protein FRS2 constitutively binds to the juxtamembrane region of the FGF receptor and becomes tyrosine phosphorylated by the activated receptor (18, 29, 30). The homolog of FRS2 in Drosophila melanogaster is not involved in FGF signaling (A. Michelson, personal communication). Instead, the adaptor molecule Dof/Sms (Downstream-of-FGFR, or Stumps) is the obligate partner of fly FGF receptors (27, 40, 41). Dof shows no sequence similarity to its vertebrate functional correlate FRS2 but is related to two other vertebrate adaptors, BCAP and BANK, which are involved in B-cell receptor signaling (2).Dof is essential in all FGF signal-mediated processes in the fly, including mesoderm formation and development of the tracheal network during embryogenesis, which we use here as an in vivo system to study signaling through Dof (40). Dof binds to both Drosophila FGF receptors, Heartless and Breathless, via its conserved DBB (Dof-BCAP-BANK) domain and becomes phosphorylated upon receptor activation at several tyrosine residues (32, 41). Corkscrew (Csw), the Drosophila homolog of SHP2, has been shown to bind to one of these phosphosites, providing a molecular link to MAPK activation (32). However, other functional structural studies show that the Csw pathway is not the only one employed by Dof to activate MAPK and indicate that different parts of the molecule might act redundantly (41).Src family kinases are also known activators of the MAPK pathway in both Drosophila and vertebrates (4, 22, 42). Furthermore, vertebrate FGF receptor signaling has been described to activate Src family kinases in many different systems (4, 5) and genetic studies of Drosophila point to an involvement of Src kinases in FGF-dependent processes (9, 36). However, there is no evidence on whether and how MAPK activation by Src can be driven directly by Drosophila FGF receptors. This prompted us to investigate whether the adaptor protein Dof might contribute to the recruitment of Src to activated FGF receptors as an alternative way of activating MAPK.Cells are able to fine-tune the strength and duration of signaling, providing a means to generate signal- or cell type-specific readouts in response to different signals, even though the signaling cascades use common components (reviewed in reference 6). For example, in PC12 cells, EGF signaling generates a transient pulse of MAPK activation, inducing cell proliferation, whereas FGF stimulation generates a sustained MAPK activation, which leads to differentiation (25, 44). One way of achieving this modulation is to be able to use several parallel links to a downstream effector. Two different types of parallel signaling connections have been observed. Adaptors can have multiple binding sites for a given signaling molecule, which allows an increase in signaling strength via the recruitment of larger numbers of the same molecule. For example, FRS2α contains four Grb2 binding sites (12), and BCAP has four PI3K interaction sites (17). Adaptor molecules can also display diverse binding sites for multiple different signaling molecules, allowing activation of the same signaling cascade along alternative routes: FRS2α contains both Grb2 and SHP2 binding sites for MAPK activation (12). In Dof we find both cases. It contains a Csw binding site, which has been shown to contribute to its function, and four Grb2 consensus binding sites, for which the relevance for MAPK activation has not been demonstrated so far (32, 41).The most frequently used binding sites in RTK signaling that allow signal-dependent transient protein-protein interactions are phosphorylatable tyrosine motifs, which provide binding surfaces for signal transducers bearing phosphotyrosine binding domains. Although all proteins containing phosphotyrosine binding domains can bind phosphotyrosines, these domains differ in their specificity for binding sites. The target recognition sites of the two most common phosphotyrosine binding domains, the PTB and SH2 domains, are determined by short stretches of amino acids upstream or downstream of the phosphorylated tyrosine (35). The motif recognized by PTB domains usually contains conserved amino acids N-terminal to a phosphotyrosine (39), whereas conserved residues C-terminal to a phosphotyrosine contribute to the binding specificity of SH2 domains (37, 38). All of the recognizable motifs surrounding phosphotyrosines in Dof suggest interactions with SH2 domains. Although the binding specificity of many SH2 domains is characterized by phosphopeptide library screens and crystallography studies (31), the interaction partners for several known phosphorylated tyrosines in signaling molecules have not yet been identified.We present here a systematic mapping of FGFR-dependent tyrosine phosphorylation of Dof by mutational analysis and show that a novel phosphotyrosine motif contributes in a redundant manner to FGF signaling by allowing Dof to utilize alternative signal transduction routes to activate the MAPK pathway.     

Memo Has a Novel Role in S1P Signaling and Crucial for Vascular Development

Memo is a conserved protein that was identified as an essential mediator of tumor cell motility induced by receptor tyrosine kinase activation. Here we show that Memo null mouse embryonic fibroblasts (MEFs) are impaired in PDGF-induced migration and this is due to a defect in sphingosine-1-phosphate (S1P) signaling. S1P is a bioactive phospholipid produced in response to multiple stimuli, which regulates many cellular processes. S1P is secreted to the extracellular milieu where it exerts its function by binding a family of G-protein coupled receptors (S1PRs), causing their activation in an autocrine or paracrine manner. The process, termed cell-autonomous S1PR signaling, plays a role in survival and migration. Indeed, PDGF uses cell-autonomous S1PR signaling to promote cell migration; we show here that this S1P pathway requires Memo. Using vascular endothelial cells (HUVECs) with Memo knock-down we show that their survival in conditions of serum-starvation is impaired. Furthermore, Memo loss in HUVECs causes a reduction of junctional VE-cadherin and an increase in sprout formation. Each of these phenotypes is rescued by S1P or S1P agonist addition, showing that Memo also plays an important role in cell-autonomous S1PR signaling in endothelial cells. We also produced conventional and endothelial cell-specific conditional Memo knock-out mouse strains and show that Memo is essential for embryonic development. Starting at E13.5 embryos of both strains display bleeding and other vascular problems, some of the phenotypes that have been described in mouse strains lacking S1PRs. The essential role of Memo in embryonic vascular development may be due in part to alterations in S1P signaling. Taken together our results show that Memo has a novel role in the S1P pathway and that Memo is needed to promote cell-autonomous S1PR activation.     

Novel Predicted Peptidases with a Potential Role in the Ubiquitin Signaling Pathway

A multi-pronged strategy including extensive sequence searches, structuralmodeling, and analysis of contextual information extracted from domainarchitectures, genetic screens, and large-scale protein-protein interaction analyseswas employed to predict previously undetected components of the eukaryoticubiquitin signaling system. Two novel groups of proteins that are likely to function asde-ubiquitinating and de-SUMOylating peptidases (DUBs) were identified. The firstgroup of putative DUBs, designated PPPDE superfamily (after Permuted Papain foldPeptidases of DsRNA viruses and Eukaryotes), consists of predicted thiol peptidaseswith a circularly permuted papain-like fold. The inference of the likely DUB functionof the PPPDE superfamily proteins is based on the fusions of the catalytic domain toUb-binding PUG (PUB)/UBA domains and a novel alpha-helical Ub-associated domain(the PLAP, Ufd3p and Lub1p or PUL domain) amongst different members of thePPPDE supefamily. The presence of the PPPDE superfamily proteins in mosteukaryotic lineages, including basal ones, such as Giardia, suggest a role indeubiquitination of highly conserved proteins involved in key cellular functions, suchas cell cycle control. In addition to eukaryotic proteins, the PPPDE superfamilyincludes predicted proteases from several groups of double-stranded RNA virusesand one single-stranded DNA virus. The apparent recruitment of DUBs for viralpolyprotein processing seems to represent a common theme in evolution of viruses.The second group of putative DUBs identified in this study is the WLM (Wss1p-likemetalloproteases) family of Zincin-like superfamily of Zn-dependent peptidases,which are linked to the Ub -system by virtue of fusions with the UB-binding PUG(PUB), ubiquitin-like and Little Finger domains. More specifically on the basis ofgenetic evidence the WLM family is implicated in de-SUMOylation. If validatedexperimentally, the WLM family proteins will represent the first case of a Zincin-likemetalloprotease involvement in Ub-signaling.     

Mononucleosis and Antigen-Driven T Cell Responses Have Different Requirements for Interleukin-2 Signaling in Murine Gammaherpesvirus Infection

Interleukin-2 (IL-2) has been implicated as being necessary for the optimal formation of primary CD8⁺ T cell responses against various pathogens. Here we have examined the role that IL-2 signaling plays in several aspects of a CD8⁺ T cell response against murine gammaherpesvirus 68 (MHV-68). Exposure to MHV-68 causes a persistent infection, along with infectious mononucleosis, providing a model for studying these processes in mice. Our study indicates that CD25 is necessary for optimal expansion of the antigen-specific CD8⁺ T cell response but not for the long-term memory response. Contrastingly, IL-2 signaling through CD25 is absolutely required for CD8⁺ T cell mononucleosis.Members of the gammaherpesvirus family are associated with significant diseases, such as nasopharyngeal carcinoma, lymphoid malignancies, and infectious mononucleosis (16). Murine gammaherpesvirus 68 (MHV-68) is a γ2-herpesvirus related to the human pathogens Epstein-Barr virus (EBV) and Kaposi''s sarcoma virus (19, 21). Intranasal (i.n.) infection of mice with MHV-68 results in acute infection of the lung epithelium, which is eventually controlled; however, the virus also establishes a latent infection in B cells, dendritic cells, and macrophages that is maintained throughout the life of the host (8, 9). Infection with MHV-68 generates a broad array of antigen-specific CD8⁺ T cells that can control the virus without eliminating persistent infection (5, 12, 13). Additionally, CD4⁺ T cells and neutralizing antibodies are thought to be critical for the prevention of virus reactivation (3, 6).A major complication of EBV infection is infectious mononucleosis (16), which occurs when infection is delayed until puberty. Signs of disease include dramatic lymph node enlargement and the presence of large numbers of activated CD8⁺ T cells in the peripheral blood. Similarly to EBV infection, MHV-68 induces a polyclonal activation of B cells upon establishment of latency. Concurrently, a CD8⁺ T cell-dominated lymphocytosis of the peripheral blood occurs, as seen with EBV. However, there are distinct differences between the two types of infectious mononucleosis. CD8⁺ T cell lymphocytosis seen with EBV consists of a broad array of T cell receptor specificities, a large proportion of which are specific for EBV epitopes. In contrast, MHV-68-induced mononucleosis is dominated by oligoclonal Vβ4⁺ CD8⁺ T cells that are not reactive to MHV-68 epitopes. With MHV-68, the expansion of this population is dramatic, with levels reaching upwards of 60% of the peripheral blood CD8⁺ T cell population (20). This occurs in different mouse strains, across at least five different major histocompatibility complex (MHC) class I haplotypes. However, it is important to note that infection of wood mice (Apodemus sylvaticus) does not induce splenomegaly, as seen with laboratory strains of mice, indicating a potential lack of Vβ4 expansion that may be species related (14). Interestingly, evidence suggests that Vβ4⁺ CD8⁺ T cell expansion does not require classical MHC class Ia antigen presentation (4). Recent studies instead implicate a secreted viral protein, M1, capable of stimulating the Vβ4+ T cell population in a novel manner, and the authors propose a role for Vβ4+ T cells in control of MHV-68 infection (7).We and others have recently shown that IL-2 signaling during the early stages of a response to acute viral and bacterial pathogens is required for optimal expansion and differentiation of CD8⁺ T cells (15, 17, 18). However, reports with other viruses have shown IL-2-independent primary CD8⁺ T cell responses (1, 22). Therefore, we wished to determine whether IL-2 signals are necessary for the expansion, maintenance, and/or recall of CD8⁺ T cell responses during murine gammaherpesvirus infection.We generated chimeric mice through lethal irradiation of C57BL/6 mice followed by adoptive transfer of mixed bone marrow from C57BL/6 wild-type (WT) and CD25^−/− donors, as previously described (17). Following previous described protocols, mice were given bone marrow in a 2:1 ratio of CD25^−/−/WT to generate equally proportioned congenic populations in recipient mice (see Fig. S1 in the supplemental material) (1, 17). The resultant mice contained CD8⁺ T cells of both WT and CD25^−/− origin, which could be distinguished by congenic markers. Chimeric mice were infected intranasally with 400 PFU of MHV-68, and the kinetics of the CD8⁺ T cell response were followed by antibody and tetramer staining of peripheral blood for CD8⁺ T cells specific for the epitopes ORF6₄₈₇ (p56) and ORF61₅₂₄ (p79), as previously described (13). While antigen-specific CD25^−/− CD8⁺ T cells were initially able to proliferate in response to infection, the peak response was significantly lower than that of the wild-type cells (Fig. ​(Fig.11 A and B). This indicates that while CD25 is dispensable for early activation of CD8⁺ T cells, IL-2 signaling is required for full expansion of the antigen-specific response to MHV-68. Despite this deficit in the acute antiviral response, the resultant memory populations were not statistically different between the groups (Fig. 1A and B). In our previous report, CD25^−/− CD8⁺ T cells were unable to fully differentiate into short-lived effector cells (SLECs), defined as KLRG1^high CD127^low (17). To determine if MHV-68-specific responses were also unable to fully differentiate, we infected chimeric mice and stained p79⁺ CD8⁺ T cells for the cell surface markers KLRG1 and CD127. At the peak of the response (14 days postinfection [p.i.]), p79⁺ WT cells had differentiated into SLEC (KLRG1^high CD127^low), memory precursor (MPEC) (KLRG1^low CD127^high), and doubly positive populations. However, the p79⁺ CD25^−/− cells failed to form the SLEC population and instead had a corresponding increase in the MPEC population, indicating that CD25 is necessary for full effector differentiation of gammaherpesvirus-specific CD8⁺ T cell responses (Fig. 1C and D).Open in a separate windowFIG. 1.IL-2 signals are necessary for the optimal expansion of MHV-68-specific CD8⁺ T cells. WT/CD25^−/− chimeric mice were infected with MHV-68 intranasally and bled at set time points. The antigen-specific responses against two dominant epitopes, p79 (A) and p56 (B), were determined via tetramer staining of peripheral blood. p79-specific CD8⁺ T cells from the WT and CD25^−/− populations were stained at the peak of the response (day 14 p.i.) for KLRG1 and CD127 to determine their ability to differentiate into short-lived and memory precursor effector cells (C and D). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Error bars represent standard deviations from the means. Four mice were used per group, and data are representative of at least two experiments.To determine whether antigen-specific CD25^−/− CD8⁺ T cells were capable of optimally responding to a secondary challenge, we infected chimeric mice with MHV-68 and waited 60 days before challenging with recombinant vaccinia virus (rVV) expressing the ORF61₅₂₄ epitope (2 × 10⁶ PFU, intraperitoneal). It is necessary to use a heterologous virus to induce a recall CD8⁺ T cell response since MHV-68 generates a robust neutralizing antibody response, preventing secondary infection. Previous studies with rVV indicate that the recall response of MHV-68-specific CD8⁺ T cells is antigen dependent, since administration of rVV expressing an irrelevant epitope had no effect upon the MHV-68-specific populations (2). WT and CD25^−/− cells were able to respond to the secondary challenge with similar kinetics (Fig. ​(Fig.22 A and B), indicating that MHV-68 memory CD8⁺ T cells are capable of a generating a recall response in the absence of IL-2 signaling. These data, together with our previous report (17), show that the dependence on CD25 for formation of the SLEC population is conserved between both persistent and acute virus infections.Open in a separate windowFIG. 2.CD25^−/− CD8⁺ T cells can respond to secondary challenge. WT/CD25^−/− chimeric mice were infected with MHV-68 i.n. After 60 days, the percentage of peripheral blood CD8⁺ T cells specific for p79 was determined. Mice were then challenged with rVV p79, and the p79⁺ CD8⁺ population was determined 5 days postchallenge (A). The numbers in the box represent the averages ± standard deviations. The average fold increase was calculated to determine the ability of WT and CD25^−/− CD8⁺ T cells to respond to a secondary challenge (B). Error bars represent standard deviations from the means. Four mice were used per group, and data are representative of at least two experiments.WT CD8⁺ T cells underwent a dramatic expansion between days 15 and 21 p.i. (Fig. ​(Fig.3A),3A), consistent with infectious mononucleosis (10). Interestingly, we did not observe a similar expansion of CD25^−/− CD8⁺ T cells, indicating a role for IL-2 signaling in the expansion of CD8⁺ T cells during mononucleosis (Fig. ​(Fig.3A).3A). Since mononucleosis is dominated by Vβ4+ CD8⁺ T cells, we analyzed these T cells from both naive and infected mice (17 days p.i.) for expression of CD25 by flow cytometry. While Vβ4⁺ CD8⁺ T cells from the spleen and peripheral blood of naive mice did not express detectable levels of CD25, mice infected with MHV-68 expressed intermediate levels of CD25 during the time period when dramatic expansion of Vβ4⁺ T cells occurs (Fig. 3B and C). Consistent with a role for IL-2 signaling in Vβ4 expansion, we observed a severe deficit in expansion in the CD25^−/− population of chimeric mice, since the percentage of WT Vβ4⁺ cells increased dramatically between days 14 and 36 p.i., accompanied by only a small expansion of the CD25^−/− Vβ4⁺ population over the same period (Fig. ​(Fig.44 A and B).Open in a separate windowFIG. 3.Vβ4⁺ CD8⁺ T cells express CD25 upon infection with MHV-68. WT/CD25^−/− chimeric mice were infected with MHV-68 i.n., and the percentage of peripheral blood cells that were CD8⁺ was determined over time for each congenic population (A). Vβ4⁺ CD8⁺ T cells from naive and MHV-68-infected mice (day 17 p.i.) were analyzed for expression of CD25 (B and C). Isotype control, filled histogram; naive mice, dashed line; infected mice, solid line (**, P < 0.01). Error bars represent standard deviations from the means. Four mice were used per group, and data are representative of at least two experiments.Open in a separate windowFIG. 4.CD8⁺ T cell-based infectious mononucleosis does not occur in the absence of IL-2 signaling in MHV-68-infected mice. WT/CD25^−/− chimeric mice were infected with MHV-68 i.n., and the percentage of Vβ4⁺ CD8⁺ T cells was determined over time for each congenic population. Representative plots from day 36 p.i. (A) or the averages over time (B) are shown. WT and CD25^−/− CD8⁺ T cells from chimeric mice were analyzed for expression of CD62L over time. Representative plots from day 24 p.i. (C) or the averages over time (D) are shown. *, P < 0.05; **, P < 0.01). Error bars represent standard deviations from the means. Four mice were used per group, and data are representative of at least two experiments.During infectious mononucleosis, CD8⁺ T cells are in a highly activated state and thus express low levels of CD62L (20). Therefore, we analyzed CD8⁺ T cells from chimeric mice for expression of CD62L. After MHV-68 infection, the majority of WT CD8⁺ T cells in the peripheral blood were CD62L^low, as previously reported (Fig. 4C and D) (20). Interestingly, CD25^−/− CD8⁺ T cells failed to develop this dominant CD62L^low population, indicating that CD25 is necessary for the activation of the CD8⁺ T cell compartment in addition to cell expansion during mononucleosis (Fig. 4C and D). When we analyzed the Vβ4⁺ CD8⁺ T cell compartment, we observed that WT cells downregulated expression of CD62L. While Vβ4⁺ cells from the CD25^−/− compartment also decreased expression of CD62L, they did so to a lesser extent both as a percentage and on a per-cell basis (see Fig. S2 in the supplemental material).In these studies, we have shown that signaling through CD25 is necessary for the generation of an optimal primary CD8⁺ T cell response against a gammaherpesvirus, since virus-specific CD8⁺ T cells were unable to expand as robustly as WT cells and did not fully differentiate into short-lived effector cells. These observations are consistent with previous results from our lab and findings of others using a variety of acute infection models (17, 18). However, not all persistent infections appear to require CD25, since the m45-specific response to murine cytomegalovirus (MCMV) infection occurs normally in the absence of IL-2 signals (1). What allows for some responses to be independent of IL-2 remains unknown. Potential explanations could involve differences in tropism, the route of infection, or the amount of proinflammatory cytokines induced by each infection. Despite the dependence on CD25 for the short-term effector response, the memory CD8⁺ T cell response remained intact in the absence of IL-2 signaling. In contrast, Vβ4 expansion and mononucleosis never attained normal levels. Unlike the antigen-specific response, which relies upon peptide/MHC interactions for induction, mononucleosis does not rely upon conventional antigen presentation (4). Instead, the M1 protein of MHV-68, expressed during the establishment and expansion of latency in the spleen, appears to drive Vβ4 expansion (7). Interestingly, our evidence shows that both antigen-dependent and -independent CD8⁺ T cell expansion require CD25. Antigen-specific T cells also undergo an apoptotic contraction phase, followed by a lower frequency of cells surviving as relatively quiescent memory cells. In contrast, during mononucleosis caused by MHV-68, CD8⁺ T cells remain in an activated state and do not undergo a marked contraction, providing a potential explanation as to why the WT and CD25^−/− Vβ4 populations continue to differ in both number and phenotype later in the response.Earlier studies have also identified CD4⁺ T cells as being critical for the development of MHV-68-induced infectious mononucleosis (11, 20). We have previously shown that CD4⁺ T cell help was critical for robust expression of CD25 on activated antigen-specific CD8⁺ T cells. Interestingly, when we measured CD25 expression on Vβ4⁺ cells from mice lacking CD4⁺ T cells, we saw a moderate decrease in the level of CD25 expressed (data not shown), indicating one potential reason why CD4-deficient mice do not experience infectious mononucleosis. However, it is likely that other factors involving CD4⁺ T cells and activation of B cells are also involved (10).In conclusion, the significance of these studies is twofold. First, they shed light on the requirements for MHV-68-induced mononucleosis. Second, our data illustrate that CD25 is required for both antigen-specific and non-antigen-specific activation of CD8⁺ T cell responses, while being dispensable for memory cell formation. This knowledge may be useful in developing new T cell-based immune therapies to enhance control of persistent gammaherpesvirus infections.      

缺氧信号在骨中的核心作用

缺氧信号在维持氧稳态和细胞生存中起着至关重要的作用。在胚胎发育时期处于快速增殖的细胞和肿瘤组织中快速生长的细胞中都能观察到缺氧现象的存在。为了应对缺氧胁迫,生物有机体形成了一系列的调节机制。在多种调节途径中,缺氧诱导因子HIF-1和HIF-2是最主要的能够应答细胞内氧气浓度的降低而对多种基因进行调控的转录因子,与生物体的生长发育及一些疾病的发病机理都存在着密切关系。最近的研究发现在骨骼发育,骨骼的形成和再生,以及关节的形成和动态平衡的调节中HIF-1和HIF-2的具有重要作用。此外,HIF-1和HIF-2的过度表达在临床上与骨肉瘤和骨关节炎明显相关。总之,这些发现预示着缺氧的信号在骨骼的生物学及其疾病中起到中心调节的作用。     

Purification and Conformational Characterization of a Novel Interleukin-2 Mutein

The Protein Journal - Toxicity of high-dose IL-2-based therapies have motivated the development of the IL-2 mutein, which has low expansion properties for regulatory T lymphocytes. The development...     

Failure to Detect the Novel Retrovirus XMRV in Chronic Fatigue Syndrome

Background

In October 2009 it was reported that 68 of 101 patients with chronic fatigue syndrome (CFS) in the US were infected with a novel gamma retrovirus, xenotropic murine leukaemia virus-related virus (XMRV), a virus previously linked to prostate cancer. This finding, if confirmed, would have a profound effect on the understanding and treatment of an incapacitating disease affecting millions worldwide. We have investigated CFS sufferers in the UK to determine if they are carriers of XMRV.

Methodology

Patients in our CFS cohort had undergone medical screening to exclude detectable organic illness and met the CDC criteria for CFS. DNA extracted from blood samples of 186 CFS patients were screened for XMRV provirus and for the closely related murine leukaemia virus by nested PCR using specific oligonucleotide primers. To control for the integrity of the DNA, the cellular beta-globin gene was amplified. Negative controls (water) and a positive control (XMRV infectious molecular clone DNA) were included. While the beta-globin gene was amplified in all 186 samples, neither XMRV nor MLV sequences were detected.

Conclusion

XMRV or MLV sequences were not amplified from DNA originating from CFS patients in the UK. Although we found no evidence that XMRV is associated with CFS in the UK, this may be a result of population differences between North America and Europe regarding the general prevalence of XMRV infection, and might also explain the fact that two US groups found XMRV in prostate cancer tissue, while two European studies did not.     

A Novel Role for MAPKAPK2 in Morphogenesis during Zebrafish Development

One of the earliest morphogenetic processes in the development of many animals is epiboly. In the zebrafish, epiboly ensues when the animally localized blastoderm cells spread, thin over, and enclose the vegetally localized yolk. Only a few factors are known to function in this fundamental process. We identified a maternal-effect mutant, betty boop (bbp), which displays a novel defect in epiboly, wherein the blastoderm margin constricts dramatically, precisely when half of the yolk cell is covered by the blastoderm, causing the yolk cell to burst. Whole-blastoderm transplants and mRNA microinjection rescue demonstrate that Bbp functions in the yolk cell to regulate epiboly. We positionally cloned the maternal-effect bbp mutant gene and identified it as the zebrafish homolog of the serine-threonine kinase Mitogen Activated Protein Kinase Activated Protein Kinase 2, or MAPKAPK2, which was not previously known to function in embryonic development. We show that the regulation of MAPKAPK2 is conserved and p38 MAP kinase functions upstream of MAPKAPK2 in regulating epiboly in the zebrafish embryo. Dramatic alterations in calcium dynamics, together with the massive marginal constrictive force observed in bbp mutants, indicate precocious constriction of an F-actin network within the yolk cell, which first forms at 50% epiboly and regulates epiboly progression. We show that MAPKAPK2 activity and its regulator p38 MAPK function in the yolk cell to regulate the process of epiboly, identifying a new pathway regulating this cell movement process. We postulate that a p38 MAPKAPK2 kinase cascade modulates the activity of F-actin at the yolk cell margin circumference allowing the gradual closure of the blastopore as epiboly progresses.     

Cross-Species Transmission and Differential Fate of an Endogenous Retrovirus in Three Mammal Lineages

Endogenous retroviruses (ERVs) arise from retroviruses chromosomally integrated in the host germline. ERVs are common in vertebrate genomes and provide a valuable fossil record of past retroviral infections to investigate the biology and evolution of retroviruses over a deep time scale, including cross-species transmission events. Here we took advantage of a catalog of ERVs we recently produced for the bat Myotis lucifugus to seek evidence for infiltration of these retroviruses in other mammalian species (>100) currently represented in the genome sequence database. We provide multiple lines of evidence for the cross-ordinal transmission of a gammaretrovirus endogenized independently in the lineages of vespertilionid bats, felid cats and pangolin ~13–25 million years ago. Following its initial introduction, the ERV amplified extensively in parallel in both bat and cat lineages, generating hundreds of species-specific insertions throughout evolution. However, despite being derived from the same viral species, phylogenetic and selection analyses suggest that the ERV experienced different amplification dynamics in the two mammalian lineages. In the cat lineage, the ERV appears to have expanded primarily by retrotransposition of a single proviral progenitor that lost infectious capacity shortly after endogenization. In the bat lineage, the ERV followed a more complex path of germline invasion characterized by both retrotransposition and multiple infection events. The results also suggest that some of the bat ERVs have maintained infectious capacity for extended period of time and may be still infectious today. This study provides one of the most rigorously documented cases of cross-ordinal transmission of a mammalian retrovirus. It also illustrates how the same retrovirus species has transitioned multiple times from an infectious pathogen to a genomic parasite (i.e. retrotransposon), yet experiencing different invasion dynamics in different mammalian hosts.     

A Novel Phosphatidic Acid-Protein-tyrosine Phosphatase D2 Axis Is Essential for ERBB2 Signaling in Mammary Epithelial Cells

We used a loss-of-function screen to investigate the role of classical protein-tyrosine phosphatases (PTPs) in three-dimensional mammary epithelial cell morphogenesis and ERBB2 signaling. The study revealed a novel role for PTPD2 as a positive regulator of ERBB2 signaling. Suppression of PTPD2 attenuated the ERBB2-induced multiacinar phenotype in three-dimensional cultures specifically by inhibiting ERBB2-mediated loss of polarity and lumen filling. In contrast, overexpression of PTPD2 enhanced the ERBB2 phenotype. We also found that a lipid second messenger, phosphatidic acid, bound PTPD2 in vitro and enhanced its catalytic activity. Small molecule inhibitors of phospholipase D (PLD), an enzyme that produces phosphatidic acid in cells, also attenuated the ERBB2 phenotype. Exogenously added phosphatidic acid rescued the PLD-inhibition phenotype, but only when PTPD2 was present. These findings illustrate a novel pathway involving PTPD2 and the lipid second messenger phosphatidic acid that promotes ERBB2 function.     

Role of Ca2+ in Drought Stress Signaling in Wheat Seedlings

Plants use complex signal transduction pathways to perceive and react to various biotic and/or abiotic stresses. As a consequence of this signaling, plants can modify their metabolism to adapt themselves to new conditions. One such change is the accumulation of proline in response to drought and salinity stresses. We have studied drought and salinity induced proline accumulation and the roles of Ca²⁺ (10 mM) and indoleacetic acid (IAA, 0.3 mM) in this response. Subjecting seedlings to both drought (6% polyethylene glycol, PEG) and salinity (150 mM NaCl) stress resulted in a dramatic increase in proline accumulation (7-fold higher than control level). However, the application of Ca²⁺ along with these stress factors had different effects. Unlike the salinity stress, Ca²⁺ prevented the drought induced proline accumulation indicating that these stress factors use distinct signaling pathways to induce similar responses. Experiments with IAA and EGTA (10 mM) supported this interpretation and suggested that Ca²⁺ and auxin participate in signaling mechanisms of drought-induced proline accumulation. Drought and salt stress-induced proline accumulation was compared on salt resistant (cv. Gerek 79) and salt sensitive (cv. Bezostaya) wheat varieties. Although proline level of the first was twofold lower than that of the second in control, relative proline accumulation was dramatically higher in the case of the salt resistant wheat variety under stress conditions.     

Novel Role for p21-activated Kinase 2 in Thrombin-induced Monocyte Migration

To understand the role of thrombin in inflammation, we tested its effects on migration of THP-1 cells, a human monocytic cell line. Thrombin induced THP-1 cell migration in a dose-dependent manner. Thrombin induced tyrosine phosphorylation of Pyk2, Gab1, and p115 RhoGEF, leading to Rac1- and RhoA-dependent Pak2 activation. Downstream to Pyk2, Gab1 formed a complex with p115 RhoGEF involving their pleckstrin homology domains. Furthermore, inhibition or depletion of Pyk2, Gab1, p115 RhoGEF, Rac1, RhoA, or Pak2 levels substantially attenuated thrombin-induced THP-1 cell F-actin cytoskeletal remodeling and migration. Inhibition or depletion of PAR1 also blocked thrombin-induced activation of Pyk2, Gab1, p115 RhoGEF, Rac1, RhoA, and Pak2, resulting in diminished THP-1 cell F-actin cytoskeletal remodeling and migration. Similarly, depletion of Gα₁₂ negated thrombin-induced Pyk2, Gab1, p115 RhoGEF, Rac1, RhoA, and Pak2 activation, leading to attenuation of THP-1 cell F-actin cytoskeletal remodeling and migration. These novel observations reveal that thrombin induces monocyte/macrophage migration via PAR1-Gα12-dependent Pyk2-mediated Gab1 and p115 RhoGEF interactions, leading to Rac1- and RhoA-targeted Pak2 activation. Thus, these findings provide mechanistic evidence for the role of thrombin and its receptor PAR1 in inflammation.     

EphA2 Signaling Following Endocytosis: Role of Tiam1

Eph receptors and their membrane‐bound ligands, the ephrins, represent a complex subfamily of receptor tyrosine kinases (RTKs). Eph/ephrin binding can lead to various and opposite cellular behaviors such as adhesion versus repulsion, or cell migration versus cell‐adhesion. Recently, Eph endocytosis has been identified as one of the critical steps responsible for such diversity. Eph receptors, as many RTKs, are rapidly endocytosed following ligand‐mediated activation and traffic through endocytic compartments prior to degradation. However, it is becoming obvious that endocytosis controls signaling in many different manners. Here we showed that activated EphA2 are degraded in the lysosomes and that about 35% of internalized receptors are recycled back to the plasma membrane. Our study is also the first to demonstrate that EphA2 retains the capacity to signal in endosomes. In particular, activated EphA2 interacted with the Rho family GEF Tiam1 in endosomes. This association led to Tiam1 activation, which in turn increased Rac1 activity and facilitated Eph/ephrin endocytosis. Disrupting Tiam1 function with RNA interference impaired both ephrinA1‐dependent Rac1 activation and ephrinA1‐induced EphA2 endocytosis. In summary, our findings shed new light on the regulation of EphA2 endocytosis, intracellular trafficking and signal termination and establish Tiam1 as an important modulator of EphA2 signaling .     

Novel Role of Src in Priming Pyk2 Phosphorylation

Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase (FAK) family of non-receptor tyrosine kinases and plays an important role in diverse cellular events downstream of the integrin-family of receptors, including cell migration, proliferation and survival. Here, we have identified a novel role for Src kinase in priming Pyk2 phosphorylation and subsequent activation upon cell attachment on the integrin-ligand fibronectin. By using complementary methods, we show that Src activity is indispensable for the initial Pyk2 phosphorylation on the Y402 site observed in response to cell attachment. In contrast, the initial fibronectin-induced autophosphorylation of FAK in the homologous Y397 site occurs in a Src-independent manner. We demonstrate that the SH2-domain of Src is required for Src binding to Pyk2 and for Pyk2 phosphorylation at sites Y402 and Y579. Moreover, Y402 phosphorylation is a prerequisite for the subsequent Y579 phosphorylation. While this initial phosphorylation of Pyk2 by Src is independent of Pyk2 kinase activity, subsequent autophosphorylation of Pyk2 in trans is required for full Pyk2 phosphorylation and activation. Collectively, our studies reveal a novel function of Src in priming Pyk2 (but not FAK) phosphorylation and subsequent activation downstream of integrins, and shed light on the signaling events that regulate the function of Pyk2.