Natural killer cell signaling pathways

Natural killer (NK) cells are lymphocytes of the innate immune system that are involved in the early defenses against foreign cells, as well as autologous cells undergoing various forms of stress, such as microbial infection or tumor transformation. NK cell activation is controlled by a dynamic balance between complementary and antagonistic pathways that are initiated upon interaction with potential target cells. NK cells express an array of activating cell surface receptors that can trigger cytolytic programs, as well as cytokine or chemokine secretion. Some of these activating cell surface receptors initiate protein tyrosine kinase (PTK)-dependent pathways through noncovalent associations with transmembrane signaling adaptors that harbor intracytoplasmic ITAMs (immunoreceptor tyrosine-based activation motifs). Additional cell surface receptors that are not directly coupled to ITAMs also participate in NK cell activation. These include NKG2D, which is noncovalently associated to the DAP10 transmembrane signaling adaptor, as well as integrins and cytokine receptors. NK cells also express cell surface inhibitory receptors that antagonize activating pathways through protein tyrosine phosphatases (PTPs). These inhibitory cell surface receptors are characterized by intracytoplasmic ITIMs (immunoreceptor tyrosine-based inhibition motifs). The tyrosine-phosphorylation status of several signaling components that are substrates for both PTKs and PTPs is thus key to the propagation of the NK cell effector pathways. Understanding the integration of these multiple signals is central to the understanding and manipulation of NK cell effector signaling pathways.     

OSBP is a cholesterol-regulated scaffolding protein in control of ERK 1/2 activation

Oxysterol-binding protein (OSBP) is the founding member of a family of sterol-binding proteins implicated in vesicle transport, lipid metabolism, and signal transduction. Here, OSBP was found to function as a cholesterol-binding scaffolding protein coordinating the activity of two phosphatases to control the extracellular signal-regulated kinase (ERK) signaling pathway. Cytosolic OSBP formed a approximately 440-kilodalton oligomer with a member of the PTPPBS family of tyrosine phosphatases, the serine/threonine phosphatase PP2A, and cholesterol. This oligomer had dual specific phosphatase activity for phosphorylated ERK (pERK). When cell cholesterol was lowered, the oligomer disassembled and the level of pERK rose. The oligomer also disassembled when exposed to oxysterols. Increasing the amount of OSBP oligomer rendered cells resistant to the effects of cholesterol depletion and decreased the basal level of pERK. Thus, cholesterol functions through its interaction with OSBP outside of membranes to regulate the assembly of an oligomeric phosphatase that controls a key signaling pathway in the cell.     

Protein tyrosine phosphatase activity of an essential virulence determinant in Yersinia

Yersinia is the genus of bacteria that is the causative agent in plague or the black death, and on several occasions this organism has killed a significant portion of the world's population. An essential virulence determinant of Yersinia was shown to be a protein tyrosine phosphatase. The recombinant 50-kilodalton Yersinia phosphatase had a specificity for removal of phosphate from Tyr-containing as opposed to Ser/Thr-containing phosphopeptides and proteins. Site-directed mutagenesis was used to show that the Yersinia phosphatase possesses an essential Cys residue required for catalysis. Amino acids surrounding an essential Cys residue are highly conserved, as are other amino acids in the Yersinia and mammalian protein tyrosine phosphatases, suggesting that they use a common catalytic mechanism.     

FBXW7 targets mTOR for degradation and cooperates with PTEN in tumor suppression

The enzyme mTOR (mammalian target of rapamycin) is a major target for therapeutic intervention to treat many human diseases, including cancer, but very little is known about the processes that control levels of mTOR protein. Here, we show that mTOR is targeted for ubiquitination and consequent degradation by binding to the tumor suppressor protein FBXW7. Human breast cancer cell lines and primary tumors showed a reciprocal relation between loss of FBXW7 and deletion or mutation of PTEN (phosphatase and tensin homolog), which also activates mTOR. Tumor cell lines harboring deletions or mutations in FBXW7 are particularly sensitive to rapamycin treatment, which suggests that loss of FBXW7 may be a biomarker for human cancers susceptible to treatment with inhibitors of the mTOR pathway.     

磁场处理对土壤磷酸酶活性的影响

本文研究了磁场处理后棕壤、黑土及白浆土中磷酸酶活性的变化。研究结果表明，磁处理对土壤的3种磷酸酶都有一定的激活作用，但以碱性磷酸酶的活性变化最明显。3种磷酸酶活性提高的大小顺序是：黑土中碱性磷酸酶＞酸性磷酸酶＞中性磷酸酶；棕壤和白浆土中碱性磷酸酶＞中性磷酸酶＞酸性磷酸酶。土壤水分对磷酸酶的磁处理效果影响不大。对于磷酸酶活性影响以＜100mT的低场强以及lrnin或10min的短时间处理效果较好。     

PEST domain-enriched tyrosine phosphatase (PEP) regulation of effector/memory T cells

Protein tyrosine kinases and phosphatases cooperate to regulate normal immune cell function. We examined the role of PEST domain-enriched tyrosine phosphatase (PEP) in regulating T cell antigen-receptor function during thymocyte development and peripheral T cell differentiation. Although normal na?ve T cell functions were retained in pep-deficient mice, effector/memory T cells demonstrated enhanced activation of Lck. In turn, this resulted in increased expansion and function of the effector/memory T cell pool, which was also associated with spontaneous development of germinal centers and elevated serum antibody levels. These results revealed a central role for PEP in negatively regulating specific aspects of T cell development and function.     

设施栽培土壤磷酸酶活性及其与土壤养分的关系

对不同种植年限的设施栽培土壤磷酸酶活性和土壤养分测定分析结果表明,设施栽培土壤磷酸酶活性和土壤养分含量均比露地栽培高,且差异均达极显著水平。随种植年限的增加,设施栽培土壤碱性磷酸酶活性、有机质、全氮、有效氮和全磷含量呈现先增后降的趋势,且不同种植年限之间的差异达极显著水平;土壤中性磷酸酶活性则呈现先降后增的趋势;土壤有效磷含量呈现波动上升趋势。设施栽培土壤碱性磷酸酶活性与土壤主要养分因子均呈极显著正相关关系,土壤中性磷酸酶活性与土壤全磷呈极显著负相关,与其它土壤主要养分因子相关性都不显著。设施栽培土壤有机质和有效磷对土壤碱性磷酸酶活性有强烈的正效应,土壤全氮对土壤中性磷酸酶活性有强烈的负效应,土壤有效磷对土壤中性磷酸酶活性有强烈的正效应,土壤全磷对土壤碱性磷酸酶和中性磷酸酶活性的影响都主要表现在间接影响上。     

Greatwall phosphorylates an inhibitor of protein phosphatase 2A that is essential for mitosis

Entry into mitosis in eukaryotes requires the activity of cyclin-dependent kinase 1 (Cdk1). Cdk1 is opposed by protein phosphatases in two ways: They inhibit activation of Cdk1 by dephosphorylating the protein kinases Wee1 and Myt1 and the protein phosphatase Cdc25 (key regulators of Cdk1), and they also antagonize Cdk1's own phosphorylation of downstream targets. A particular form of protein phosphatase 2A (PP2A) containing a B55δ subunit (PP2A- B55δ) is the major protein phosphatase that acts on model CDK substrates in Xenopus egg extracts and has antimitotic activity. The activity of PP2A-B55δ is high in interphase and low in mitosis, exactly opposite that of Cdk1. We report that inhibition of PP2A-B55δ results from a small protein, known as α-endosulfine (Ensa), that is phosphorylated in mitosis by the protein kinase Greatwall (Gwl). This converts Ensa into a potent and specific inhibitor of PP2A-B55δ. This pathway represents a previously unknown element in the control of mitosis.     

Protein phosphatases: properties and role in cellular regulation

Protein phosphorylation is a principal regulatory mechanism in the control of almost all cellular processes. The nature of the protein phosphatases that participate in these reactions has been a subject of controversy. Four enzymes, termed protein phosphatases 1, 2A, 2B, and 2C, account for virtually all of the phosphatase activity toward phosphoproteins involved in controlling glycogen metabolism, glycolysis, gluconeogenesis, fatty acid synthesis, cholesterol synthesis, and protein synthesis. The properties, physiological roles, and mechanisms for regulating the four protein phosphatases are reviewed.     

PDGFRA activating mutations in gastrointestinal stromal tumors

Most gastrointestinal stromal tumors (GISTs) have activating mutations in the KIT receptor tyrosine kinase, and most patients with GISTs respond well to Gleevec, which inhibits KIT kinase activity. Here we show that approximately 35% (14 of 40) of GISTs lacking KIT mutations have intragenic activation mutations in the related receptor tyrosine kinase, platelet-derived growth factor receptor alpha (PDGFRA). Tumors expressing KIT or PDGFRA oncoproteins were indistinguishable with respect to activation of downstream signaling intermediates and cytogenetic changes associated with tumor progression. Thus, KIT and PDGFRA mutations appear to be alternative and mutually exclusive oncogenic mechanisms in GISTs.     

Phosphoproteomic analysis identifies Grb10 as an mTORC1 substrate that negatively regulates insulin signaling

The evolutionarily conserved serine-threonine kinase mammalian target of rapamycin (mTOR) plays a critical role in regulating many pathophysiological processes. Functional characterization of the mTOR signaling pathways, however, has been hampered by the paucity of known substrates. We used large-scale quantitative phosphoproteomics experiments to define the signaling networks downstream of mTORC1 and mTORC2. Characterization of one mTORC1 substrate, the growth factor receptor-bound protein 10 (Grb10), showed that mTORC1-mediated phosphorylation stabilized Grb10, leading to feedback inhibition of the phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated, mitogen-activated protein kinase (ERK-MAPK) pathways. Grb10 expression is frequently down-regulated in various cancers, and loss of Grb10 and loss of the well-established tumor suppressor phosphatase PTEN appear to be mutually exclusive events, suggesting that Grb10 might be a tumor suppressor regulated by mTORC1.     

The genetic landscape of the childhood cancer medulloblastoma

Medulloblastoma (MB) is the most common malignant brain tumor of children. To identify the genetic alterations in this tumor type, we searched for copy number alterations using high-density microarrays and sequenced all known protein-coding genes and microRNA genes using Sanger sequencing in a set of 22 MBs. We found that, on average, each tumor had 11 gene alterations, fewer by a factor of 5 to 10 than in the adult solid tumors that have been sequenced to date. In addition to alterations in the Hedgehog and Wnt pathways, our analysis led to the discovery of genes not previously known to be altered in MBs. Most notably, inactivating mutations of the histone-lysine N-methyltransferase genes MLL2 or MLL3 were identified in 16% of MB patients. These results demonstrate key differences between the genetic landscapes of adult and childhood cancers, highlight dysregulation of developmental pathways as an important mechanism underlying MBs, and identify a role for a specific type of histone methylation in human tumorigenesis.     

Specific expression of a tyrosine kinase gene, blk, in B lymphoid cells

Several pathways of transmembrane signaling in lymphocytes involve protein-tyrosine phosphorylation. With the exception of p56lck, a tyrosine kinase specific to T lymphoid cells that associates with the T cell transmembrane proteins CD4 and CD8, the kinases that function in these pathways are unknown. A murine lymphocyte complementary DNA that represents a new member of the src family has now been isolated and characterized. This complementary DNA, termed blk (for B lymphoid kinase), specifies a polypeptide of 55 kilodaltons that is related to, but distinct from, previously identified retroviral or cellular tyrosine kinases. The protein encoded by blk exhibits tyrosine kinase activity when expressed in bacterial cells. In the mouse and among cell lines, blk is specifically expressed in the B cell lineage. The tyrosine kinase encoded by blk may function in a signal transduction pathway that is restricted to B lymphoid cells.     

水稻根际和非根际土磷酸酶活性对碳、磷添加的响应

【目的】研究外源养分添加对稻田土壤磷酸酶活性影响的特征,明确水稻根际和非根际土壤胞外磷酸酶活性对碳、磷添加的响应过程,为稻田土壤水肥管理,实现农业可持续利用提供理论指导。【方法】选取湖南长期种植水稻的典型缺磷水稻土,进行盆栽试验。试验设置4个处理,分别为不添加碳磷（CK）、添加碳（C）、添加磷（P）和添加碳磷（CP）。采用96微孔荧光法测定根际土与非根际土的酸性磷酸酶（ACP）和碱性磷酸酶（ALP）活性,同时基于生物可利用性的磷分级方法（BBP法）测量4种磷组分（CaCl₂-P、Citrate-P、Enzyme-P和HCl-P）,探讨碳、磷添加对4种生物有效性的磷组分的影响和土壤磷酸酶活性的响应特征。【结果】与CK相比,C、P添加和CP配施处理水稻地上部分生物量分别增加29.76%、84.03%和87.94%（P＜0.05）,地下部分生物量分别减少20.13%、增加57.49%和56.53%（P＜0.05）;植物全磷（TP）含量与生物量变化规律一致,C、P和CP添加处理地上部分TP含量比CK分别增加57.23%、95.21%和95.91%（P＜0.05）,地下部分TP含量比CK分别减少26.12%、增加45.45%和38.01%（P＜0.05）。根际土pH、NH₄⁺-N和Olsen-P的含量低于非根际土,CP配施处理中根际土微生物量磷（MBP）含量高于非根际土;碳、磷添加对4种基于生物有效性磷组分具有显著调控作用（P＜0.05）;Olsen-P和MBP与ALP呈极显著负相关（P＜0.05）,与ACP无显著相关性,表明微生物对速效养分利用明显。冗余分析表明非根际土壤中的酶活性变化主要受Olsen-P、MBP、CaCl₂-P和Citrate-P含量影响;而土壤中含水量、pH、NH₄⁺-N、根系生物量、HCl-P和Enzyme-P含量主要影响水稻根际土壤中的酶活性。【结论】P和CP配施处理能提高缺磷水稻土微生物活性,显著增加水稻生物量,提升根际微生物效应,改善土壤环境,有利于稻田生态系统的健康。     

Selective inhibition of a regulatory subunit of protein phosphatase 1 restores proteostasis

Many biological processes are regulated through the selective dephosphorylation of proteins. Protein serine-threonine phosphatases are assembled from catalytic subunits bound to diverse regulatory subunits that provide substrate specificity and subcellular localization. We describe a small molecule, guanabenz, that bound to a regulatory subunit of protein phosphatase 1, PPP1R15A/GADD34, selectively disrupting the stress-induced dephosphorylation of the α subunit of translation initiation factor 2 (eIF2α). Without affecting the related PPP1R15B-phosphatase complex and constitutive protein synthesis, guanabenz prolonged eIF2α phosphorylation in human stressed cells, adjusting the protein production rates to levels manageable by available chaperones. This favored protein folding and thereby rescued cells from protein misfolding stress. Thus, regulatory subunits of phosphatases are drug targets, a property used here to restore proteostasis in stressed cells.     

Rapid destruction of human Cdc25A in response to DNA damage

To protect genome integrity and ensure survival, eukaryotic cells exposed to genotoxic stress cease proliferating to provide time for DNA repair. Human cells responded to ultraviolet light or ionizing radiation by rapid, ubiquitin- and proteasome-dependent protein degradation of Cdc25A, a phosphatase that is required for progression from G1 to S phase of the cell cycle. This response involved activated Chk1 protein kinase but not the p53 pathway, and the persisting inhibitory tyrosine phosphorylation of Cdk2 blocked entry into S phase and DNA replication. Overexpression of Cdc25A bypassed this mechanism, leading to enhanced DNA damage and decreased cell survival. These results identify specific degradation of Cdc25A as part of the DNA damage checkpoint mechanism and suggest how Cdc25A overexpression in human cancers might contribute to tumorigenesis.     

人乳腺良恶性病变组织中端粒酶活性的检测

目的：探讨端粒酶活性与人乳腺良、恶性病变组织之间的关系。方法：用ＴＲＡＰ－ＥＬＩＳＡ检测法,对１５例乳腺良性病变和７种乳腺癌组织进行端粒酶活性水平检测。结果：７例乳腺浸润性导管癌中,５例端粒酶阳性,而１５例乳腺良性疾病中仅２例端粒酶阳性,两组差异有显著性（Ｐ〈０．０５）。此外,有淋巴结转移与无淋巴结转移的乳腺癌组织中端粒酶活性水平差异无显著性（Ｐ〉０．０５）。结论：端粒酶的激活在人类乳腺癌的发生过