PTP及其在植物MAPK途径中的作用

蛋白酪氨酸磷酸酶（protein tyrosine phosphatases,PTPs）是一个结构多样的磷酸酶家族,含有高度保守的催化结构域。在植物体内,PTP主要的靶蛋白是促细胞分裂剂激活性蛋白激酶（mitogen-activated protein kinase,MAPK）。MAPK级联途径参与有机体的发育、细胞增殖、激素调节以及逆境胁迫的信号转导,PTP在MAPK级联途径中主要起负调控作用。本文就PTP的结构和功能、MAPK在植物中的作用及PTP在MAPK级联途径中的功能进行综述,并着重介绍PTP在拟南芥中的研究进展。     

Regulation of gene expression,growth,and cell survival by IL—4：Contribution of multiple signaling pathways

Interleukin-4 is a cytokine produced by activated T cells,mast cells,and basophils that elicits many important biological responses[1](see Tab 1).These responses range from the regulation of helper T cell differentiation[2] and the production of IgE[3] to the regulation of the adhesive properties of endothelial cells via VCAM-1[4],In keeping with these diverse biological effects,high-affinity binding sites for IL-4(Kd 20 to 300pM) have been detected on many hematopoietic and non-hematopoietic cell types at levels ranging from 50 to 5000 sites per cell[5].This review will focus on the discrete signal transduction pathways activated by the IL-4 recxeptor and the coordination of these individual pathways in the regulation of a final biological outcome.     

酪氨酸蛋白磷酸酶参与脱氢抗坏血酸的信号途径

研究酪氨酸蛋白磷酸酶（PTPase）的抑制剂氧化苯肿（PAO）、NaVO3和Zn2＋在脱氢抗坏血酸（DHA）调控烟草气孔运动中的作用。结果表明,0．01mmol·L-1PAO、1mmol．L-1NaVO3和2mmol·L-1Zn2＋抑制黑暗和DHA诱导的气孔关闭,而对光诱导的烟草气孔开度的影响不大。据此推测PTPase参与DHA诱导的气孔关闭信号途径。     

S(yp) Y279和Y304介导了BcrAbl与Grb2等蛋白的结合

利用体外定点突变技术获得Ｓｙｐ Ｙ２７９Ｆ、Ｙ３０４Ｆ和Ｙ５４６Ｆ突变的ｃＤＮＡ, 将这些突变体和野生型Ｓｙｐ 分别构建入ｐＸＭ 真核表达载体, 转入Ｋ５６２ 细胞。经Ｗｅｓｔｅｒｎ 印迹证明, 各转染Ｋ５６２ 细胞中都有Ｓｙｐ 蛋白的表达。免疫沉淀与免疫印迹结果发现ＷＴ、Ｙ２７９Ｆ、Ｙ３０４Ｆ和Ｙ５４６Ｆ等４ 种Ｓｙｐ 在胞内均能直接与ＢｃｒＡｂｌ 结合。体外结合实验结果表明Ｙ３０４Ｆ突变导致了Ｓｙｐ 不能与Ｓｈｃ 结合,Ｙ２７９Ｆ突变则导致了Ｓｙｐ 不能与Ｇｒｂ２ 结合。结论是： 作为“接头蛋白”,Ｓｙｐ 可以介导ＢｃｒＡｂｌ 与Ｓｈｃ 和Ｇｒｂ２ 之间的结合;Ｇｒｂ２ 结合在Ｓｙｐ 的Ｙ２７９ 上,Ｓｈｃ 则结合在Ｓｙｐ的Ｙ３０４ 上     

酪氨酸蛋白磷酸酶(PTPase)参与水杨酸调控蚕豆气孔运动的信号转导

研究酪氨酸蛋白磷酸酶(PTPase)的抑制剂氧化苯胂(PAO)、钒酸钠(NaVO3)和Zn2 对水杨酸(SA)调控蚕豆气孔运动影响的结果表明,0~1mmol·L-1 PAO、0~4mmol·L-1 NaVO3和0~4mmol·L-1Zn2 对光诱导蚕豆气孔开度变化的影响不大,但都可以抑制黑暗或SA诱导的气孔关闭,据此推测,PTPase可能参与SA诱导气孔关闭的信号转导过程。     

Phosphorylation of human interleukin-2 (IL-2)

Human interleukin-2 (IL-2) is a lymphokine which is capable of activating lymphocytes and supporting the long-term in vitro growth of activated T cell clones. Recombinant human IL-2, expressed in either E. coli or cos cells, was shown to be phosphorylated by protein kinase C. Phosphorylated IL-2 synthesized in E. coli was analyzed by SDS-PAGE, reverse phase HPLC, and tryptic peptide mapping. The phosphorylated tryptic peptide was identified as the N-terminal fragment containing a single phosphorylation site at the serine residue at position 7. There was no difference in biological activity between non-phosphorylated and phosphorylated IL-2, as determined by a T cell growth assay. Although the physiological role of phosphorylation of IL-2 is unclear, IL-2 can be labeled with [-^32p] ATP and protein kinase C to a high specific radioactivity, and the synthesis of biologically active ^32p-labeled IL-2 may be useful for receptor-binding studies of the cells containing low level of phosphoprotein phosphotases.Abbreviations IL-2 Interleukin-2 - rIL-2 recombinant IL-2 - SDS-PAGE Sodium Dodecylsulfate Polyacrylamide Gel Electrophoresis, Tris tris (hydroxymethyl)-amino methane - RP-HPLC Reverse Phase High Pressure Liquid Chromatography - PTH Phenylthiohydantoin - IFN- Leukocyte Interferon - IFN- Fibroblast Interferon - IFN- Immune Interferon By acceptance of this article, the publisher or recipient acknowledges the right of the U.S. Government to retain a nonexclusive, royalty-free license in and to any copyright covering the articleResearch being carried out at the Frederick Cancer Research Facility, Frederick, Maryland     

Negative regulation of Ros receptor tyrosine kinase signaling. An epithelial function of the SH2 domain protein tyrosine phosphatase SHP-1

Male "viable motheaten" (me(v)) mice, with a naturally occurring mutation in the gene of the SH2 domain protein tyrosine phosphatase SHP-1, are sterile. Known defects in sperm maturation in these mice correlate with an impaired differentiation of the epididymis, which has similarities to the phenotype of mice with a targeted inactivation of the Ros receptor tyrosine kinase. Ros and SHP-1 are coexpressed in epididymal epithelium, and elevated phosphorylation of Ros in the epididymis of me(v) mice suggests that Ros signaling is under control of SHP-1 in vivo. Phosphorylated Ros strongly and directly associates with SHP-1 in yeast two-hybrid, glutathione S-transferase pull-down, and coimmunoprecipitation experiments. Strong binding of SHP-1 to Ros is selective compared to six other receptor tyrosine kinases. The interaction is mediated by the SHP-1 NH(2)-terminal SH2 domain and Ros phosphotyrosine 2267. Overexpression of SHP-1 results in Ros dephosphorylation and effectively downregulates Ros-dependent proliferation and transformation. We propose that SHP-1 is an important downstream regulator of Ros signaling.     

Effects of interleukin-2 (IL-2) on human plasma lipid,lipoprotein, and C-reactive protein

Six patients with confirmed malignant disease received four consecutive weekly cycles of human recombinant interleukin-2 (IL-2) 4 days/week, continuous iv. infusion, 3 × 10⁶ U/m²/day. Plasma cholesterol decreased a mean of 7% within 24 hours after IL-2 infusion and decreased by 33% within 4 days. Plasma cholesterol was significantly lower than baseline concentration by day 21 (–21%), and day 25 (–41%) was significantly lower than day 21. Decreased plasma cholesterol was the result of decreased HDL and LDL cholesterol concentrations. Plasma triglyceride demonstrated a mean increase of 46% after 4 days of therapy and remained greater than baseline concentrations at all time points analyzed. Apolipoprotein AI and AII decreased concomitantly with HDL-cholesterol concentrations, whereas apolipoprotein B after an initial mean decrease of 17% during the first cycle was not significantly different from baseline during the fourth cycle. Apolipoprotein E and Lp(a) were not significantly affected by IL-2 treatment. Plasma C-reactive protein (CRP) increased by 79% within 24 hours of therapy, increased by 254% on day 4, then decreased to baseline concentrations by day 21 after 3 days off of IL-2. Day 25 CRP was elevated compared to both baseline and day 21 concentrations. IL-2 induced plasma lipoprotein changes may be due in part to the induction of interferon gamma.     

Immunomodulated signaling in macrophages: Studies on activation of Raf-1, MAPK, cPLA_2 and secretion of IL-12

Little is known about the mechanism and signal transduction by LPS-mediated immunomodulation of murine peritoneal macrophages. It is found that the signal molecules of the down-stream of Ras, Raf-1, MAPK p44, and MAPK p42 are phosphorylated, and cPLA2 is activated with a significant increase of the release of [ H3 ] AA by macrophages in response to LPS and PMA. Compared with the very recent finding that LPS and PMA trigger the activation and translocation of PKC-α and PKC-ε, these findings suggest that there is a connection between PKC signaling pathway and the Raf-1/MAPK pathway and that the activation of these main signaling events may be closely related to the secretion of IL-12 during LPS-induced modulation of macrophages.     

EphA2 Receptor Unliganded Dimers Suppress EphA2 Pro-tumorigenic Signaling

The EphA2 receptor tyrosine kinase promotes cell migration and cancer malignancy through a ligand- and kinase-independent distinctive mechanism that has been linked to high Ser-897 phosphorylation and low tyrosine phosphorylation. Here, we demonstrate that EphA2 forms dimers in the plasma membrane of HEK293T cells in the absence of ephrin ligand binding, suggesting that the current seeding mechanism model of EphA2 activation is incomplete. We also characterize a dimerization-deficient EphA2 mutant that shows enhanced ability to promote cell migration, concomitant with increased Ser-897 phosphorylation and decreased tyrosine phosphorylation compared with EphA2 wild type. Our data reveal a correlation between unliganded dimerization and tumorigenic signaling and suggest that EphA2 pro-tumorigenic activity is mediated by the EphA2 monomer. Thus, a therapeutic strategy that aims at the stabilization of EphA2 dimers may be beneficial for the treatment of cancers linked to EphA2 overexpression.     

Altered MAP kinase phosphorylation and impaired motor coordination in PTPRR deficient mice

The neuronal protein tyrosine phosphatases encoded by mouse gene Ptprr (PTPBR7, PTP-SL, PTPPBSgamma-42 and PTPPBSgamma-37) have been implicated in mitogen-activated protein (MAP) kinase deactivation on the basis of transfection experiments. To determine their physiological role in vivo, we generated mice that lack all PTPRR isoforms. Ptprr-/- mice were viable and fertile, and not different from wildtype littermates regarding general physiology or explorative behaviour. Highest PTPRR protein levels are in cerebellum Purkinje cells, but no overt effects of PTPRR deficiency on brain morphology, Purkinje cell number or dendritic branching were detected. However, MAP kinase phosphorylation levels were significantly altered in the PTPRR-deficient cerebellum and cerebrum homogenates. Most notably, increased phospho-ERK1/2 immunostaining density was observed in the basal portion and axon hillock of Ptprr-/- Purkinje cells. Concomitantly, Ptprr-/- mice displayed ataxia characterized by defects in fine motor coordination and balance skills. Collectively, these results establish the PTPRR proteins as physiological regulators of MAP kinase signalling cascades in neuronal tissue and demonstrate their involvement in cerebellum motor function.     

Helicobacter pylori CagA: Analysis of Sequence Diversity in Relation to Phosphorylation Motifs and Implications for the Role of CagA as a Virulence Factor

CagA is transported into host target cells and subsequently phosphorylated. Clearly this is a mechanism by which Helicobacter pylori could take control of one or more host cell signal transduction pathways. Presumably the end result of this interaction favors survival of H. pylori, irrespective of eventual damage to the host cell. CagA is noted for its amino acid (AA) sequence diversity, both within and outside the variable region of the molecule. The primary purpose of this review is to examine how variation in the type and number of CagA phosphorylation sites might determine the outcome of infection by different strains of H. pylori. The answer to this question could help to explain the widely disparate results obtained when H. pylori CagA status has been compared to type and severity of disease outcome in different populations, that is in different countries. Analysis of all available CagA sequences revealed that CagA contains both tyrosine phosphorylation motifs (TPMs) and cyclic-AMP-dependent phosphorylation motifs (CPMs). There are two potential CPMs near the N-terminus of CagA and at least two in the repeat region; these are not all equally well conserved. We also defined a 48-residue AA sequence, which includes the N-terminal TPM at tyrosine (Y)-122, which distinguishes between Eastern (Hong Kong-Taiwan-Japan-Thailand) H. pylori isolates and those from the West (Europe-Africa-the Americas-Australia). All 28 of the Eastern type CagA proteins have a functional N-terminal TPM whereas 11 of 47 (23.4%) of the Western type contain an inactive motif, with threonine (T) replacing the critical aspartic acid (D) residue. Only 13 of 24 (54%) known CagA sequences have an active TPM in the repeat region and only one has two TPMs in this region. The potential TPM near the C-terminus of CagA is not likely to be important since only 3 of 24 (12.5%) sequences were found to be intact. Protein database searches revealed that the AA sequence immediately following the TPM at Y-122 in CagA is homologous with a pair of PDZ domains which are common in signal transducing proteins, particularly tyrosine phosphatases. This provides a theoretical link between CagA and many of the observed responses of host cells to H. pylori. In summary, not all CagA proteins are equal in their potential for initiating host cell responses via signal transduction pathways. The degree of functional diversity of this protein depends upon which phosphorylation motifs are critical to the biological activity of CagA.     

Receptor tyrosine kinases and their activation in melanoma

Receptor tyrosine kinases (RTKs) and their downstream signalling pathways have long been hypothesized to play key roles in melanoma development. A decade ago, evidence was derived largely from animal models, RTK expression studies and detection of activated RAS isoforms in a small fraction of melanomas. Predictions that overexpression of specific RTKs implied increased kinase activity and that some RTKs would show activating mutations in melanoma were largely untested. However, technological advances including rapid gene sequencing, siRNA methods and phospho-RTK arrays now give a more complete picture. Mutated forms of RTK genes including KIT, ERBB4, the EPH and FGFR families and others are known in melanoma. Additional over- or underexpressed RTKs and also protein tyrosine phosphatases (PTPs) have been reported, and activities measured. Complex interactions between RTKs and PTPs are implicated in the abnormal signalling driving aberrant growth and survival in malignant melanocytes, and indeed in normal melanocytic signalling including the response to ultraviolet radiation. Kinases are considered druggable targets, so characterization of global RTK activity in melanoma should assist the rational development of tyrosine kinase inhibitors for clinical use.     

Role of protein phosphatase 2C from bovine adrenal chromaffin cells in the dephosphorylation of phospho-serine 40 tyrosine hydroxylase

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the synthesis of catecholamines. It is dephosphorylated by protein phosphatase (PP) 2A and PP2C. In this study we used a fixed amount of bacterially expressed rat TH (5 microM), phosphorylated only at serine 40 (pSer40TH), to determine the PP activities against this site that are present in extracts from the bovine adrenal cortex, adrenal medulla, adrenal chromaffin cells and rat striatum. We found that PP2C was the main TH phosphatase activity in extracts from the adrenal medulla and adrenal chromaffin cells. In adrenal cortex extracts PP2C and PP2A activities toward pSer40TH did not differ significantly. PP2A was the main TH phosphatase activity in extracts from rat striatum. Kinetic studies with extracts from adrenal chromaffin cells showed that when higher concentrations of pSer40TH (> 5 microM) were used the activity of PP2C increased more than the activity of PP2A. PP2C was maximally activated by 1.25 mM Mn2+ and by 5 mM Mg2+ but was inhibited by calcium. Our data suggest a more important role for PP2C than was previously suggested in the dephosphorylation of serine 40 on TH.     

白细胞介素－2活化DNA结合因子的研究

用凝胶阻滞分析的方法, 发现鼠T淋巴细胞系CTLL-2在白细胞介素-2(IL-2)刺激下可活化一个DNA结合因子, 它与γ-干扰素活化序列(GAS)专一性结合, 命名这个DNA结合因子为白细胞介素-2活化核因子(IL-2-NAF).IL-2-NAF的活化非常迅速, 不需要新的蛋白质合成, 并且它的活化程度随着IL-2刺激细胞的时间的不同而发生相应的变化. 进一步研究表明, IL-2-NAF的活化过程是通过酪氨酸激酶的信号传递途径, 并且它本身的酪氨酸残基也被磷酸化, 酪氨酸残基的磷酸化为其结合DNA所必需. IL-4、γ-IFN刺激CTLL-2细胞不活化与GAS专一性结合的因子. 而在Hut-102细胞中, IL-2、IL-4均可活化GAS结合因子, 但活化程度较弱.