急性淋巴细胞性白血病患者外周血与骨髓细胞蛋白酪氨酸激酶活性研究

目的：研究淋巴细胞性白血病外周血与骨髓细胞蛋白酪氨酸激酶（PTK）活性，为临床诊断、治疗白血病提供基础数据。方法：采用γ^-32P标记及生化方法对正常人及白血病患的淋巴细胞进行PTK活性测定。结果：外周血PTK活性淋巴细胞白血病各组均高于对照组，骨髓PTK活性白血病各组与对照组比较结果差异较大，同型白血病外周血与骨髓比较结果亦存在差异。该实验的各组白血病PTK活性质膜高于胞浆。结论：白血病分型不同，PTK活性存在差异。该实验可用于临床作为白血病诊断、药物疗效观察的指标之一，并且有更深入研究白血病发病机制的实验价值。     

阻断酪氨酸蛋白激酶Src表达对蛋白磷酸酯酶2A活性和tau蛋白磷酸化的影响

目的:研究细胞内酪氨酸蛋白激酶Src对蛋白磷酸酯酶2A(PP2A)酪氨酸307位点(Y307)磷酸化和活性以及tau蛋白磷酸化的影响,为阿尔茨海默病脑内PP2A活性下调和tau蛋白异常磷酸化机制提供实验依据。方法:体外培养小鼠成神经瘤N2a细胞,转染特异性阻断Src表达的SiRNA,检测转染不同时间点PP2A Y307的磷酸化水平、PP2A的活性以及tau蛋白在不同位点的磷酸化水平。结果:转染Src SiRNA 12 h时Src蛋白水平显著降低,PP2A Y307的磷酸化水平下降,但总的PP2A蛋白水平也降低,伴随PP2A活性下调,tau蛋白在Ser198/199/202位点发生过度磷酸化。结论:细胞内PP2A活性的调节受多种因素的影响,单纯阻断上游Src的表达可降低PP2A的活性,并导致tau蛋白的过度磷酸化。     

蛋白酪氨酸激酶在骨肉瘤治疗中的研究进展

骨肉瘤是一种常见的恶性骨肿瘤,其治疗方法目前有化疗、外科手术以及靶向治疗等。蛋白酪氨酸激酶（PTK）是一类具有酪氨酸激酶活性的蛋白质,在细胞生长、增殖、分化中具有重要作用,以蛋白酪氨酸激酶为靶点进行靶向治疗成为目前国际上抗肿瘤药物研究的热点。文章就蛋白酪氨酸激酶在骨肉瘤中作用的研究进展进行综述。     

蛋白酪氨酸激酶活力增高在恶性血液病发病中的作用

蛋白酪氨酸激酶(PTK)通过调节底物蛋白的活化状态参与细胞的信号传递,最终调节细胞的分化、生长和激活。PTK活力异常与多种恶性血液病的发生发展有密切关系。已经明确,单纯BCR-ABL融合基因导致ABL所表达的酪氨酸激酶活力持续增高,即可引发慢性髓系白血病(CML)。正在研制的PTK酶抑制剂系列中,STI571作为BCR-ABL酪氨酸激酶相对特异的抑制剂,已在Ⅰ-Ⅱ期临床试验中取得良好的疗效,将会作为第一个特异性药物用于CML的治疗。     

人类GM-CSF受体胞外结构域及功能的研究进展

人类粒-巨噬细胞集落刺激因子(granulocyte-macrophage colony-stimulating factor,GM-CSF)通过细胞膜表面特异性受体(GM-CSF receptor,GMR)介导维持细胞存活,刺激细胞增殖与分化,调节成熟细胞功能。GMR为跨膜受体由α链和β链共同组成,结构上分为胞外域、跨膜部分和胞内域。α链和β链胞外域均含有一种造血生长因子受体超家族的共同结构模体(cytokine receptor modules,CRM)并依靠保守的半胱氨酸残基形成的二硫键维持胞外空间结构。GM-CSF和α链通过电荷识别结合并诱导β链加入,形成完整的配体受体复合物。在二硫键的作用下,GMR多聚化使两条以上β链胞内部分相互接近活化JAK2,完成GM-CSF对细胞的激活过程。     

葡萄糖-6-磷酸脱氢酶缺乏时红细胞膜带3蛋白酪氨酸磷酸化的改变

目的：从红细胞膜蛋白磷酸化改变的角度探讨葡萄糖－6－磷酸脱氢酶（G6PD）缺乏症溶血的机制。方法：Western blot法检测G6PD缺乏症的红细胞膜蛋白磷酸化的改变以及二硫苏糖醇（DTT）对蛋白磷酸化的影响；以对硝基苯磷酸（PNPP）为底物，测磷酸酪氨酸磷酸酶（PTPs)活性以探讨磷酸化改变的可能成因。结果：G6PD缺乏的红细胞膜带3（Band 3)蛋白酪氨酸的磷酸化水平较正常对照明显增多，而PTPs活性检测较正常对照组明显减弱；DTT处理的G6PD缺乏红细胞，其膜Band 3蛋白酪氨酸的磷酸化与未处理者无明显差异，PTPs活性检测结果与未处理者亦无显著差异。结论：氧化致使G6PD缺乏红细胞的PTPs活性减弱，膜Band 3蛋白酪氨酸磷酸化增强，成为红细胞溶血的一个重要原因。但PTPs巯基的改变并不是影响PTPs活性的唯一因素。     

归肝经中药作用于人肝癌细胞差异表达 蛋白酪氨酸激酶的筛选与分析

目的 应用蛋白芯片技术筛选归肝经中药作用于人肝癌细胞蛋白酪氨酸激酶(PTKs)系统的特征性差异表达蛋白,并分析不同归肝经中药与PTKs调节的差异性.方法 选择40只BALB/C裸小鼠,采用将皮下瘤组织块植入肝左叶实质内的方法复制原位移植瘤模型,模型复制10 d确定成瘤后分为4组,各组分别腹腔注射相应的中药提取物,归肝经化瘀消癥药对组给予三棱和莪术(给药量为含生药4.5 g·kg-1·d-1)、归肝经攻毒散结药对组给予蜈蚣和全蝎(给药量为含生药0.3 g·kg-1·d-1)、归脾经对照药对组给予黄芪和白术(给药量为含生药6.3 g·kg-1·d-1),模型组腹腔注射等量0.9%生理盐水.药物处理3周后处死小鼠取肝癌组织,采用蛋白芯片技术筛选归肝经药对作用于肝癌细胞的差异表达PTKs并进行聚类分析.结果 实验结束后各组动物数分别为化瘀消癥药对组6只,攻毒散结药对组5只,对照药对组5只,模型组7只.蛋白芯片筛选结果显示:以调节倍数大于1.50或小于0.67判定为差异有统计学意义,与模型组比较,化瘀消癥药对组42种表达均为上调〔包括29种受体酪氨酸激酶(RTKs)和13种非受体酪氨酸激酶(nrPTKs)〕,其中调节倍数大于5.0的有促红细胞生成素产生肝细胞受体B1(EphB1)、表皮生长因子受体(ErbB2、ErbB4)等3种RTKs;调节倍数为3.0~5.0的包含7种RTKs,分别为EphA1、EphA3、EphA7、成纤维细胞生长因子受体2-α(FGFR2-α)、肝细胞生长因子受体(HGFR)、巨噬细胞集落刺激因子受体(M-CSFR)、血管内皮细胞生长因子2(VEGFR2);2种nrPTKs分别为非受体酪氨酸激酶BMX(BMX)、Janus激酶(JAK1).攻毒散结药对组表达上调23种(RTKs 15种、nrPTKs 8种),6种下调,其中调节倍数为3.0~5.0的RTKs分别为ErbB4、M-CSFR,1种nrPTKs为巨核细胞相关酪氨酸激酶(MATK).对照药对组表达上调28种,下调8种.聚类分析结果显示,符合筛选条件的PTKs差异表达在化瘀消癥药对组有17种,其中9种RTKs〔受体酪氨酸激酶样孤儿受体(ROR2)、干细胞因子受体(SCFR)、间变性淋巴瘤激酶(ALK)、血小板源性生长因子受体-β(PDGFR-β)、胰岛素样生长因子-IR(IGF-IR)、ErbB2、ErbB3、EphB1、EphA2〕,1种nrPTKs〔fps/fes相关酪氨酸激酶(FER)〕,7种PTKs,包括3种RTKs(M-CSFR、FGFR2-α、EphA3)和4种nrPTKs〔乙酸激酶1(ACK1)、布鲁顿酪氨酸蛋白激酶(Btk)、非受体酪氨酸激酶ABL1(ABL1)、BMX〕;在攻毒散结药对组有7种,包括5种RTKs(M-CSFR、FGFR1、ROR2、EphB1、ErbB2)和2种nrPTKs〔缺乏C端调节与N端烷基化位点的Src相关激酶(SRMS)、FER〕.结论 具有攻毒散结作用的蜈蚣和全蝎药对比具有化瘀消癥作用的三棱和莪术药有更好的抗肝癌作用,其机制可能是通过调节PTKs信号通路.归肝经活血化瘀药对三棱和莪术可能存在独立于PTKs信号系统外的抗肝癌效应途径.     

As2O3诱导K562细胞凋亡过程中酪氨酸蛋白激酶活性的变化

目的：阐明Ａｓ２Ｏ３诱导Ｋ５６２细胞凋亡的可能机制，为Ａｓ２Ｏ３治疗白血病的推广应用提供一定的理论基础。方法：采用免疫沉淀、Ｗｅｓｔｅｒｎｂｌｏｔ及生物化学等方法，观察在Ａｓ２Ｏ３诱导Ｋ５６２细胞凋亡过程中，细胞胞浆和胞膜蛋白及ＡＢＬ蛋白酪氨酸激酶（ＰＴＫ）活性及某些内源性蛋白酪氨酸磷酸化的变化。结果：在Ａｓ２Ｏ３诱导Ｋ５６２细胞凋亡过程中，细胞胞浆和胞膜蛋白及ＡＢＬ蛋白ＰＴＫ活性降低，分子量为１８万和１２．５万的蛋白酪氨酸磷酸化减少。结论：Ａｓ２Ｏ３可能通过降低细胞内某些蛋白，尤其ＢＣＲ／ＡＢＬ蛋白的ＰＴＫ活性，减少蛋白酪氨酸磷酸化，从而阻断ＢＣＲ／ＡＢＬ抗凋亡信号的传导，诱导Ｋ５６２细胞凋亡     

蛋白酪氨酸激酶对吸烟大鼠肺泡巨噬细胞诱导型一氧化氮合酶表达的影响

【目的】探讨蛋白酪氨酸激酶(PTK)在吸烟大鼠肺泡巨噬细胞(AM)诱导型一氧化氮合酶(iNOS)mRNA和蛋白表达中的作用。【方法】选用Wistar大鼠30只随机分为对照组,被动吸烟组和PTK抑制剂Genistein干预组。用RTPCR检测iNOSmRNA的表达,用Westernblot检测iNOS蛋白的表达,用Griess法测定NO2-/NO3-含量。【结果】吸烟大鼠AM中iNOSmRNA和蛋白的表达与对照组相比显著增加(P<0.01),吸烟组AM培养上清液中的NO2-/NO3-含量较对照组显著增加(P<0.01)。在体实验发现,Genistein显著降低AM中iNOSmRNA及其蛋白的表达,同时使培养上清液中NO2-/NO3-含量下降(P<0.01)。【结论】蛋白酪氨酸激酶抑制剂Genistein显著抑制了吸烟所致的AM中iNOS的表达,说明蛋白酪氨酸激酶参与吸烟诱导的AM中iNOS表达的信号传导。     

高脂饮食诱导肥胖模型大鼠骨骼肌组织蛋白酪氨酸磷酸酯酶1B和胰岛素受体底物2的表达

背景:外周组织的胰岛素抵抗是2型糖尿病的主要病因。目的:观察高脂饮食诱导的肥胖大鼠骨骼肌中蛋白酪氨酸磷酸酯酶1B和胰岛素受体底物2的表达。方法:将20只SD大鼠随机等分为对照组和高脂组,分别给予常规饲料和高脂饲料喂养12周。结果和结论:与对照组相比,高脂组大鼠胰岛素敏感指数显著降低(P<0.01),大鼠葡萄糖耐量受损,胰岛素释放试验提示葡萄糖刺激的胰岛素第一时相分泌受损,骨骼肌组织中蛋白酪氨酸磷酸酯酶1B蛋白表达水平明显增加(P<0.01),骨骼肌中胰岛素诱导的胰岛素受体底物2磷酸化程度降低(P<0.01)。提示高脂饮食诱导的肥胖大鼠骨骼肌中蛋白酪氨酸磷酸酯酶1B蛋白表达量升高,使胰岛素诱导的胰岛素受体底物2磷酸化程度降低,可能是肥胖导致胰岛素抵抗的机制之一。     

Involvement of p59fynT in interleukin-5 receptor signaling [published erratum appears in J Exp Med 1995 Oct 1;182(4):1179]

Previous studies implicate the nonreceptor protein tyrosine kinase (PTK) p59fyn in the propagation of signals from the B cell antigen receptor. To elucidate the functions of this kinase, we examined B cell responsiveness in mice engineered to lack the hematopoietic isoform of p59fyn. Remarkably, antigen receptor signaling was only modestly defective in fynTnull B cells. In contrast, signaling from the interleukin (IL)-5 receptor which ordinarily provides a comitogenic stimulus with antiimmunoglobulin, was completely blocked. Our results document the importance of p59fynT in IL-5 responses in B cells, and they support a general model for cytokine receptor signal transduction involving the simultaneous recruitment of at least three families of PTK.     

TGF-beta abrogates TCR-mediated signaling by upregulating tyrosine phosphatases in T cells

TGF-beta is known to inhibit many of the immune cell functions including T cell proliferation and IL-2 production. The mechanism of such TGF-beta-mediated inhibition of T cell functions is poorly understood. The present study examined the effects of TGF-beta on the activation of protein tyrosine kinases (PTK) P56lck, P59fyn, and Zap-70, and protein tyrosine phosphatases (PTP) SHP-1 and SHP-2. A balance between the actions of PTK and PTP is critical for appropriate T cell activation. These studies were carried out using nylon wool-purified splenic T cells from healthy Sprague-Dawley rats. Results from these studies showed that incubation of T cells with TGF-beta inhibited the activation of P56lck, P59fyn and Zap-70. The decrease in these three protein tyrosine kinases was accompanied by an increase in the activation of the protein tyrosine phosphatase SHP-1. There was no change in the phosphorylation of SHP-2 with and without pretreatment of T cells with TGF-beta. The decrease in P56lck, P59fyn kinase activity, and Zap-70 phosphorylation was prevented when T cells were stimulated with anti-CD3 in the presence of pervanadate, an inhibitor of PTP. These results suggested that TGF-beta-mediated inhibition of P56lck, P59fyn, and Zap-70 is likely due to an up-regulation of protein tyrosine phosphatases such as SHP-1.     

Tyrosine phosphorylation of mitogen-activated protein kinases is necessary for activation of murine macrophages by natural and synthetic bacterial products

The purpose of these studies was to determine the intracellular signal transduction pathways of bacterial products in murine macrophages from lipopolysaccharide (LPS)-responder C3H/HeN and LPS-nonresponder C3H/HeJ mice. Both LPS and synthetic lipopeptide CGP 31362 (LPP) induced production of tumor necrosis factor alpha (TNF-alpha) in C3H/HeN macrophages. In C3H/HeJ macrophages, however, TNF-alpha was induced only by incubation with LPP. Both LPS and LPP induced tyrosine phosphorylation on proteins with apparent molecular masses of 39, 41, and 45 kD (p35, p41, and p45) in C3H/HeN macrophages, whereas in C3H/HeJ macrophages, tyrosine phosphorylation was induced only by LPP. 20-h incubation with LPS or LPP downregulated TNF-alpha production/secretion and tyrosine phosphorylation in C3H/HeN macrophages induced by additional LPS or LPP. In C3H/HeJ macrophages, however, the downregulation of TNF-alpha production and tyrosine phosphorylation were observed only with LPP. Protein kinase assays, Western blotting analyses, phenyl-Sepharose chromatography, and immunocomplex kinase assay suggested that p45 and p39 were similar or identical to mitogen-activated protein (MAP) kinase 1 and 2, respectively. Pretreatment of macrophages with LPS or LPP did not change the amount of kinase proteins but inhibited the stimulation of kinase activity by the agents. These data suggest that MAP kinases are among target proteins involved in the transduction of LPS and LPP signals that lead to activation of murine macrophages to produce/secrete TNF.     

Differential regulation of T cell antigen responsiveness by isoforms of the src-related tyrosine protein kinase p59fyn

Recent observations suggest that the src-related tyrosine protein kinase p59fyn may be involved in antigen-induced T lymphocyte activation. As a result of alternative splicing, p59fyn exists as two isoforms that differ exclusively within a short sequence spanning the end of the Src Homology 2 (SH2) region and the beginning of the tyrosine protein kinase domain. While one p59fyn isoform (fynB) is highly expressed in brain, the alternative product (fynT) is principally found in T lymphocytes. To further understand the role of p59fyn in T cell activation and to test the hypothesis that p59fynT serves a tissue-specific function in T lymphocytes, we have examined the effects of expression of activated versions (tyrosine 528 to phenylalanine 528 mutants) of either form of p59fyn on the physiology of an antigen-specific mouse T cell hybridoma. Our results demonstrated that the two forms of fyn, expressed in equivalent amounts, efficiently enhanced antibody-induced T cell receptor (TCR)-mediated signals. In contrast, only p59fynT increased interleukin 2 production in response to antigen stimulation. This finding implies that the distinct p59fyn isoform expressed in T lymphocytes regulates the coupling of TCR stimulation by antigen/major histocompatibility complex to lymphokine production.     

Tyrosine phosphorylation and association with phospholipase C gamma-1 of the GAP-associated 62-kD protein after CD2 stimulation of Jurkat T cell

Numerous substrates are tyrosine phosphorylated upon CD2 stimulation of human Jurkat T cells using a mitogenic pair of CD2 monoclonal antibodies, including the phospholipase C (PLC)gamma-1-p35/36 complex. Most of these substrates are identically tyrosine phosphorylated after CD3 ligation, suggesting that both stimuli share the same biochemical pathway. We show, however, in this report that a 63-kD protein is specifically phosphorylated on tyrosine residues after ligation of the CD2 molecule. The tyrosine phosphorylation of p63 can be induced independently of other substrates when using a single CD2 mAb recognizing the D66 epitope of the molecule. Importantly, this CD2- induced tyrosine phosphorylation of p63 can also occur in the absence of the CD3 zeta chain membrane expression, and is also distinct from the protein tyrosine kinases p56lck and p59fyn. We demonstrate, moreover, that p63 is physically linked with PLC gamma-1 and p35/36 upon CD2 stimulation. Finally, we also show that a 62-kD protein coimmunoprecipitating with the p21ras GTPase activating protein (GAP) is heavily tyrosine phosphorylated only after CD2 stimulation. This ultimately suggests that p63 may represent in fact the 62-kD protein that associates with GAP after tyrosine phosphorylation. Taken together, these results demonstrate the occurrence in Jurkat cells of a tyrosine kinase pathway specifically coupled to the CD2 molecule. They also suggest a function of the p62-GAP-associated protein as a link between PLC gamma-1 and p21ras activation pathways after CD2 activation.     

T cell receptor zeta/CD3-p59fyn(T)-associated p120/130 binds to the SH2 domain of p59fyn(T)

Intracellular signaling from the T cell receptor (TCR)zeta/CD3 complex is likely to be mediated by associated protein tyrosine kinases such as p59fyn(T), ZAP-70, and the CD4:p56lck and CD8:p56lck coreceptors. The nature of the signaling cascade initiated by these kinases, their specificities, and downstream targets remain to be elucidated. The TCR- zeta/CD3:p59fyn(T) complex has previously been noted to coprecipitate a 120/130-kD doublet (p120/130). This intracellular protein of unknown identity associates directly with p59fyn(T) within the receptor complex. In this study, we have shown that this interaction with p120/130 is specifically mediated by the SH2 domain (not the fyn-SH3 domain) of p59fyn(T). Further, based on the results of in vitro kinase assays, p120/130 appears to be preferentially associated with p59fyn(T) in T cells, and not with p56lck. Antibody reprecipitation studies identified p120/130 as a previously described 130-kD substrate of pp60v- src whose function and structure is unknown. TCR-zeta/CD3 induced activation of T cells augmented the tyrosine phosphorylation of p120/130 in vivo as detected by antibody and GST:fyn-SH2 fusion proteins. p120/130 represents the first identified p59fyn(T):SH2 binding substrate in T cells, and as such is likely to play a key role in the early events of T cell activation.     

Signal transduction via CD40 involves activation of lyn kinase and phosphatidylinositol-3-kinase, and phosphorylation of phospholipase C gamma 2

CD40 is a 50-kD glycoprotein that plays an important role in B cell survival, memory, and immunoglobulin isotype switch. Engagement of the CD40 antigen by monoclonal antibodies (mAbs) results in increased protein tyrosine kinase (PTK) activity, which plays an important role in mediating the biologic effects of CD40. We demonstrate, using an in situ phosphorylation technique, that CD40 cross-linking by the anti- CD40 mAb 626.1 resulted within 1 min in increased phosphorylation of the src type kinase, lyn, in Daudi B cell lines and remained sustained for up to 20 min. The activity of lyn kinase, as measured by immune complex kinase assay, was also increased after CD40 engagement, with similar kinetics. In contrast, the phosphorylation and activity of fyn, fgr, and lck kinases demonstrated minimal changes following stimulation of Daudi cells with mAb 626.1 over this same time period. CD40 engagement also resulted in phosphorylation of phospholipase C gamma 2 of phosphatidylinositol (PLC gamma 2) and phosphatidylinositol (PI)-3- kinase. Phosphorylation of PI-3-kinase was shown to be associated with an increase in its enzymatic activity. These results suggest that lyn plays an important role in CD40-mediated PTK activation and identify PLC gamma 2 and PI-3-kinase targets for CD40-mediated phosphorylation, suggesting a role for these two enzymes in CD40 signal transduction.     

Lck regulates the tyrosine phosphorylation of the T cell receptor subunits and ZAP-70 in murine thymocytes

The Src-family and Syk/ZAP-70 family of protein tyrosine kinases (PTK) are required for T cell receptor (TCR) functions. We provide evidence that the Src-family PTK Lck is responsible for regulating the constitutive tyrosine phosphorylation of the TCR zeta subunit in murine thymocytes. Moreover, ligation of the TCR expressed on thymocytes from Lck-deficient mice largely failed to induce the phosphorylation of TCR- zeta, CD3 epsilon, or ZAP-70. In contrast, we find that the TCR-zeta subunit is weakly constitutively tyrosine phosphorylated in peripheral T cells isolated from Lck-null mice. These data suggest that Lck has a functional role in regulation of TCR signal transduction in thymocytes. In peripheral T cells, other Src-family PTKs such as Fyn may partially compensate for the absence of Lck.     

The Src-family kinase, Fyn, regulates the activation of phosphatidylinositol 3-kinase in an interleukin 2-responsive T cell line

The proliferation of antigen-activated T cells is mediated by the T cell-derived growth factor, interleukin 2 (IL-2). The biochemical signaling cascades initiating IL-2-induced growth are dependent upon protein tyrosine kinase (PTK) activity. One IL-2-regulated PTK implicated in this cascade is the Src-family kinase, Fyn. Previous studies have described a physical association between Fyn and a potential downstream substrate, phosphatidylinositol 3-kinase (PI3- kinase) as well as the IL-2-dependent activation of PI3-kinase in T cells; however, the role of Fyn in IL-2-induced PI3-kinase activation remains unclear. In this report, we demonstrate that IL-2 stimulation triggers tyrosine phosphorylation of the p85 subunit of PI3-kinase in the murine T cell line, CTLL-2. Lysates prepared from growth factor- deprived and IL-2-stimulated T cells reconstituted both the binding of CTLL-2 cell-derived Fyn to and the IL-2-inducible tyrosine phosphorylation of exogenously added recombinant p85. Furthermore, overexpression of wild-type Fyn in these cells enhanced both the basal and IL-2-mediated activation of PI3-kinase. Additional studies of the Fyn-PI3-kinase interaction demonstrated that the Src homology 3 (SH3) domain of Fyn constitutes a direct binding site for the p85 subunit of PI3-kinase. These results support the notion that Fyn may be directly involved in the activation of the downstream signaling enzyme, PI3- kinase, in IL-2-stimulated T cells.