Src激酶的功能研究新进展

Src激酶家族是具有酪氨酸蛋白激酶活性的蛋白质,作为连接许多细胞外和细胞内重要信号途径的膜结合开关分子,Src激酶在受体介导的信号传递及细胞间通讯中具中心调节作用。最近发现它在淋巴因子介导的细胞存活及血管内皮生长因子介导的血管发生中也具有重要作用。     

Src介导骨桥蛋白诱导的血管平滑肌细胞黏附和迁移过程

为阐明酪氨酸激酶Src在整合素被骨桥蛋白(OPN)激活所触发的细胞黏附和迁移信号途径中所起的作用,应用Src特异性抑制剂PP2阻断Src,观察OPN诱导的血管平滑肌细胞(VSMC)黏附和迁移活性的改变,并利用免疫沉淀检查PP2对整合素下游信号分子黏着斑激酶(FAK)和整合素偶联激酶(ILK)磷酸化及其相互作用的影响。结果显示,PP2可明显抑制OPN诱导的VSMC黏附和伤口愈合(黏附和迁移活性分别为对照组的76.6%和33.8%);OPN可显著诱导FAK磷酸化(磷酸化水平达对照组的1.9倍),促进ILK去磷酸化,并使FAK与ILK的结合减少(降至对照组的46.4%)。10μmol/LPP2可明显抑制OPN诱导的FAK磷酸化、拮抗OPN诱导对ILK的去磷酸化作用、促进FAK与ILK之间的结合。研究结果表明,Src作为OPN-整合素-FAK信号途径中的信号分子,通过影响FAK和ILK的磷酸化以及两者之间的相互作用来调节VSMC的黏附和迁移活性。     

Src和Abl酪氨酸蛋白激酶家族参与病原微生物感染的研究进展

Src和Abl家族激酶属于非受体型酪氨酸激酶(Nonreceptor tyrosine kinase,NRTK)家族重要成员,广泛存在于各种细胞中,参与细胞内信号传递并调节细胞生理过程,它们在维持细胞、组织和器官稳态功能中发挥着至关重要的作用。研究表明,Src和Abl家族激酶通过多种机制参与病原微生物的感染(如与病原微生物的脯氨酸基序-PXXP互作)。因此,从Src和Abl家族激酶角度出发探究病原微生物感染机制逐渐成为一个热点。本文就Src和Abl家族激酶的结构特点以及参与病原微生物感染的研究报道进行综述,以期为病原微生物感染的致病机制、防控和药物研发提供参考。     

PP2 对人胆管癌细胞株QBC939 侵袭能力的影响

目的：探讨Src 激酶特异性抑制剂PP2 对人胆管癌QBC939 细胞侵袭能力的影响和机制。方法：通过Western Blotting 技术 检测PP2 对人胆管癌QBC939细胞中Src 激酶活化的影响;用Transwell 小室法观察PP2 对QBC939细胞的影响;用RT-PCR 和 Western Blotting 技术检测PP2对QBC939 细胞侵袭能力相关分子的作用。结果：实验组p-Src 蛋白表达水平明显低于对照组,差 异具有统计学意义（P<0.05）;实验组QBC939 细胞体外侵袭能力较对照组显著降低,差异具有统计学意义（P<0.05）;与对照组相 比,实验组E-cadherin 表达显著增强,CD44表达明显减弱,差异具有统计学意义（P<0.05）。结论：PP2 通过抑制Src 激酶活化,增 强E-cadherin 表达、减弱CD44 表达,抑制人胆管癌QBC939 细胞侵袭能力。     

酪氨酸激酶Fyn的生物学特性及其在肿瘤发生发展中的作用

酪氨酸激酶Fyn是Src家族激酶的成员之一,其持续活化被认为与肿瘤细胞的代谢、增殖和迁移有密切的联系.Fyn在肿瘤的发生发展中起着重要的调控作用.该文从结构特征、相关信号通路及基本生理功能等方面阐述了Fyn的生物学特性,总结了近年来围绕Fyn展开的研究,重点归纳了Fyn在脑胶质瘤、血液系统肿瘤、胰腺癌等肿瘤中发挥的促癌...     

TrkA介导电针抑制局灶性脑缺血再灌注大鼠模型神经元Src磷酸化的作用

目的探讨电针通过TrkA通路对脑缺血再灌注损伤诱导的Src磷酸化的影响。方法采用改良的血管内线栓技术制备大鼠局灶性脑缺血再灌注模型,电针大鼠"水沟"、"承浆"穴,侧脑室注射TrkA受体及下游信号通路的拮抗剂,K252a拮抗TrkA的作用、Wortmannin拮抗PI-3K的作用、U0126拮抗MEK的作用;免疫组织化学技术检测缺血侧大脑皮层、海马Src磷酸化。结果脑缺血再灌注损伤诱导大鼠皮层和海马神经元Src磷酸化水平异常增加,与对照组相比,差异显著(P0.05);电针抑制脑缺血再灌注损伤引起的Src磷酸化异常增加水平,与缺血再灌注组相比电针能明显减少Src磷酸化水平(P0.05);分别脑室注射TrkA抑制剂、PI-3K抑制和MEK抑制剂预处理后,能有效翻转电针对皮层神经元Src磷酸化异常增加的抑制作用,统计学处理有显著性差异(P0.05)。结论电针减少缺血再灌注导致的Src磷酸化,通过激活TrkA/PI-3K和TrkA/MAPK通路抑制Src的活性,发挥神经保护作用。     

NIH3T3细胞中PTPα基因高表达激活Src的早期细胞表型变化

利用蛋白质酪氨酸磷酸酶α(PTPα)基因转染NIH3T3细胞 ,研究PTPα诱导前后细胞生物学行为的变化 ,为肿瘤形成早期机制的研究提供细胞模型。用携带PTPα基因的四环素调控体系转染NIH3T3细胞并诱导 2 4h后 ,用RT PCR和蛋白质印迹法证实PTPα在诱导细胞中的表达高于未诱导细胞 ,用RT PCR及蛋白质印迹法发现内源性Src的表达水平在诱导与未诱导细胞中没有变化 ,而Src激酶的活性在诱导细胞中增高 ,并且在诱导细胞中Src的酪氨酸磷酸化水平降低。再用透射式电子显微镜和流式细胞技术观察到诱导细胞的表型已发生变化 ,实验结果表明PTPα诱导 2 4h使细胞的表型开始发生转化 ,这种变化很可能与PTPα表达水平升高使SrcC末端的pTyr52 7去磷酸化而激活Src相关     

Src蛋白激酶活性的调节机制

Src蛋白激酶在人类多种肿瘤细胞中被激活并在肿瘤发生、发展过程中起重要作用.Src活性的调节主要包括共价修饰、异构调节,但基因突变和其他一些方式也可以调节其活性.Src共价修饰主要是磷酸化,Tyr530、Tyr419、Thr34、Thr46、Ser72、Tyr138和Tyr213等都是Src的磷酸化位点,其中Tyr530位点和Tyr419位点是Src最重要的磷酸化位点.异构调节包括SH3、SH2等区域结合的调节,分别涉及黏着斑激酶(focal adhesion kinase,FAK)、孕酮受体(progesterone receptor,PR)、雌激素受体(estrogen receptor,ER)、雄激素受体(androgen receptor,AR)、P130Cas、血小板源生长因子(the platelet-derived growth factor,PDGF)、血小板衍生生长因子受体(platelet-derived growth factor receptor,PDGFR)、表皮生长因子受体(epidermal growth factor receptor,EGFR,HER1/erb B1)、人类表皮生长因子受体2(human epidermal growth factor receptor-2,ERBB2/HER2/NEU)、胰岛素样生长因子1受体(insulin-like growth factor-1 receptor,IGF-1R)、纤维母细胞生长因子受体1(fibroblast growth factor receptor,FGFR1)、肝细胞生长因子受体(hepatocyte growth factor receptor c-Met)、人类1型T细胞白血病病毒编码的辅助蛋白p13、HIV-1毒力因子Nef和Sin.本文就Src蛋白激酶的调节机制作一简要综述.     

GST-pull down方法鉴定Src激酶与PSD-95的直接结合

脚手架蛋白PSD-95通过募集多种蛋白质在包括缺血性脑中风在内的多种神经系统疾病中具有重要的作用。Src蛋白激酶家族是膜相关非受体酪氨酸蛋白激酶中最大的家族,该家族激酶含有与突触后致密蛋白PSD-95相结合的结构域。Src激酶是其家族中主要成员之一,在脑组织中表达丰富。脑缺血/再灌注引起缺血敏感区Src激酶活性的显著增强。之前的研究表明,Src激酶参与调节PSD-95酪氨酸磷酸化。本实验主要通过GST-pull down实验体外鉴定Src与PSD-95之间的直接结合。     

癌基因Src在骨肉瘤细胞侵袭伪足形成中的作用

目的:探讨癌基因Src在体外培养骨肉瘤细胞侵袭伪足形成中的作用。方法:构建Src sh RNA慢病毒表达载体,在HEK293T细胞中包装慢病毒,感染HT-1080骨肉瘤细胞,经嘌呤霉素加压筛选,获得稳定沉默Src基因的骨肉瘤细胞系HT-1080-sh Src;实时定量PCR和Western Blot法检测基因沉默效率;采用原位明胶酶谱法检测侵袭伪足形成;采用侵袭小室实验检测下调Src基因表达对HT-1080细胞侵袭力的影响。结果:成功构建稳定沉默Src基因的骨肉瘤细胞系HT-1080-sh Src及对照细胞系HT-1080-shluc,经实时定量PCR和Western Blot检测,与对照细胞系相比,HT-1080-sh Src细胞中Src基因表达下调3倍以上;下调HT-1080细胞中Src基因表达能显著抑制HT-1080细胞侵袭伪足形成及其对细胞外基质的降解能力;下调Src基因表达能显著抑制骨肉瘤细胞侵袭力。结论:癌基因Src参与调节骨肉瘤细胞HT-1080侵袭伪足形成,促进肿瘤侵袭、转移。     

Medicinal Chemistry Perspectives on Recent Advances in Src Kinase Inhibitors as a Potential Target for the Development of Anticancer Agents: Biological Profile,Selectivity, Structure-Activity Relationship

The physiological Src proto-oncogene is a protein tyrosine kinase receptor that served as the essential signaling pathway in different types of cancer. Src kinase receptor is divided into different domains: a unique domain, an SH3 domain, an SH2 domain, a protein tyrosine kinase domain, and a regulatory tail, which runs from the N-terminus to the C-terminus. Src kinase inhibitors bind in the kinase domain and are activated by phosphorylation. The etiology of cancer involved various signaling pathways and Src signaling pathways are also involved in those clusters. Although the dysregulation of Src kinase resulted in cancer being discovered in the late 19^th century it is still considered a cult pathway because it is not much explored by different medicinal chemists and oncologists. The Src kinase regulated through different kinase pathways (MAPK, PI3K/Akt/mTOR, JAK/STAT3, Hippo kinase, PEAK1, and Rho/ROCK pathways) and proceeded downstream signaling to conduct cell proliferation, angiogenesis, migration, invasion, and metastasis of cancer cells. There are numerous FDA-approved drugs flooded the market but still, there is a huge demand for the creation of novel anticancer drugs. As the existing drugs are accompanied by several adverse effects and drug resistance due to rapid mutation in proteins. In this review, we have elaborated about the structure and activation of Src kinase, as well as the development of Src kinase inhibitors. Our group also provided a comprehensive overview of Src inhibitors throughout the last two decades, including their biological activity, structure-activity relationship, and Src kinase selectivity. The Src binding pocket has been investigated in detail to better comprehend the interaction of Src inhibitors with amino acid residues. We have strengthened the literature with our contribution in terms of molecular docking and ADMET studies of top compounds. We hope that the current analysis will be a useful resource for researchers and provide glimpse of direction toward the design and development of more specific, selective, and potent Src kinase inhibitors.     

MG53 inhibits angiogenesis through regulating focal adhesion kinase signalling

Mitsugumin 53 (MG53), which is expressed predominantly in striated muscle, has been demonstrated to be a myokine/cardiokine secreted from striated muscle under specific conditions. The important roles of MG53 in non-striated muscle tissues have also been examined in multiple disease models. However, no previous study has implicated MG53 in the control of endothelial cell function. In order to explore the effects of MG53 on endothelial cells, human umbilical vein endothelial cells (HUVECs) were stimulated with recombinant human MG53 (rhMG53). Then, rhMG53 uptake, focal adhesion kinase (FAK)/Src/Akt/ERK1/2 signalling pathway activation, cell migration and tube formation were determined in vitro. The efficacy of rhMG53 in regulating angiogenesis was also detected in postnatal mouse retinas. The results demonstrated that rhMG53 directly entered into endothelial cells in a cholesterol-dependent manner. The uptake of rhMG53 directly bound to FAK in endothelial cells, which resulted in a significant decrease in FAK phosphorylation at Y397. Accompanied by the dephosphorylation of FAK, rhMG53 uncoupled FAK-Src interaction and reduced the phosphorylation of Src at Y416. Consequently, the activation of FAK/Src downstream signalling pathways, such as Akt and ERK1/2, was also significantly inhibited by rhMG53. Furthermore, rhMG53 remarkably decreased HUVEC migration and tube formation in vitro and postnatal mouse retinal angiogenesis in vivo. Taken together, these data indicate that rhMG53 inhibits angiogenesis through regulating FAK/Src/Akt/ERK1/2 signalling pathways. This may provide a novel molecular mechanism for the impaired angiogenesis in ischaemic diseases.     

Suppression of IL‐8‐Src signalling axis by 17β‐estradiol inhibits human mesenchymal stem cells‐mediated gastric cancer invasion

Epidemiologic data show the incidence of gastric cancer in men is twofold higher than in women worldwide. Oestrogen is reported to have the capacity against gastric cancer development. Endogenous oestrogen reduces gastric cancer incidence in women. Cancer patients treated with oestrogens have a lower subsequent risk of gastric cancer. Accumulating studies report that bone marrow mesenchymal stem cells (BMMSCs) might contribute to the progression of gastric cancer through paracrine effect of soluble factors. Here, we further explore the effect of oestrogen on BMMSCs‐mediated human gastric cancer invasive motility. We founded that HBMMSCs notably secrete interleukin‐8 (IL‐8) protein. Administration of IL‐8 specific neutralizing antibody significantly inhibits HBMMSCs‐mediated gastric cancer motility. Treatment of recombinant IL‐8 soluble protein confirmed the role of IL‐8 in mediating HBMMSCs‐up‐regulated cell motility. IL‐8 up‐regulates motility activity through Src signalling pathway in human gastric cancer. We further observed that 17β ‐estradiol inhibit HBMMSCS‐induced cell motility via suppressing activation of IL8‐Src signalling in human gastric cancer cells. 17β‐estradiol inhibits IL8‐up‐regulated Src downstream target proteins including p‐Cas, p‐paxillin, p‐ERK1/2, p‐JNK1/2, MMP9, tPA and uPA. These results suggest that 17β‐estradiol significantly inhibits HBMMSCS‐induced invasive motility through suppressing IL8‐Src signalling axis in human gastric cancer cells.     

The Helicobacter pylori CagA protein induces tyrosine dephosphorylation of ezrin

Helicobacter pylori is one of the most wide-spread bacterial pathogens and infects the human stomach to cause diseases, such as gastritis, gastric ulceration, and gastric cancer. A major virulence determinant is the H. pylori CagA protein (encoded by the cytotoxin-associated gene A) which is translocated from the bacteria into the cytoplasm of host cells by a type IV secretion system. In the host cell, CagA is phosphorylated on tyrosine residues and induces rearrangements of the actin cytoskeleton. We have previously shown that tyrosine-phosphorylated CagA inhibits the catalytic activity of Src family kinases and induces tyrosine dephosphorylation of several host cell proteins. Here, we identified one of these proteins as ezrin by a combination of preparative gel electrophoresis, two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). Specific pharmacological inhibition of Src family kinases also induces ezrin dephosphorylation. Therefore, ezrin dephosphorylation appears to be induced by CagA-mediated Src inactivation. Ezrin is the founding member of the ezrin-radixin-moesin (ERM) family of proteins which are signalling integrators at the cell cortex. Since ezrin is a component of microvilli and a linker protein between actin filaments and membrane proteins, this observation has important implications for H. pylori pathogenesis and might also help to explain the development of gastric cancer.     

Frequent amplification of PTP1B is associated with poor survival of gastric cancer patients

The protein tyrosine phosphatase 1B (PTP1B), a non-transmembrane protein tyrosine phosphatase, has been implicated in gastric pathogenesis. Several lines of recent evidences have shown that PTP1B is highly amplified in breast and prostate cancers. The aim of this study was to investigate PTP1B amplification in gastric cancer and its association with poor prognosis of gastric cancer patients, and further determine the role of PTP1B in gastric tumorigenesis. Our data demonstrated that PTP1B was significantly up-regulated in gastric cancer tissues as compared with matched normal gastric tissues by using quantitative RT-PCR (qRT-PCR) assay. In addition, copy number analysis showed that PTP1B was amplified in 68/131 (51.9%) gastric cancer cases, whereas no amplification was found in the control subjects. Notably, PTP1B amplification was positively associated with its protein expression, and was significantly related to poor survival of gastric cancer patients. Knocking down PTP1B expression in gastric cancer cells significantly inhibited cell proliferation, colony formation, migration and invasion, and induced cell cycle arrested and apoptosis. Mechanically, PTP1B promotes gastric cancer cell proliferation, survival and invasiveness through modulating Src-related signaling pathways, such as Src/Ras/MAPK and Src/phosphatidylinositol-3-kinase (PI3K)/Akt pathways. Collectively, our data demonstrated frequent overexpression and amplification PTP1B in gastric cancer, and further determined the oncogenic role of PTP1B in gastric carcinogenesis. Importantly, PTP1B amplification predicts poor survival of gastric cancer patients.     

Expanding the role of Src and protein-tyrosine phosphatases balance in modulating osteoblast metabolism: Lessons from mice

The widespread nature of protein phosphorylation/dephosphorylation underscores its key role in cell signaling metabolism, growth and differentiation. Tyrosine phosphorylation of cytoplasmic proteins is a critical event in the regulation of intracellular signaling pathways activated by external stimuli. An adequate balance in protein phosphorylation is a major factor in the regulation of osteoclast and osteoblast activities involved in bone metabolism. However, although phosphorylation is widely recognized as an important regulatory pathway in skeletal development and maintenance, the mechanisms involved are not fully understood. Among the putative protein-tyrosine kinases (ptk) and protein-tyrosine phosphatases (ptp) involved in this phenomenon there is increasing evidence that Src and low molecular weight-ptps play a central role in a range of osteoblast activities, from adhesion to differentiation. A role for Src in bone metabolism was first demonstrated in Src-deficient mice and has since been confirmed using low molecular weight Src inhibitors in animal models of osteoporosis. Several studies have shown that Src is important for cellular proliferation, adhesion and motility. In contrast, few studies have assessed the importance of the ptk/ptp balance in driving osteoblast metabolism. In this review, we summarize our current knowledge of the functional importance of the ptk/ptp balance in osteoblast metabolism, and highlight directions for future research that should improve our understanding of these critical signaling molecules.     

Down-regulation of the tumor suppressor gene C-terminal Src kinase: an early event during premalignant colonic epithelial hyperproliferation

Hyperproliferation of the premalignant epithelium is critical for colonic carcinogenesis; however the mechanisms remain largely unexplored. We report herein that prior to occurrence of neoplastic lesions in the azoxymethane-rat model of colon carcinogenesis; the tumor suppressor gene C-terminal Src kinase (Csk) was down-regulated with a concomitant increase in Src activity. Furthermore, pharmacological or genetic (RNA interference) inhibition of Csk resulted in increased proliferation in colon cancer cell lines through the mitogen-activated protein kinase dependent pathway. Thus, we demonstrate, for the first time, that Csk suppression is an important early event in colorectal cancer pathogenesis.