纤维连接蛋白对人结肠癌细胞侵袭力的影响

目的研究纤维连接蛋白（fibronectin，FN）对人结肠癌细胞侵袭力的影响，并探讨其中的信号传导机制。方法以递增浓度的FN刺激结肠癌细胞株Colo320，以免疫沉淀和蛋白质印迹法检测结肠癌细胞内黏着斑激酶（focal adhesion kinase，FAK）第397位酪氨酸（tyr-397）磷酸化的状况．以改良Boyden小室法检测相应的细胞侵袭力变化。设计反义寡核苷酸阻断FAK蛋白质表达，再次观察接受FN刺激后．结肠癌细胞内FAK tyr-397磷酸化状况及细胞侵袭力的改变。结果FN能够促进Colo320 FAK tyr-397磷酸化，在一定范围内具有剂量依赖性。FN浓度达10nmol／L时．此作用最显著，继续增加FN浓度．FAK tyr-397磷酸化不再呈现继续增强趋势．当浓度达到100nmol／L时，磷酸化程度反而有所下降；FN可以增强细胞侵袭力，同样在一定范围内具有剂量依赖性；反义寡核苷酸干预后．结肠癌细胞内FAK tyr-397磷酸化及细胞侵袭力均有显著降低（P〈0.01）。结论FN可以有效增强结肠癌细胞的侵袭力，其作用是通过FN—FAK信号通路实现的，FAK的活性形式是其酪氨酸位点的磷酸化．阻断FAK的表达町以减弱FN促进细胞侵袭的作用。     

胃泌素对结肠癌细胞中粘着斑激酶通路下游E-钙粘蛋白/β-连环蛋白复合物的影响

目的探讨胃泌素对结肠癌细胞CoLo320WT中粘着斑激酶（FAK）通路下游E-钙粘蛋白／β-连环蛋白（E-cadherin／β-catenin）复合物分布的影响；方法脂质体转染表达胃泌索受体CCK-2R的pCR3．1／OR质粒于结肠癌细胞CoLo320中。G418筛选出稳定表达CCK-2R的阳性克隆，RT-PCR鉴定，转染成功命为CoLo320WT。应用10^-8mmol／L 胃泌素（G17）以时间梯度（0h、1h、6h、12h、24h、48h）干预CoLo320WT细胞，同时应用10^-6mmol／L胃泌素受体拮抗剂L365，260干预CoLo320WT细胞30min，再予10^-8mmol／L胃泌素干预。采用免疫印迹法检测磷酸化的FAK Tyr397和总FAK的表达。采用免疫共沉淀和免疫印迹法检测CoLo320WT中TX-100溶解和未溶部分中的E-钙粘蛋白和β-连环蛋白的表达。用免疫细胞化学法观察E-钙粘蛋白和β-连环蛋白的在胞膜、胞质和胞核的分布。结果随着胃泌素干预时间的延长，细胞中磷酸化的FAK Tyr397的表达量呈增加趋势，12h达最大值。胃泌素受体拮抗剂L365，260阻断后磷酸化的FAK Tyr397表达减少。而胃泌素对总FAK没有明显影响。TX-100可溶性部分中E-钙粘蛋白和酽连环蛋白的量在胃泌素干预后表达减少，拮抗剂L365，260阻断后又增加。而TX-100不溶解部分中表达却相反。免疫细胞化学观察到在胃泌素干预下CoLo320WT细胞中E-钙粘蛋白和β-连环蛋白的分布发生胞质和胞核转移。结论胃泌素与其受体CCK-2受体结合，磷酸化的FAK Tyr397、激活FAK通路进而影响结肠癌细胞中E-钙粘蛋白和β-连环蛋白的分布，促进结肠癌细胞侵袭和转移。     

hFRNK基因对胃泌素诱导的人结肠癌细胞侵袭力的影响

目的 观察腺病毒介导hFRNK基因对胃泌素所诱导的人结肠癌Colo320WT细胞侵袭力的影响.方法 试验分为胃泌素组、hFRNK组和对照组,胃泌素组用100 μmol/L胃泌素诱导结肠癌Col0320WT细胞12 h;hFRNK组,首先用脂质体瞬时转染腺病毒受体pCR3.1-CAR于Col0320WT细胞48 h,然后用100 μmol/L胃泌素干预结肠癌Colo320WT细胞12 h,再用重组腺病毒(pAdhFRNK)感染细胞;对照组为未经处理的Colo320WT细胞.用免疫印迹检测hFRNK基因黏着斑激酶(FAK)397位酪氨酸(FAKTyr397)的磷酸化表达,激光共聚焦显微镜观察FAKTyr397在细胞板状伪足的表达情况,免疫共沉淀检测hFRNK基因对四联信号复合物FAK-Src-Doek180一p130Cas形成的影响,Pull-down法检测hFRNK对Rac蛋白活性的影响.结果 胃泌素诱导后,磷酸化FAKTyrr397明显增强;与胃泌素组相比,hFRNK组中FAKTyr397表达下降,FAKTyr397定位到细胞板状伪足的量明显减少,FAK、Src、Dockl80和p130Cas 四联信号复合物没有形成,Rac的活性降低.结论 hFRNK基因可阻断胃泌素引起的FAK的磷酸化,阻断FAKTyr397摹积到细胞的板状伪足,阻止四联信号复合物FAK-Src-Dock180-p130Cas的形成以及Rac的活化,为hFRNK基因防治肿瘤的侵袭和转移提供理论依据.     

黏着斑激酶对血小板源性生长因子刺激的血管平滑肌细胞迁移和黏附的调控

目的探讨黏着斑激酶(FAK)信号分子在雷帕霉素抑制血小板源性生长因子(PDGF)诱导血管平滑肌细胞(VSMC)迁移、黏附中的调控作用。方法将培养的大鼠VSMC分为对照组、PDGF组、雷帕霉素+PDGF组(雷帕霉素组)和FAK反义寡核苷酸+PDGF组(FAK组)。用PDGF诱导VSMC的迁移和黏附,计数贴壁细胞;采用Boyden检测细胞迁移;采用RT-PCR、Western blot、免疫沉淀方法分别检测FAK基因、蛋白及蛋白磷酸化表达量。将FAK反义寡核苷酸经脂质体转染VSMC,观察FAK mRNA及蛋白磷酸化、细胞迁移和黏附的变化。结果与对照组比较,PDGF组明显诱导细胞迁移和黏附,上调FAK mRNA的表达,提高FAK蛋白和磷酸化FAK表达,差异有统计学意义(P0.05),与PDGF组比较,FAK组和雷帕霉素组细胞迁移、黏附能力减弱,FAK mRNA、FAK蛋白和磷酸化FAK表达水平明显降低,差异有统计学意义(P0.05)。结论 PDGF诱导细胞迁移和黏附可能是FAK介导的,雷帕霉素可能是通过抑制FAK蛋白和磷酸化FAK来抑制VSMC的迁移和黏附。     

磷酸化黏着斑激酶在结肠癌中表达及意义

目的:观察磷酸化黏着斑激酶(phosphorylatedfocaladhesionkinase,phospho-FAK)在结肠癌组织和对应癌旁组织中的表达,探讨其在结肠癌发病的可能机制.方法:采用Westernbloting方法检测20例新鲜结肠癌及相对应的癌旁组织FAK表达水平,并在调平每对组织FAK的含量后再进行FAKTyr397磷酸化蛋白的检测.结果:20例结肠癌组织FAK阳性表达率为95%,对应癌旁组织FAK阳性表达率为60%(c2=5.16,P<0.05);癌组织表达平均值为0.482±0.150,癌旁表达平均值为0.269±0.015(t=6.39,P<0.01).20例结肠癌组织18例有FAKTyr397磷酸化蛋白表达,表达率为90%,而对应癌旁组织仅有4例有FAKTyr397磷酸化蛋白表达,表达率为20%(c2=17.1,P<0.01);癌组织表达平均值为0.385±0.021,癌旁表达平均值为0.110±0.005(t=54.23,P<0.01).结论:FAK特别是FAKTyr397磷酸化蛋白的表达水平增加在结肠癌的发生、发展中可能起重要作用.     

腺病毒介导人FRNK基因对结肠癌细胞Colo320WT中p190RhoGAP磷酸化和RhoA活性的影响

目的利用腺病毒载体表达人FRNK基因,体外观察其对胃泌素干预下的结肠癌细胞Colo320WT中p190RhoGAP磷酸化和RhoA活性的影响。方法2005年10月至2006年9月,武汉大学人民医院消化内科在中国科学院,武汉病毒所病毒学国家重点实验室,利用AdEasyTM系统在大肠埃希菌内同源重组构建表达人FRNK基因的腺病毒载体pAdhFRNK。脂质体转染pCR3.1-GR质粒于结肠癌细胞Colo320中,G418抗生素筛选出稳定表达胆囊收缩素-2受体/胃泌素受体(CCK-2R)的阳性克隆,逆转录-聚合酶链反应(RT-PCR)鉴定。用10~8mol/L胃泌素干预Colo320WT细胞12 h和pAdhFRNK体外感染Colo320WT细胞2 d后,再用10~8 mol/L的胃泌素干预细胞12 h,然后用免疫沉淀方法检测磷酸化的p190RhoGAP的表达,pull-down测定RhoA的活性。结果在胃泌素干预12 h后的磷酸化的p190RhoGAP的表达量明显增加,RhoA活性降低,而用pAdhFRNK感染后胃泌素干预的细胞中磷酸化p190RhoGAP表达又降低,RhoA活性却增加。结论hFRNK基因可明显阻断外源性胃泌素引起Colo320WT细胞中p190RhoGAP磷酸化的表达和RhoA的活性。     

不同转移潜能人肝癌细胞系酪氨酸磷酸化蛋白质差异分析

目的研究不同转移潜能人肝癌细胞系中酪氨酸磷酸化蛋白质表达的差异并筛查与肝癌转移相关的酪氨酸磷酸化蛋白质分子。方法应用双电泳(2-D E)、免疫印迹法及基质辅助激光解析电离飞行时间质谱分析,对三种不同转移潜能人肝癌细胞系Hep 3B、MHCC97L和MHCC97H进行酪氨酸磷酸化蛋白质组分析。结果对照2-DE胶和免疫印迹发光胶片,Hep3B检测到10个点,MHCC 9 7L检测到19个点, MHCC97H检测到17个点。经质谱鉴定,得到膜连蛋白Ⅰ等19个差异点。结论细胞内酪氨酸磷酸化蛋白的表达差异与肝癌的侵袭转移有关。     

胃泌素促FAK-Src-ERK1/2通路的活化对大肠癌细胞运动和侵袭的影响

大肠癌的侵袭和转移是影响疗效、预后和导致死亡的重要因素。胃泌素通过与肿瘤细胞表面的胃泌素受体（CCK-BR）结合而刺激肿瘤细胞的生长和增殖。我们前期研究证明胃泌素通过胃液素-胃液素受体．黏着斑激酶（FAK）信号传导通路引起结肠癌细胞侵袭力的增加。本研究观察了胃泌素及其受体对大肠癌细胞FAK-Src-细胞外信号调节蛋白激酶（ERK1／2）通路的影响以进一步了解胃泌素及其受体对大肠癌细胞运动、侵袭和转移的作用机制。     

黏着斑激酶在结肠癌组织中的表达及意义

目的探讨黏着斑激酶（FAK）在结肠癌发生、发展中的作用。方法采用RT-PCR法检测30例新鲜结肠癌及与之相对应的癌旁组织的FAK mRNA表达；同时用Western印迹法检测20例新鲜结肠癌及相对应的癌旁组织FAK蛋白表达水平，调平每对组织FAK蛋白含量后再行FAK Tyr397磷酸化位点蛋白检测。结果30例结肠癌组织FAK mRNA阳性率为90．0％，其表达值为0．745±0．530，对应癌旁组织阳性率为43．3％，其表达值为0．241±0．131（P〈0．01）；20例结肠癌组织FAK蛋白阳性率为95．0％，表达值为0．482±0．150；对应癌旁组织阳性率为60．0％，表达值为0．269±0．015；P均〈0．01。20例结肠癌组织中FAK Tyr397磷酸化蛋白阳性率为90．0％（18／20），表达值为0．385±0．021；而对应癌旁组织阳性率为20％（4／20），表达值为0．110±0．005，P均〈0．01。结论FAK特别是FAK Tyr397磷酸化蛋白表达增加在结肠癌的发生、发展中可能起重要作用。     

IL-1β和TNF-α诱导RA成纤维样滑膜细胞ICAM-1和VCAM-1表达调控的机制研究

目的探讨酪氨酸激酶在白细胞介素(IL)-1β和肿瘤坏死因子(TNF)-α诱导类风湿关节炎(RA)成纤维样滑膜细胞(FLS)细胞间黏附分子(ICAM-1)和血管细胞黏附分子(VCAM-1)表达中的作用。方法原代培养RA成纤维样滑膜细胞，用Western blot方法检测IL-1β和TNF-α短时间内引起RA FLS蛋白质酪氨酸磷酸化状态改变，并应用genistein，蛋白质酪氨酸激酶(PTK)抑制剂观察对ICAM-1和VCAM-1表达影响。结果IL-1β和TNF—α可以瞬时引起RA FLS蛋白质酪氨酸磷酸化程度增加：IL-1β和TNF-α在1—100U／ml之间呈浓度依赖性促进ICAM-1和VCAM-1的表达；genistein显著抑制两种炎性因子刺激下的VCAM-1表达，而对。ICAM-1的表达仅起中度抑制作用。结论IL-1β和TNF-α在RAFLS信号转导中，可以瞬时导致蛋白质酪氨酸磷酸化程度增加．在炎性因子诱导ICAM-1和VCAM-1的表达调控中蛋白酪氨酸激酶作用不同。     

Effects of gastrin 17 on β-catenin/Tcf-4 pathway in Colo320WT colon cancer cells

AIM: To explore the effect of gastrin 17 (G17) on β-catenin/T cell factor-4 (Tcf-4) signaling in colonic cancer cell line Colo320WT.METHODS: The pCR3.1/GR plasmid, which expresses gastrin receptor, cholecystokinin-2 receptor (CCK-2R),was transfected into a colonic cancer cell line Colo320 by Lipofectamine TM2000 and the stably expressing CCK-2R clones were screened by G418. The expression levels of gastrin receptor in the Colo320 and the transfected Colo320WT cell line were assayed by RTPCR. Colo320WT cells were treated with G17 in a timedependent manner (0, 1, 6, 12, 24 and 48 h), then with L365,260 (Gastrin17 receptor blocker) for 30 min, and with G17 again for 12 h or L365,260 for 12 h. Expression levels of β-catenin in a TX-100 soluble fraction and TX-100 insoluble fraction of Colo320WT cells treated with G17 were detected by co-immuniprecipation and Western blot. Immunocytochemistry was used to examine the distribution of β-catenin in CoLoWT320 cells. Expression levels of c-myc and cyclin D1 in Colo320WT cells treated with G17 were assayed by Western blot.RESULTS: Expression levels of β-catenin in the TX-100 solution fraction decreased apparently in a timedependent fashion and reached the highest level after G17 treatment for 12 h, while expression levels of β-catenin in the TX-100 insoluble fraction were just on the contrary. Immunocytochemistry showed that β-catenin was translocated from the cell membranes into the cytoplasm and nucleus under G17 treatment.Expression levels of c-myc and cyclin D1 in the G17-treated Colo320WT cells were markedly higher compared to the untreated Colo320WT cells. In addition, the aforementioned G17-stimulated responses were blocked by L365,260.CONCLUSION: Gastrin17 activates β-catenin/Tcf-4 signaling in Colo320WT cells, thereby leading to overexpression of c-myc and cyclin D1.     

Effects of gastrin 17 on β-catenin/Tcf-4 pathway in Colo320WT colon cancer cells

AIM: To explore the effect of gastrin 17 (G17) on beta-catenin/T cell factor-4 (Tcf-4) signaling in colonic cancer cell line Colo320WT. METHODS: The pCR3.1/GR plasmid, which expresses gastrin receptor, cholecystokinin-2 receptor (CCK-2R), was transfected into a colonic cancer cell line Colo320 by Lipofectamine (TM)2000 and the stably expressing CCK-2R clones were screened by G418. The expression levels of gastrin receptor in the Colo320 and the transfected Colo320WT cell line were assayed by RT-PCR. Colo320WT cells were treated with G17 in a time-dependent manner (0, 1, 6, 12, 24 and 48 h), then with L365,260 (Gastrin(17) receptor blocker) for 30 min, and with G17 again for 12 h or L365,260 for 12 h. Expression levels of beta-catenin in a TX-100 soluble fraction and TX-100 insoluble fraction of Colo320WT cells treated with G17 were detected by co-immuniprecipation and Western blot. Immunocytochemistry was used to examine the distribution of beta-catenin in CoLoWT320 cells. Expression levels of c-myc and cyclin D1 in Colo320WT cells treated with G17 were assayed by Western blot. RESULTS: Expression levels of beta-catenin in the TX-100 solution fraction decreased apparently in a time-dependent fashion and reached the highest level after G17 treatment for 12 h, while expression levels of beta-catenin in the TX-100 insoluble fraction were just on the contrary. Immunocytochemistry showed that beta-catenin was translocated from the cell membranes into the cytoplasm and nucleus under G17 treatment. Expression levels of c-myc and cyclin D1 in the G17-treated Colo320WT cells were markedly higher compared to the untreated Colo320WT cells. In addition, the aforementioned G17-stimulated responses were blocked by L365,260. CONCLUSION: Gastrin17 activates beta-catenin/Tcf-4 signaling in Colo320WT cells, thereby leading to over-expression of c-myc and cyclin D1.     

Gastrin suppresses growth of CCK2 receptor expressing colon cancer cells by inducing apoptosis in vitro and in vivo

BACKGROUND & AIMS: The role of amidated gastrin17 (G17) and the gastrin/CCKB/CCK2 receptor in colorectal carcinogenesis is still a controversial issue. Here, we investigated the effect of G17 on proliferation and apoptosis of CCK2 receptor-expressing human colon cancer cell lines in vitro and in vivo. METHODS: Proliferation was determined by cell counting and cell cycle analysis. Apoptosis was analyzed by annexin V staining, TUNEL staining, caspase-3/7 assay, and JC1 (delta psi) assay. Signal-transduction pathways were analyzed by Western blotting and gel-shift and luciferase assays. An in vivo tumor model with subcutaneously inoculated colon cancer cells in SCID mice was used, and systemic hypergastrinemia was induced by omeprazole. RESULTS: In Colo320 cells stably transfected with the wild-type CCK2 receptor (Colo320wt) or in Lovo cells endogenously expressing CCK2 receptors, G17 treatment inhibited proliferation along with a G2/M cell cycle arrest. Furthermore, the administration of G17 significantly augmented apoptosis of CCK2 receptor-expressing cells. In contrast, G17 had no effect on proliferation and apoptosis in Colo320 cells stably transfected with a tumor-derived CCK2 receptor mutant (Colo320mut) or in cells lacking CCK2 receptor expression. Systemic hypergastrinemia in severe combined immunodeficiency (SCID) mice suppressed the growth of Colo320wt tumors accompanied by enhanced apoptosis as compared with untreated tumors. In contrast, omeprazole did not affect Colo320mut tumors reflecting a loss-of-function state of the CCK2(mut) receptor. This is supported by the observation that, in Colo320wt cells, but not in Colo320mut cells, G17 treatment induced the MAPK/ERK/AP-1 pathway and inhibited the activity of NF-kappaB. CONCLUSIONS: G17 exerts an antiproliferative and proapoptotic effect on human colon cancer cells expressing the wild-type CCK2 receptor. This supports the view that amidated gastrin prevents rather than promotes colorectal carcinogenesis.     

PTEN在HepG2细胞中诱导凋亡发生及上调p53表达

目的 研究肿瘤抑制基因PTEN对HepG2细胞凋亡及p53蛋白表达的影响,并探讨相关机制。方法 将携带有野生型PTEN基因及突变型G129E-PTEN,C124A-PTEN基因的真核表达载体转染HepG2细胞,利用western印迹杂交检测PTEN表达、蛋白激酶B(PKB/Akt)和焦点粘附激酶(FAK)的磷酸化状态,以及野生型p53蛋白表达水平的变化,并应用流式细胞仪,激光共聚焦技术检测细胞周期及细胞凋亡情况。结果 与对照细胞相比,转染野生型PTEN和G129E-PTEN的HepG2细胞中磷酸化FAK(-65％,-65％)与磷酸化Akt(-93％,-35％)表达均存在不同程度的下调,而细胞凋亡率分别增加至19.8％±1.2％和9.2％±0.6％,并且p53蛋白表达上调( 120%,, 50％);然而转染C124A-PTEN的细胞中各项检测指标均无明显变化。 结论 PTEN依赖其蛋白磷酸酶活性抑制FAK的磷酸化;并主要通过脂质磷酸酶活性抑制Akt的磷酸化,并诱导HepG2细胞凋亡和p53蛋白表达上调。     

Bradykinin enhances insulin receptor tyrosine kinase in 32D cells reconstituted with bradykinin and insulin signaling pathways

We have previously shown that bradykinin potentiated insulin-induced glucose uptake through GLUT4 translocation in canine adipocytes and skeletal muscles. The aim of this study was to determine the molecular mechanism of bradykinin enhancement of the insulin signal. For this purpose, 32D cells, which express a limited number of insulin receptors and lack endogenous bradykinin B2 receptor (BK2R) or insulin receptor substrate (IRS)-1 were transfected with BK2R cDNA and/or insulin receptor cDNA and/or IRS-1 cDNA, and analyzed. In 32D cells that expressed BK2R and insulin receptor (32D-BKR/IR), bradykinin alone had no effect on the phosphorylation of the insulin receptor, but it enhanced insulin-stimulated tyrosine phosphorylation of the insulin receptor. In 32D cells that expressed BK2R, insulin receptor and IRS-1 (32D-BKR/IR/IRS1), bradykinin also enhanced insulin-stimulated tyrosine phosphorylation of the insulin receptor and IRS-1. An increase in insulin-stimulated phosphorylation of IRS-1 by treatment with bradykinin in 32D-BKR/IR/IRS1 cell was associated with increased binding of 85 kD subunit of phosphatidylinositol 3 (PI 3)-kinase and increased IRS-1 associated PI 3-kinase activity. These effects of bradykinin were not observed in 32D cells which lack the expression of BK2R (32D-IR/IRS1) or insulin receptor (32D-BKR/IRS1). Furthermore, tyrosine phosphatase activity against insulin receptor beta-subunit in plasma membrane fraction of 32D-BKR/IR cells was significantly reduced by bradykinin, suggesting that the effect of bradykinin was in part mediated by inhibition of protein tyrosine phosphatase(s). Our results clearly demonstrated that bradykinin enhanced insulin-stimulated tyrosine kinase activity of the insulin receptor and downstream insulin signal cascade through the BK2R mediated signal pathway.     

Characterization of RAFTK, a novel focal adhesion kinase, and its integrin-dependent phosphorylation and activation in megakaryocytes

We have recently isolated a cDNA encoding a novel human intracellular tyrosine kinase, termed RAFTK (for a related adhesion focal tyrosine kinase). The RAFTK cDNA, which encodes a polypeptide of 1,009 amino acids, shares 65% homology to the focal adhesion kinase (FAK), including several consensus motifs. In this report, we describe the biochemical characterization and functional analysis of the RAFTK protein. Coexpression of RAFTK and FAK proteins in megakaryocytic cells and blood platelets was observed. Using a specific antibody to RAFTK and the monoclonal antibody 2A7 to FAK, FAK and RAFTK could be distinguished antigenically. RAFTK had intrinsic tyrosine kinase and autokinase activities. It was phosphorylated on tyrosine in growing cultures of COS cells transfected with the pCDNAIII/flag-RAFTK expression vector containing the RAFTK cDNA ligated with the 8 amino acid flag peptide sequence. Similar to FAK, dephosphorylation of RAFTK was observed when adherent transfected COS cells were detached. Phosphorylation was regained upon replating of these cells on the fibronectincoated dishes. Analysis of tyrosine-phosphorylated RAFTK from adherent transfected COS cells showed that the Src homology 2 (SH2) domains of the Src and Fyn protein kinases as well as the Grb2 adaptor protein were able to specifically associate with RAFTK. Tyrosine phosphorylation of endogenous RAFTK was observed upon fibronectin-induced activation of human megakaryocytic cells. Furthermore, colocalization of RAFTK protein with vinculin, a focal adhesion protein, was observed by confocal microscopy in focal adhesion- like structures in adherent CMK cells and in transfected pCDNAIII/flag- RAFTK COS cells upon fibronectin activation. These data suggest that RAFTK is a novel member of the FAK family, that it localizes to focal adhesion-like structures in CMK megakaryocytic cells, that it participates in integrinmediated signaling pathways in megakaryocytes, and that it is able to associate with the tyrosine kinases Src and Fyn as well as the adaptor protein Grb2 via SH2-phosphotyrosine interactions.     

Characterization of gastrin-cholecystokinin 2 receptor interaction in relation to c-fos induction

The interaction of gastrin with the cholecystokinin 2 (CCK2)/gastrin receptor has been studied extensively in relation to gastric acid secretion. However, not much is known about the contribution of individual amino acids of gastrin interacting with the CCK2 receptor, when gastrin is acting as a tumor growth factor. The purpose of the present study was to determine the significance of each individual amino acid residue of human gastrin-17 with respect to CCK2 receptor-mediated cell proliferation. Activation of this receptor was assessed using an in vitro bioassay based on gastrin-induced expression of a c-fos-luciferase reporter, transfected in AR42JB13 and Colo 320 cells, a rat pancreatic and human colorectal cell line respectively. Gastrin-17 dose dependently increased c-fos induction in both cancer cell lines. L365,260, a known CCK2 receptor antagonist, completely blocked the gastrin signal, demonstrating the specificity of this assay. We demonstrated for the first time that four carboxy-terminal amino acids of gastrin-17 are essential for activation of the CCK2 receptor with respect to c-fos induction. Also other residues of gastrin-17, notably glycine-2 for the rat CCK2 receptor and glutamic acid 8-10 and tyrosine-12 for the human receptor, were found to be important, although to a lesser extent. Alanine-substitution variants of each of the four carboxy-terminal amino acids of gastrin-17 showed strongly reduced receptor activation but did not act as competitive inhibitors of gastrin-17. Identification of the essential role of the carboxy-terminal tetrapeptide of gastrin-17 in CCK2 receptor-mediated c-fos induction indicates that gastrin inhibitory therapeutic strategies should mainly be targeted toward this region of gastrin.     

Shp2 negatively regulates growth in cardiomyocytes by controlling focal adhesion kinase/Src and mTOR pathways

The aim of this study was to investigate whether Shp2 (Src homology region 2, phosphatase 2) controls focal adhesion kinase (FAK) activity and its trophic actions in cardiomyocytes. We show that low phosphorylation levels of FAK in nonstretched neonatal rat ventricular myocytes (NRVMs) coincided with a relatively high basal association of FAK with Shp2 and Shp2 phosphatase activity. Cyclic stretch (15% above initial length) enhanced FAK phosphorylation at Tyr397 and reduced FAK/Shp2 association and phosphatase activity in anti-Shp2 precipitates. Recombinant Shp2 C-terminal protein tyrosine phosphatase domain (Shp2-PTP) interacted with nonphosphorylated recombinant FAK and dephosphorylated FAK immunoprecipitated from NRVMs. Depletion of Shp2 by specific small interfering RNA increased the phosphorylation of FAK Tyr397, Src Tyr418, AKT Ser473, TSC2 Thr1462, and S6 kinase Thr389 and induced hypertrophy of nonstretched NRVMs. Inhibition of FAK/Src activity by PP2 {4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine} abolished the phosphorylation of AKT, TSC2, and S6 kinase, as well as the hypertrophy of NRVMs induced by Shp2 depletion. Inhibition of mTOR (mammalian target of rapamycin) with rapamycin blunted the hypertrophy in NRVMs depleted of Shp2. NRVMs treated with PP2 or depleted of FAK by specific small interfering RNA were defective in FAK, Src, extracellular signal-regulated kinase, AKT, TSC2, and S6 kinase phosphorylation, as well as in the hypertrophic response to prolonged stretch. The stretch-induced hypertrophy of NRVMs was also prevented by rapamycin. These findings demonstrate that basal Shp2 tyrosine phosphatase activity controls the size of cardiomyocytes by downregulating a pathway that involves FAK/Src and mTOR signaling pathways.