Identification of an IGF-1R kinase regulatory phosphatase using the fission yeast Schizosaccharomyces pombe and a GFP tagged IGF-1R in mammalian cells
Identification of an IGF-1R kinase regulatory phosphatase using the fission yeast Schizosaccharomyces pombe and a GFP tagged IGF-1R in mammalian cells
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DOI：
10.1136/mp.55.1.46
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发表时间：
2002-02-01
期刊：
影响因子：
--
通讯作者：
O'Connor, R
中科院分区：
文献类型：
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作者：
Buckley, DA;Loughran, G;O'Connor, R
目的：研究裂殖酵母及其绿色荧光蛋白全长胰岛素样生长因子受体(IGF-1R)对I型胰岛素样生长因子受体(IGF-1R)酪氨酸激酶活性的调节。方法：在裂解酵母S中诱导表达IGF-1R的β链。用抗酪氨酸磷酸化抗体进行蛋白印迹分析，以评估Betawt的激酶活性。构建了绿色荧光蛋白(GFP)标记的胰岛素样生长因子-1受体(GFP-IGF-1R)，用于研究全长IGF-1R的酪氨酸激酶活性。研究了瞬时转基因成纤维细胞中GFP-IGF-1R对IGF-1刺激的信号传递能力。免疫荧光染色检测细胞内磷酸化酪氨酸含量，检测GFP-IGF1R的定位和酪氨酸激酶活性。结果：该蛋白在S庞贝和磷酸化的内源酵母蛋白中具有功能性酪氨酸激酶活性。在IGF-1刺激下，GFP-IGF-1R发生自磷酸化，并激活磷脂酰肌醇3-激酶和丝裂原激活的蛋白激酶通路。GFP-IGF-1R的酪氨酸磷酸化和激酶活性可以通过抗磷酸酪氨酸抗体的免疫荧光来观察。哺乳动物酪氨酸磷酸酶PTP 1B与Betawt的共表达可完全抑制酵母中的酪氨酸激酶活性，并降低GFP-IGF-1R转基因COS细胞的酪氨酸磷酸化水平。结论：裂殖酵母可用于分析IGF-1Rβ链的酪氨酸激酶活性及其酪氨酸磷酸酶的调节作用。此外，可以用标记了IGF-1R的GFP来研究IGF-1R酪氨酸激酶活性的调节。使用这两种方法，IGF-1R激酶活性都被蛋白质酪氨酸磷酸酶PTP 1B所抑制。
Aims: To study the regulation of type 1 insulin like growth factor receptor (IGF-1 R) tyrosine kinase activity using the fission yeast Schizosaccharomyces pombe and a green fluorescent protein (GFP) tagged, full length IGF-1 R.Methods: The beta chain of the IGF-1 R (betawt) was expressed under inducible conditions in the fission yeast S pombe. Western blot analysis with antiphosphotyrosine antibodies was used to assess the kinase activity of betawt. A GFP tagged IGF-1 R (GFP-IGF-1 R) was constructed to study the tyrosine kinase activity of the full length IGF-1 R. The signalling capabilities of GFP-IGF-1 R in response to IGF-1 stimulation were investigated in transiently transfected fibroblasts. Immunofluorescent staining for cellular phosphotyrosine content was used to assess the localisation and tyrosine kinase activity of GFP-IGF-1 R.Results: The betawt protein displayed functional tyrosine kinase activity in S pombe and phosphorylated endogenous yeast proteins. In response to IGF-1 stimulation, the GFP-IGF-1 R became autophosphorylated and also activated the phosphatidylinositol 3-kinase and mitogen activated protein kinase pathways. Tyrosine phosphorylation and kinase activity of the GFP-IGF-1 R could be visualised by immunofluorescence with antiphosphotyrosine antibodies. Coexpression of a mammalian tyrosine phosphatase PTP 1 B with betawt completely inhibited this tyrosine kinase activity in yeast and also reduced the tyrosine phosphorylation in COS cells transfected with the GFP-IGF-1 R.Conclusions: Schizosaccharomyces pombe can be used to analyse the tyrosine kinase activity of the IGF-1 R beta chain and its regulation by tyrosine phosphatases. In addition, the regulation of IGF-1 R tyrosine kinase activity can be studied using a GFP tagged IGF-1 R. Using both of these methods, IGF-1 R kinase activity was shown to be inhibited by the protein tyrosine phosphatase, PTP 1 B.