al-Sakkaf K A, Dobson P R, Brown B L
Cellular Signalling Section, University of Sheffield Medical School, UK.
J Mol Endocrinol. 1997 Dec;19(3):347-50. doi: 10.1677/jme.0.0190347.
Our previous studies indicated that PI3-kinase is involved in prolactin (PRL) signalling. We have now examined the involvement of the src tyrosine kinase, fyn, in PRL-induced the activation of PI3-kinase in the rat lymphoma cell line, Nb2. Cells were stimulated with increasing doses of PRL, lysed and immunoprecipitated with anti-fyn specific antibody. Then PI3-kinase activity was measured as the increase in the phosphorylation of phosphatidylinositol to phosphatidylinositol 3-phosphate separated by TLC. Our data indicated that, in PRL treated cells, co-precipitation of PI3-kinase with anti-fyn antiserum led to time and dose-dependent activation of PI3-kinase in vitro and that this activation was blocked by the addition of LY294002. However, LY294002 appeared to have no effect on fyn autophosphorylation. Furthermore, the physical association of PI3-kinase with fyn was confirmed by Western blot analysis employing the same specific antisera. These data provide evidence that PRL-induced activation of PI3-kinase may be mediated by the tyrosine phosphorylation of fyn in Nb2 cells.
我们之前的研究表明，PI3激酶参与催乳素（PRL）信号传导。我们现在研究了src酪氨酸激酶fyn在大鼠淋巴瘤细胞系Nb2中PRL诱导的PI3激酶激活过程中的作用。用递增剂量的PRL刺激细胞，裂解后用抗fyn特异性抗体进行免疫沉淀。然后通过薄层层析（TLC）测定PI3激酶活性，即磷脂酰肌醇磷酸化为磷脂酰肌醇3磷酸过程中磷酸化水平的增加。我们的数据表明，在PRL处理的细胞中，PI3激酶与抗fyn抗血清的共沉淀导致体外PI3激酶的时间和剂量依赖性激活，并且这种激活被LY294002的添加所阻断。然而，LY294002似乎对fyn自身磷酸化没有影响。此外，使用相同的特异性抗血清通过蛋白质印迹分析证实了PI3激酶与fyn的物理结合。这些数据提供了证据，表明PRL诱导的PI3激酶激活可能是由Nb2细胞中fyn的酪氨酸磷酸化介导的。
