Molecular mechanism underlying cAMP-induced amplification of IGF mitogenic activity through PI3-kinase binding protein, PI3KAP/XB130.

我们之前证明，用TSH或camp生成试剂长期预处理大鼠FRTL-5甲状腺细胞可增强igf - i依赖性DNA合成。在这种情况下，cAMP处理增加了125 kDa蛋白（p125）的酪氨酸磷酸化及其与磷脂酰肌醇3-激酶（p85 PI3K）的p85调节亚基的关联，这被认为是增强DNA合成的重要因素。本研究旨在鉴定p125并阐明其在IGF-I诱导的DNA合成增强中的作用。在camp刺激的FRTL-5细胞中免疫沉淀的磷酸酪氨酸p125，通过MALDI-TOF质谱分析显示，是人类XB130的大鼠同源物，我们将其命名为磷脂酰肌醇3激酶相关蛋白（PI3KAP）。cAMP处理提高了PI3KAP/XB130 mRNA和蛋白水平，酪氨酸磷酸化和PI3KAP/XB130与p85 PI3K的相互作用，导致PI3K活性增加。重要的是，FRTL-5细胞中PI3KAP/XB130的敲低减弱了igf -i诱导的camp依赖性DNA合成增强。在cAMP治疗期间，添加Src家族激酶抑制剂PP1或PP2，可消除PI3KAP/XB130的酪氨酸磷酸化及其与p85 PI3K的相互作用。此外，c-Src与PI3KAP/XB130相关，并在cAMP作用下被激活。总之，这些数据表明，camp依赖性诱导PI3KAP/XB130及其与PI3K的关联是增强IGF有丝分裂活性所必需的。

We previously demonstrated that long-term pretreatment of rat FRTL-5 thyroid cells with TSH or cAMP-generating reagents potentiated IGF-I-dependent DNA synthesis. Under this condition, cAMP treatment increased tyrosine phosphorylation of a 125 kDa protein(p125) and its association with a p85 regulatory subunit of phosphatidylinositol 3-kinase(p85 PI3K), which were suggested to be important for potentiation of DNA synthesis. This study was undertaken to identify p125 and to elucidate its roles in potentiation of DNA synthesis induced by IGF-I. Immunoprecipitated phosphotyrosyl p125 in cAMP-stimulated FRTL-5 cells, was shown by MALDI-TOF MS analysis, to be a rat orthologue of human XB130, which we named phosphatidylinositol 3-kinase-associated protein(PI3KAP). cAMP treatment elevated PI3KAP/XB130 mRNA and protein levels as well as tyrosine phosphorylation and PI3KAP/XB130 interaction with p85 PI3K, leading to increased PI3K activities. Importantly, PI3KAP/XB130 knockdown in FRTL-5 cells attenuated cAMP-dependent potentiation of IGF-I-induced DNA synthesis. Addition of Src family kinase inhibitors, PP1 or PP2, during cAMP treatment abolished tyrosine phosphorylation of PI3KAP/XB130 and its interaction with p85 PI3K. In addition, c-Src was associated with PI3KAP/XB130 and was activated in response to cAMP. Together, these data indicate that cAMP-dependent induction of PI3KAP/XB130 and its association with PI3K are required for enhancement of IGF mitogenic activities.