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

我们先前证明，用TSH或cAMP生成试剂长期预处理大鼠FRTL-5甲状腺细胞可以增强IGF-I依赖的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)调节亚基(P85 PI3K)的联系，这被认为对DNA合成的增强是重要的。本研究旨在鉴定p125并阐明其在IGF-I诱导的DNA合成中的作用。MALDI-TOF MS分析表明，cAMP刺激的FRTL-5细胞中免疫沉淀的磷酸化酪氨酰p125是人XB130的大鼠同源蛋白，我们将其命名为磷脂酰肌醇3-激酶相关蛋白(PI3KAP)。CAMP处理使PI3KAP/XB130基因和蛋白水平升高，酪氨酸磷酸化以及PI3KAP/XB130与P85 PI3K的相互作用增强，导致PI3K活性增强。重要的是，PI3KAP/XB130在FRTL-5细胞中的敲除减弱了IGF-I诱导的DNA合成中cAMP依赖的增强。在cAMP处理过程中加入Src家族激酶抑制剂PP1或PP2，可阻断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，并被cAMP激活。综上所述，这些数据表明，依赖cAMP的PI3KAP/XB130的诱导及其与PI3K的结合是增强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.