Valverde A M, Teruel T, Lorenzo M, Benito M
Departamento de Bioquímica y Biología Molecular II, Facultad de Farmacia, Universidad Complutense, Madrid, Spain.
Endocrinology. 1996 Sep;137(9):3832-41. doi: 10.1210/endo.137.9.8756554.
In the present study we have examined the signaling cascades involved in insulin-like growth factor I (IGF-I)-induced mitogenesis in fetal rat brown adipocyte primary cultures, a model that constitutively expresses a high number of IGF-I receptors, where IGF-I is a complete mitogen at physiological concentrations. IGF-I rapidly stimulated beta-chain IGF-I receptor autophosphorylation, which peaked at a physiological/mitogenic concentration (1.4 nM) and also stimulated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1). Tyrosine-phosphorylated IRS-1 bound and subsequently activated phosphatidylinositol 3-kinase by 3.5-fold, whereas the tyrosine-phosphorylated IGF-I receptor was not directly associated with the p85 subunit of the phosphatidylinositol 3-kinase. Moreover, mitogenic concentrations of IGF-I enhanced glucose transport by 2.5-fold. In addition, tyrosine phosphorylation of the 46- and 52-kDa SHC proteins was high in the basal state and doubled after IGF-I treatment, whereas IGF-I enhanced by 4-fold tyrosine phosphorylation of the 66-kDa SHC band. Furthermore, a 2-fold increase in the Ras. GTP active form was induced upon IGF-I stimulation. Downstream from Ras, IGF-I increased both Raf kinase and protein kinase C (PKC) zeta activities by 3.5-fold. (Bu)2cAMP, an inhibitor of IGF-I-induced mitogenesis in fetal brown adipocyte primary cultures, did not block the very early steps of the IGF-I-induced mitogenic cascade, such as IGF-I receptor autophosphorylation, IRS-1 or SHC tyrosine phosphorylation, and Ras activation to its GTP active form. However, (Bu)2cAMP disrupted IGF-I-Raf and IGF-I-PKC zeta signaling pathways by preventing IGF-I-induced Raf-1 kinase and PKC zeta enzymatic activities, respectively. Our results show the first characterization in situ of an IGF-I mitogenic signaling cascade that downstream Ras diverges to the nucleus through two different serine/threonine kinases (Raf-1 kinase and PKC zeta) in mammalian fetal primary cells under physiological conditions. Both kinases represent a point of regulation primarily described for IGF-I-induced, cAMP-inhibited mitogenic pathways.
在本研究中，我们检测了胎鼠棕色脂肪细胞原代培养物中胰岛素样生长因子I（IGF-I）诱导有丝分裂过程中涉及的信号级联反应，该模型组成性表达大量IGF-I受体，在该模型中IGF-I在生理浓度下是一种完全的促有丝分裂原。IGF-I迅速刺激β链IGF-I受体自身磷酸化，其在生理/促有丝分裂浓度（1.4 nM）时达到峰值，并且还刺激胰岛素受体底物-1（IRS-1）的酪氨酸磷酸化。酪氨酸磷酸化的IRS-1结合并随后使磷脂酰肌醇3-激酶激活3.5倍，而酪氨酸磷酸化的IGF-I受体并不直接与磷脂酰肌醇3-激酶的p85亚基相关联。此外，IGF-I的促有丝分裂浓度使葡萄糖转运增加2.5倍。另外，46 kDa和52 kDa的SHC蛋白的酪氨酸磷酸化在基础状态下较高，在IGF-I处理后增加一倍，而IGF-I使66 kDa的SHC条带的酪氨酸磷酸化增加4倍。此外，IGF-I刺激后Ras的GTP活性形式增加了2倍。在Ras的下游，IGF-I使Raf激酶和蛋白激酶C（PKC）ζ活性均增加3.5倍。（Bu）2cAMP是胎鼠棕色脂肪细胞原代培养物中IGF-I诱导有丝分裂的抑制剂，它并不阻断IGF-I诱导的有丝分裂级联反应的非常早期步骤，如IGF-I受体自身磷酸化、IRS-1或SHC酪氨酸磷酸化以及Ras激活为其GTP活性形式。然而，（Bu）2cAMP分别通过阻止IGF-I诱导的Raf-1激酶和PKCζ酶活性来破坏IGF-I-Raf和IGF-I-PKCζ信号通路。我们的结果首次在原位表征了IGF-I促有丝分裂信号级联反应，在生理条件下，在哺乳动物胎鼠原代细胞中，该级联反应在Ras下游通过两种不同的丝氨酸/苏氨酸激酶（Raf-1激酶和PKCζ）发散至细胞核。这两种激酶代表了主要针对IGF-I诱导的、cAMP抑制的有丝分裂途径所描述的一个调控点。
