Ugi Satoshi, Imamura Takeshi, Maegawa Hiroshi, Egawa Katsuya, Yoshizaki Takeshi, Shi Kun, Obata Toshiyuki, Ebina Yousuke, Kashiwagi Atsunori, Olefsky Jerrold M
Division of Endocrinology and Metabolism, Department of Medicine, Shiga University of Medical Science, Otsu, Japan.
Mol Cell Biol. 2004 Oct;24(19):8778-89. doi: 10.1128/MCB.24.19.8778-8789.2004.
Protein phosphatase 2A (PP2A) is a multimeric serine/threonine phosphatase which has multiple functions, including inhibition of the mitogen-activated protein (MAP) kinase pathway. Simian virus 40 small t antigen specifically inhibits PP2A function by binding to the PP2A regulatory subunit, interfering with the ability of PP2A to associate with its cellular substrates. We have reported that the expression of small t antigen inhibits PP2A association with Shc, leading to augmentation of insulin and epidermal growth factor-induced Shc phosphorylation with enhanced activation of the Ras/MAP kinase pathway. However, the potential involvement of PP2A in insulin's metabolic signaling pathway is presently unknown. To assess this, we overexpressed small t antigen in 3T3-L1 adipocytes by adenovirus-mediated gene transfer and found that the phosphorylation of Akt and its downstream target, glycogen synthase kinase 3beta, were enhanced both in the absence and in the presence of insulin. Furthermore, protein kinase C lambda (PKC lambda) activity was also augmented in small-t-antigen-expressing 3T3-L1 adipocytes. Consistent with this result, both basal and insulin-stimulated glucose uptake were enhanced in these cells. In support of this result, when inhibitory anti-PP2A antibody was microinjected into 3T3-L1 adipocytes, we found a twofold increase in GLUT4 translocation in the absence of insulin. The small-t-antigen-induced increase in Akt and PKC lambda activities was not inhibited by wortmannin, while the ability of small t antigen to enhance glucose transport was inhibited by dominant negative Akt (DN-Akt) expression and Akt small interfering RNA (siRNA) but not by DN-PKC lambda expression or PKC lambda siRNA. We conclude that PP2A is a negative regulator of insulin's metabolic signaling pathway by promoting dephosphorylation and inactivation of Akt and PKC lambda and that most of the effects of PP2A to inhibit glucose transport are mediated through Akt.
蛋白磷酸酶2A（PP2A）是一种多聚体丝氨酸/苏氨酸磷酸酶，具有多种功能，包括抑制丝裂原活化蛋白（MAP）激酶途径。猿猴病毒40小t抗原通过与PP2A调节亚基结合特异性抑制PP2A功能，干扰PP2A与其细胞底物结合的能力。我们曾报道小t抗原的表达抑制PP2A与Shc的结合，导致胰岛素和表皮生长因子诱导的Shc磷酸化增强，同时Ras/MAP激酶途径的激活增强。然而，PP2A在胰岛素代谢信号通路中的潜在作用目前尚不清楚。为了评估这一点，我们通过腺病毒介导的基因转移在3T3-L1脂肪细胞中过表达小t抗原，发现无论有无胰岛素，Akt及其下游靶点糖原合酶激酶3β的磷酸化均增强。此外，在表达小t抗原的3T3-L1脂肪细胞中蛋白激酶Cλ（PKCλ）活性也增强。与此结果一致，这些细胞中基础葡萄糖摄取和胰岛素刺激的葡萄糖摄取均增强。为支持这一结果，当将抑制性抗PP2A抗体显微注射到3T3-L1脂肪细胞中时，我们发现在无胰岛素的情况下葡萄糖转运蛋白4（GLUT4）易位增加了两倍。小t抗原诱导的Akt和PKCλ活性增加不受渥曼青霉素抑制，而小t抗原增强葡萄糖转运的能力受显性负性Akt（DN-Akt）表达和Akt小干扰RNA（siRNA）抑制，但不受DN-PKCλ表达或PKCλ siRNA抑制。我们得出结论，PP2A通过促进Akt和PKCλ的去磷酸化和失活而成为胰岛素代谢信号通路的负调节因子，并且PP2A抑制葡萄糖转运的大多数作用是通过Akt介导的。
