Protein Phosphatase 2A Negatively Regulates Insulin's Metabolic Signaling Pathway by Inhibiting Akt (Protein Kinase B) Activity in 3T3-L1 Adipocytes

Abstract

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 3␤, were enhanced both in the absence and in the presence of insulin. Furthermore, protein kinase C (PKC ) activity was also augmented in small-t-antigenexpressing 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 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 expression or PKC siRNA. We conclude that PP2A is a negative regulator of insulin's metabolic signaling pathway by promoting dephosphorylation and inactivation of Akt and PKC and that most of the effects of PP2A to inhibit glucose transport are mediated through Akt.Protein phosphorylation plays a key role in many cellular processes, including insulin signal transduction (24), and the phosphorylation state of a target protein is regulated by opposing kinase and phosphatase activities (24). Thus, the balance of enzyme activity between kinases and phosphatases is critical for the mediation of insulin's effects and, in turn, for the pathogenesis of insulin-resistant states.Tyrosine phosphorylation is essential for insulin action, and several lines of evidence have demonstrated that protein tyrosine phosphatases can play a role in insulin-resistant states (3, 4). For example, protein tyrosine phosphatase 1B (PTP1B) directly interacts with the activated insulin receptor and exhibits high specific activity for IRS-1 (22, 49). It has been reported previously that hyperglycemia can impair insulin-stimulated tyrosine phosphorylation of the insulin receptor and IRS-1, at least in part because of the increased expression and activity of PTP1B (37,41), and that overexpression of PTP1B inhibits insulin-stimulated glucose metabolism in 3T3-L1 adipocytes and L6 myocytes (12,18,51).Serine/thre...

Turn motif phosphorylation negatively regulates activation loop phosphorylation in Akt

“…To determine which of these two phenomena is occurring, we tried to inhibit the A-loop phosphatase in vivo. We used okadaic acid (OA) to inhibit the A-loop phosphatase as protein phosphatase 2A has been reported to be an Aloop phosphatase in mammalian cells (Andjelkovicé t al., 1996;Sato et al, 2000;Borgatti et al, 2003;Ugi et al, 2004;Kuo et al, 2008). Microinjection of OA into unstimulated oocytes induced A-loop phosphorylation in the absence of 1-MeAde (Figure 5a), as expected from the constitutive activity of PDK1 (Alessi et al, 1997a, b;Hiraoka et al, 2004).…”

Negative regulation of Pim-1 protein kinase levels by the B56β subunit of PP2A

“…Figure 8b demonstrates (Qian et al, 2005b). It is interesting that Pim-1 has been shown to transform cells in concert with both c-Myc (Verbeek et al, 1991;Shirogane et al, 1999;Ellwood-Yen et al, 2003) or Akt (Amaravadi and Thompson, 2005), and that the levels of both of these proteins are regulated by PP2A (Sears, 2004;Ugi et al, 2004;Van Kanegan et al, 2005). Using a model in which hematopoietic cells are grown in an IL-3-dependent fashion, we find that knocking down a PP2A B subunit increases viability only in Pim containing, but not in Pim knockout cells.…”

Akt2 and acid ceramidase cooperate to induce cell invasion and resistance to apoptosis

“…25 Furthermore, a major role of ceramide is to activate the protein phosphatases PP2A and PP1, [43][44][45][46] and the upregulation of ASAH1 often seen in cancer would decrease ceramide and, therefore, PP1/PP2A activities. PP2A is known to act as a tumor suppressor via dephosphorylating T308 in Akt, 47,48 RalA, 49 SPHK1, 50 but also anti-apoptotic Bcl-2 51 and pro-apoptotic Bax. 52 Similarly, PP1 plays anti-proliferative and (ASAH1 α subunit, #612302), Covance (GAPDH, #MMS-580S) and SigmaAldrich (vinculin, #V9131).…”