One of the major metabolic actions of insulin involves stimulation of GLUT4 translocation and glucose transport. The PI 3-kinase dependency of this stimulation has been clearly demonstrated in a variety of insulin-responsive tissues and cell lines (29
). The molecular events subsequent to PI 3-kinase, which cause glucose transport stimulation, have also been extensively studied. Akt and PKC λ are serine/threonine kinases activated downstream of PI 3-kinase, and both have been suggested to be mediators of glucose transport stimulation (14
). PDK-1 is a PI 3-kinase-dependent serine/threonine kinase which is upstream of Akt and PKC λ, and the ability of PDK-1 to phosphorylate and activate Akt and PKC λ has been extensively studied (13
). Although phosphorylation clearly activates these kinases, the process of dephosphorylation and deactivation is less well understood.
PP2A is a multimeric ubiquitously expressed serine/threonine phosphatase consisting of scaffolding A, regulatory B, and catalytic C subunits. SV40-derived small t antigen inhibits PP2A activity by interfering with the regulatory B subunit recognition of PP2A target substrates, and it was previously shown that small t antigen expression augments insulin's mitogenic effects (60
In the present study, we demonstrate that the inhibition of PP2A, either by expression of small t antigen or by microinjection of anti-PP2A antibody or PP2A siRNA, caused increased GLUT4 translocation and glucose transport in 3T3-L1 adipocytes. These changes in glucose transport were accompanied by the elevation of Akt and PKC λ phosphorylation and activity levels, and these effects (small-t-antigen-induced Akt and PKC λ activation, glucose transport, and GLUT4 translocation) were not inhibited by wortmannin. Furthermore, in vitro, recombinant PP2A was able to dephosphorylate and inactivate Akt and PKC λ, and this effect was inhibited by preincubating the recombinant PP2A with anti-PP2A antibody. Thus, these results indicate that the augmentation of glucose transport by inhibition of PP2A is due to direct actions of PP2A downstream of PI 3-kinase, most likely at the level of Akt and PKC λ.
The precise roles of Akt and PKC λ in glucose transport are incompletely understood. With respect to Akt, a number of previous studies report both positive and negative evidence for its role in glucose transport (7
). The expression of two different types of constitutively active forms of Akt augments glucose transport both in the basal state and after insulin stimulation (32
) in 3T3-L1 adipocytes and L6 myotubes, and the expression of a constitutively active form of PKC λ showed similar results in L6 myotubes (8
), 3T3-L1 adipocytes (33
), and rat adipocytes (58
). Furthermore, the expression of DN forms of Akt (28
) and PKC λ (8
) partially inhibited glucose uptake in 3T3-L1 adipocytes and L6 myotubes. Interestingly, the DN constructs completely inhibited endogenous enzymatic activities, even though glucose transport was only partially blocked (28
). Thus, it is quite possible that both Akt and PKC λ contribute to insulin-stimulated glucose transport and that both are necessary for full activation of this process. Furthermore, the relative importance of these molecules may be cell context dependent.
Both Akt and PKC λ activities are elevated in the basal and insulin-stimulated states in small-t-antigen-expressing cells, and this result parallels the glucose transport results. Each of these kinases may contribute to insulin-stimulated glucose transport, because both DN-Akt and DN-PKC λ, as well as Akt and PCK λ siRNA, partially inhibited the effect of insulin on GLUT4 translocation and glucose transport. However, whereas the inhibition of Akt and PKC λ decreased insulin-stimulated glucose uptake to the same extents, when PP2A was suppressed by small t antigen or PP2A siRNA, only Akt inhibition reversed the effects of PP2A suppression on glucose transport. These results indicate that PP2A exerts its inhibitory effects on glucose transport at the level of Akt and not PKC λ. While Akt activation is an important component of glucose transport stimulation, these studies do not rule out a role for PKC λ in this process. Indeed, DN-PKC λ and PKC λ siRNA both inhibited insulin-stimulated GLUT4 translocation and glucose transport, and it is possible that the magnitude of PKC λ activation induced by PP2A inhibition was insufficient to generate a glucose transport stimulatory signal.
While the Akt activation mechanism has been extensively investigated, the inactivation mechanisms are less well understood. We found that the inhibition of PP2A led to an elevation of the Akt phosphorylation level without affecting PI 3-kinase or PDK-1 activity, and these results suggest that PP2A may directly modulate Akt function. In support of this idea, it has been reported that Akt can associate with PP2A and is dephosphorylated by PP2A (6
). PP2A was eluted in the same fraction as Akt by ion-exchange chromatography (47
), and the coexpression of Akt with the catalytic subunit of PP2A inhibits Akt activity (26
). In fact, it was previously reported that Akt was coimmunoprecipitated with PP2A and that this coimmunoprecipitation was decreased by small t antigen expression, a finding consistent with the idea that small t antigen displaces the PP2A regulatory B subunit, inhibiting the ability of PP2A to associate with its cellular targets (60
). Thus, we conclude that PP2A is a physiologically relevant Akt phosphatase.
Interestingly, GSK-3 is one of the cellular targets of Akt (14
), and Akt phosphorylates GSK-3α and GSK-3β at specific serine residues (serine 21 and serine 8, respectively), leading to enzyme inactivation (15
). Our studies showed that small t antigen expression resulted in enhanced GSK-3β phosphorylation, consistent with the idea that PP2A can function as an Akt phosphatase.
Although PKC λ activity is enhanced in small-t-antigen-expressing cells, the present studies do not definitively identify a mechanism for this effect. PKC λ is directly activated by PDK-1-mediated phosphorylation at threonine 410 (13
). The expression of small t antigen did not affect PDK-1 activity in our study, suggesting that the effects of PP2A on PKC λ may be direct. On the other hand, we did not observe coimmunoprecipitation of PKC λ with PP2A (data not shown). However, these negative results are inconclusive, since coimmunoprecipitation cannot detect all interactions of cellular proteins. Thus, it remains possible that PP2A directly dephosphorylates and inactivates PKC λ. Interestingly, it has been reported that expression of small t antigen led to increased PKC ζ activity in CV-1 cells and that small-t-antigen-induced MEK activation and cell growth were inhibited by the coexpression of a DN form of PKC ζ (55
). However, all of these small t antigen effects were PI 3-kinase dependent in CV-1 cells (55
), and these disparate results suggest that small t antigen can modulate PKC ζ/λ activity by different mechanisms in a cell context-specific manner. Similarly, several different mechanisms whereby PP2A negatively regulates the Ras/MAP kinase pathway have been reported (39
). In EGF-treated adipocytes or PC12 cells, PP2A may directly dephosphorylate MAP kinase (5
). On the other hand, in small-t-antigen-transfected CV-1 cells, the activation of MEK and MAP kinase by inhibition of PP2A was dependent on PI 3-kinase and PKC ζ (55
). Whereas small t antigen did not affect Raf-1 activity in CV-1 cells (54
), it has been reported that PP2A can associate with Raf-1 and that it positively regulates its activity in COS7 cells (1
). Furthermore, it has been demonstrated that PP2A associates with Shc and inhibits its tyrosine phosphorylation in HIRc B cells (60
). These different results may be explained by unique PP2A subunits. There are multiple targeting B subunits for PP2A, and different forms of this trimeric protein may be responsible for its diverse actions, since each B subunit has unique substrate specificity and displays tissue- and cell type-specific distribution (39
). Thus, it is possible that PP2A can act at multiple points, not only in the Ras/MAP kinase pathway, but also on the PI 3-kinase pathway, depending on cell context.
Another important issue concerning the involvement of serine/threonine phosphatases in insulin's metabolic actions involves phosphorylation of IRS proteins. In the basal state, IRS-1 is highly phosphorylated on serine and threonine residues and becomes more heavily phosphorylated after insulin stimulation. Reports have shown that serine/threonine phosphorylation of IRS-1 can serve as either a positive or a negative modulator of insulin signal transduction (20
). Several serine/threonine kinases responsible for phosphorylation of IRS-1 have been reported, including Akt (45
), PKC ζ (36
), JNK (2
), IKKβ (21
), and GSK-3 (20
). Treatment of cells with okadaic acid increases IRS-1 serine/threonine phosphorylation and attenuates insulin action (40
). Thus, phosphatases must be involved in the regulation of serine/threonine phosphorylation of IRS-1, but a specific phosphatase has not been identified. In this study, we did not observe any change in IRS-1 function in small-t-antigen-expressing cells, which suggests that PP2A does not modulate serine/threonine residues that affect IRS-1 tyrosine phosphorylation or PI 3-kinase association.
In summary, our results show that the inhibition of PP2A by small t antigen expression stimulates GLUT4 translocation and glucose transport, most likely by causing increased Akt phosphorylation and activation. These effects of small t antigen are PI 3-kinase independent, suggesting that PP2A acts directly on Akt. These results indicate that the serine/threonine phosphatase PP2A is a physiologic negative regulator of insulin's metabolic signaling pathway, which exerts its effects by dephosphorylating and inactivating Akt. As such, this result raises the possibility that abnormalities in PP2A function may be a cause of insulin resistance.