Serine 474 phosphorylation is essential for maximal Akt2 kinase activity in adipocytes

The Ser/Thr protein kinase Akt regulates essential biological processes such as cell survival, growth, and metabolism. Upon growth factor stimulation, Akt is phosphorylated at Ser474; however, how this phosphorylation contributes to Akt activation remains controversial. Previous studies, which induced loss of Ser474 phosphorylation by ablating its upstream kinase mTORC2, have implicated Ser474 phosphorylation as a driver of Akt substrate specificity. Here we directly studied the role of Akt2 Ser474 phosphorylation in 3T3-L1 adipocytes by preventing Ser474 phosphorylation without perturbing mTORC2 activity. This was achieved by utilizing a chemical genetics approach, where ectopically expressed S474A Akt2 was engineered with a W80A mutation to confer resistance to the Akt inhibitor MK2206, and thus allow its activation independent of endogenous Akt. We found that insulin-stimulated phosphorylation of four bona fide Akt substrates (TSC2, PRAS40, FOXO1/3a, and AS160) was reduced by ∼50% in the absence of Ser474 phosphorylation. Accordingly, insulin-stimulated mTORC1 activation, protein synthesis, FOXO nuclear exclusion, GLUT4 translocation, and glucose uptake were attenuated upon loss of Ser474 phosphorylation. We propose a model where Ser474 phosphorylation is required for maximal Akt2 kinase activity in adipocytes.

The serine/threonine kinase Akt (protein kinase B) is a key regulatory node in a range of cell signaling networks that con-trol essential biological processes such as cell survival, proliferation, and metabolism (1). Upon growth factor stimulation, there is an increase in plasma membrane phosphatidylinositol (3,4,5) triphosphate (PIP 3 ) 7 (2,3). Akt then binds PIP3 via its pleckstrin homology (PH) domain and accumulates at the plasma membrane (4,5). This interaction causes a conformational change, allowing PDK1 to phosphorylate Akt within its kinase domain at Thr 309 (in Akt2; Thr 308 in Akt1 and Thr 305 in Akt3) and mTORC2 to phosphorylate Akt within its hydrophobic motif (HM) at Ser 474 (in Akt2; Ser 473 in Akt1 and Ser 472 in Akt3) (6,7). These phosphorylation events enhance Akt kinase activity, allowing Akt to phosphorylate a number of substrates and facilitate several biological processes. For example, phosphorylation of TSC2 and PRAS40 activates mTORC1 and protein synthesis (8 -10), phosphorylation of AS160/TBC1D4 facilitates GLUT4 translocation and glucose uptake (11,12), and phosphorylation of FOXO proteins regulates their transcriptional activity via nuclear exclusion (13,14).
Although there is consensus that Akt Thr 309 phosphorylation is required for its activation (6,(15)(16)(17), the role of Ser 474 phosphorylation remains controversial. Initial studies that assessed Akt activity in vitro reported that loss of Ser 474 phosphorylation severely diminished Akt kinase activity (6,17). Accordingly, Akt crystal structures show that an active kinase conformation is only possible by mimicking Ser 474 phosphorylation with a S474D substitution (18,19). From this, a model arose whereby Ser 474 phosphorylation was integral to a high level of Akt kinase activity.
However, the accuracy of this model has been disputed in more recent studies in cells, where Ser 474 phosphorylation was required for Akt activity only toward specific substrates. These studies abolished Ser 474 phosphorylation through genetic or pharmacological ablation of its upstream kinase, mTORC2. For example, in mouse embryonic fibroblasts lacking SIN1, one of the subunits of mTORC2, there is loss of FOXO1/3a phosphorylation, but other Akt substrates (TSC2 and GSK3) and mTORC1 effectors (S6K and 4EBP1) are unaffected (20). Similarly, phosphorylation of FOXO3a and AS160 is impaired, but GSK3␤ phosphorylation is unaltered, in adipocytes from mice lacking the mTORC2 subunit RICTOR (21). Further, rat adipocytes treated with small-molecule inhibitors of mTORC2 exhibit decreased phosphorylation of PRAS40 but not GSK3␤ (22). These studies have implicated Ser 474 phosphorylation as a driver of Akt substrate specificity. Alternatively, a recent study using RICTOR knockout mice indicates that Ser 474 phosphorylation is dispensable for maximal phosphorylation of AS160, PRAS40, FOXO1, and GSK3␤ in adipose tissue (23). Nevertheless, use of mTORC2 deletion or inhibition to study the role of Akt Ser 474 phosphorylation may be confounded by several factors, including the induction of compensatory mechanisms because of a chronic absence of mTORC2 (24), lack of specificity of mTORC2 inhibitors (25), and interference from other downstream actions of mTORC2 that are independent of Akt Ser 474 phosphorylation, such as other phosphorylation sites on Akt (26 -28).
A chemical genetics approach originally designed by Green et al. (29) overcomes these disadvantages by facilitating acute prevention of Ser 474 phosphorylation in cells without perturbing mTORC2 activity. An Akt phosphomutant can be engineered to contain a W80A mutation in its PH domain, which confers resistance to the allosteric pan-Akt inhibitor MK2206 without compromising its activation. Treatment of cells ectopically expressing this mutant with MK2206 acutely eliminates endogenous Akt activity, allowing specific analysis of the W80A Akt mutant (29 -31). We utilized this system to address how loss of Ser 474 phosphorylation influences Akt2 activity toward a number of substrates and insulin-regulated processes in 3T3-L1 adipocytes. We show that insulin-stimulated phosphorylation of four Akt substrates was reduced by ϳ50% in the absence of Ser 474 phosphorylation. Similarly, several insulinregulated processes were partially impaired following loss of Ser 474 phosphorylation. These findings support a model where Ser 474 phosphorylation is essential for maximal Akt2 kinase activity in adipocytes.

The Akt2 W80A mutation confers MK2206 resistance and is a powerful tool to study Akt2 mutants
Studying the activity of ectopically expressed mutant proteins in cells can be confounded by the presence of their endogenous counterparts. To circumvent this and examine the role of Akt2 Ser 474 phosphorylation in 3T3-L1 adipocytes (hereafter referred to as adipocytes), we employed a chemical genetics approach originally designed by Green et al. (29). MK2206 is an Akt inhibitor that works by preventing Akt recruitment to the plasma membrane (PM), which is essential for its activation (5,32,33). However, Akt with the W80A mutation is resistant to MK2206 inhibition. Treatment of cells ectopically expressing W80A Akt with MK2206 prevents activation of endogenous Akt, allowing specific assessment of the W80A Akt mutant upon insulin stimulation (Fig. 1A) (29 -31).
To validate the W80A system, we assessed insulin-stimulated PM recruitment of TagRFP-T-WT and W80A Akt2 in adipocytes using live-cell total internal reflection fluorescence (TIRF) microscopy. There was no significant difference in the kinetics of insulin-stimulated WT and W80A Akt2 PM recruitment (Fig. 1, B and C). Aligning with previous studies (34,35), both WT and W80A Akt2 exhibited an overshoot in PM local-ization in response to insulin. However, pretreatment with 10 M MK2206 abolished PM accumulation of WT Akt2, whereas robust accumulation of W80A Akt2 was observed under identical conditions (Fig. 1, D and E). These data are consistent with the Akt2 W80A mutation conferring MK2206 resistance but having negligible impact on Akt2 recruitment to the PM.
We next generated a series of N-terminally FLAG-tagged Akt2 constructs that were engineered to express Akt2 with Thr 309 or Ser 474 mutated to alanine to prevent phosphorylation; WT Akt2 (WT), Akt2 with the W80A mutation (W80A), Akt2 with W80A and T309A mutations (W80A-T309A), and Akt2 with W80A and S474A mutations (W80A-S474A) (Fig.   Figure 1. The Akt2 W80A mutation confers MK2206 resistance and has negligible impact on Akt2 activation at the plasma membrane in 3T3-L1 adipocytes. A, the Akt W80A mutation confers resistance to the Akt inhibitor MK2206. Treatment of cells ectopically expressing W80A Akt with MK2206 prevents activation of endogenous Akt, allowing specific activation of the W80A Akt mutant upon insulin stimulation. IR, insulin receptor; P, phosphorylation site. B, 3T3-L1 adipocytes were electroporated with TagRFP-T-WT or W80A Akt2, and plasma membrane recruitment was assessed using live-cell TIRF microscopy. Adipocytes were exposed to 1 nM insulin. Representative images for 3-7 independent experiments are presented. C, quantification of B (WT, 41 cells from three independent experiments; W80A, 60 cells from seven independent experiments; mean Ϯ S.E.). D, 3T3-L1 adipocytes were electroporated with TagRFP-T WT or W80A Akt2, and plasma membrane recruitment was assessed using live-cell TIRF microscopy. Adipocytes were exposed to 10 M MK2206 for 5 min, followed by 1 nM insulin. Representative images for two independent experiments are presented. E, quantification of D (WT, 15 cells from two independent experiments; W80A, 59 cells from two independent experiments; mean Ϯ S.E.). 2A). These were stably expressed in adipocytes to similar degrees at ϳ75% of endogenous Akt2 (Fig. 2, B-D). Ectopic FLAG-Akt2 could be separated from endogenous Akt2 (Fig.  S1). As expected, treatment of adipocytes expressing WT Akt2 with 10 M MK2206 blocked insulin-stimulated phosphorylation of Akt at Thr 309 and Ser 474 and phosphorylation of its substrates TSC2, PRAS40, FOXO1/3a, and AS160 (Fig. 2, E and F). MK2206 did not completely block insulin-stimulated phosphorylation of the Akt substrate GSK3␤ at Ser 9 (Fig. S2), suggesting GSK3␤ is also phosphorylated by another kinase at this site. Therefore, we did not use GSK3␤ phosphorylation as a readout of Akt activity in this study because we could not confidently attribute changes in GSK3␤ phosphorylation to Akt. In contrast, adipocytes expressing W80A Akt2 displayed robust insulin-stimulated phosphorylation of Akt and its substrates in the presence of 10 M MK2206, again implicating W80A Akt2 as being MK2206-resistant (Fig. 2, E and F). Consistent with previous reports (6,(15)(16)(17), Thr 309 phosphorylation was required for Akt2 activity because loss of phosphorylation at this site abolished insulin-stimulated phosphorylation of Akt substrates (Fig. 2, E and F). Intriguingly, loss of Thr 309 phosphorylation resulted in hyperphosphorylation of Akt at Ser 474 upon insulin stimulation, indicating interdependence between the two phosphorylation sites. These data highlight that the Akt2 W80A mutation confers resistance to MK2206 and that this can be used to analyze the activity of Akt2 mutants in the absence of endogenous Akt activity in adipocytes.

Akt2 Ser 474 phosphorylation is required for maximal phosphorylation of TSC2, PRAS40, FOXO1/3a, and AS160
How Ser 474 phosphorylation regulates Akt kinase activity remains controversial. MK2206-resistant W80A Akt was a valuable tool to assess the role of Ser 474 phosphorylation in the activation of Akt2 in adipocytes through direct mutation of this phosphorylation site. We first assessed the role of Ser 474 phosphorylation in Akt2 activity toward its canonical substrates TSC2, PRAS40, FOXO1/3a, and AS160. We did so by measuring the temporal dynamics by which these substrates were phosphorylated following insulin stimulation in adipocytes stably expressing W80A Akt2 or W80A-S474A Akt2. This was conducted in the presence of MK2206 to specifically activate the ectopic mutant. We have reported previously that a small degree of Akt phosphorylation is sufficient to achieve maximal substrate phosphorylation (36,37); thus, we utilized a submaximal insulin dose (1 nM) to ensure that changes in Akt kinase activity were detectable.
In adipocytes expressing W80A Akt2, there was a time-dependent increase in phosphorylation of all substrates following insulin stimulation (Fig. 3, A and B). Distinct phosphorylation kinetics were observed between each substrate, aligning with

Regulation of Akt2 kinase activity by Ser 474 phosphorylation
previous reports of a lack of concordance in the behavior of Akt substrates (36,38). Nevertheless, in adipocytes expressing W80A-S474A Akt2, substrate phosphorylation plateaued at ϳ50% of that observed in adipocytes expressing W80A Akt2 (Fig. 3, A and B). These data suggest that Ser 474 phosphorylation plays an important role in maximizing Akt2 kinase activity toward TSC2, PRAS40, FOXO1/3a, and AS160 in adipocytes, with no evidence for substrate specificity.
Throughout the insulin time course, total phosphorylation of Akt at Thr 309 was not significantly different between adipocytes expressing W80A and W80A-S474A Akt2 (Fig. 3, A and B). This finding is corroborated by previous studies where loss of Ser 474 phosphorylation did not affect Thr 309 phosphorylation (6,20), supporting the notion that defective Akt2 substrate phosphorylation upon loss of Ser 474 phosphorylation is not due to decreased Thr 309 phosphorylation.

Akt2 Ser 474 phosphorylation is required for maximal insulinstimulated mTORC1 activation and protein synthesis
We next assessed whether loss of Akt2 Ser 474 phosphorylation alters the activation of the insulin signaling network beyond the immediate phosphorylation of Akt substrates. In response to insulin, Akt activates mTORC1 via phosphorylation of its substrates TSC2 and PRAS40 (8,9). mTORC1 then phosphorylates its substrates, which facilitate a variety of responses, including an increase in the rate of protein synthesis ( Fig. 4A) (10,39,40). Thus, we first assessed whether loss of Ser 474 phosphorylation affects insulin-stimulated activation of mTORC1 and protein synthesis in adipocytes. We assessed mTORC1 activation by the phosphorylation status of its substrate, P70 S6K. In adipocytes expressing WT or kinase-dead W80A-T309A Akt2, MK2206 blocked the ability of Akt2 to activate mTORC1 in response to insulin (Fig. 4, B and C). Sim- Figure 3. Akt2 Ser 474 phosphorylation is required for maximal insulin-stimulated phosphorylation of PRAS40, AS160, FOXO1/3a, and TSC2 in 3T3-L1 adipocytes. 3T3-L1 adipocytes stably expressing FLAG-Akt2 mutants were treated with 10 M MK2206 for 5 min, followed by 1 nM insulin for the times specified. A, lysates were immunoblotted with antibodies as specified, with 14-3-3 as a loading control. A representative blot for three to four independent experiments is presented. Band 1 and Band 2 indicate shifts in Akt2 gel migration. p, phosphorylated. B, quantification of A (n ϭ 3-4, mean Ϯ S.E., two-way analysis of variance; *, p Ͻ 0.05; ns, not significant). AU, arbitrary unit; p, phosphorylated.

Regulation of Akt2 kinase activity by Ser 474 phosphorylation
ilarly, following MK2206 treatment, adipocytes expressing WT Akt2 could not facilitate insulin-stimulated protein synthesis (Fig. 4D), aligning with these events being activated downstream of Akt. In the presence of MK2206, adipocytes expressing W80A-S474A Akt2 exhibited partially impaired P70 S6K phosphorylation, S6 phosphorylation and protein synthesis compared with adipocytes expressing W80A Akt2 (Fig. 4,  B-D). These data, together with diminished TSC2 and PRAS40 phosphorylation (Fig. 3, A and B), suggest that Akt2 Ser 474 phosphorylation is essential for maximal insulin-stimulated mTORC1 activation and protein synthesis.

Regulation of Akt2 kinase activity by Ser 474 phosphorylation
Therefore, we next assessed the functional consequence of Aktmediated FOXO phosphorylation by examining whether Akt2 Ser 474 phosphorylation is required for insulin-stimulated FOXO1 nuclear exclusion. In adipocytes expressing WT Akt2, MK2206 inhibited insulin-stimulated FOXO1 nuclear exclusion (Fig. 4, E and F), consistent with it being Akt-dependent. In adipocytes expressing W80A-S474A Akt2, the rate of FOXO1 nuclear exclusion was reduced compared to adipocytes expressing W80A Akt2 (Fig. 4, E and F). These observations, together with impaired FOXO1/3a phosphorylation (Fig. 3, A  and B), suggest that Akt2 Ser 474 phosphorylation is required for maximal insulin-stimulated FOXO nuclear exclusion.

Akt2 Ser 474 phosphorylation is required for maximal insulinstimulated GLUT4 translocation and glucose uptake
Finally, we assessed whether Akt2 Ser 474 phosphorylation is required for insulin-stimulated GLUT4 translocation and glucose uptake in adipocytes. Akt activation is both necessary and sufficient for insulin-stimulated glucose uptake (41). This is predominantly achieved through the phosphorylation of AS160, which is required for translocation of GLUT4 to the PM (Fig. 4A) (30,42). Consistent with this, MK2206 blocked insulin-stimulated GLUT4 translocation and glucose uptake in adipocytes expressing WT Akt2 (Fig. 4, G-I). In the presence of MK2206, both GLUT4 translocation and glucose uptake were activated by insulin in adipocytes expressing W80A Akt2; however, only partial activation was achieved in adipocytes expressing W80A-S474A Akt2 (Fig. 4, G-I). These observations, together with impaired AS160 phosphorylation (Fig. 3, A and  B), suggest that Akt2 Ser474 phosphorylation is essential for maximal insulin-stimulated GLUT4 translocation and glucose uptake.

Discussion
Akt is a major regulatory node for numerous biological processes, but the mechanism by which it is activated remains controversial. It is widely accepted that Thr 309 phosphorylation is essential for Akt activation (6,(15)(16)(17); however, the role of Ser 474 phosphorylation is unclear. Here we examined how loss of Ser 474 phosphorylation influences Akt2 activity toward a number of substrates and insulin-regulated processes in 3T3-L1 adipocytes. Insulin-stimulated phosphorylation of four canonical Akt substrates was reduced by ϳ50% in the absence of Ser 474 phosphorylation. Accordingly, we measured reduced efficiency of several insulin-regulated processes in the absence of Ser 474 phosphorylation. Overall, our findings suggest that Ser 474 phosphorylation regulates the full activation of Akt2 toward a cadre of substrates and biological processes in adipocytes.
Previous studies have proposed an alternate model where Ser 474 phosphorylation is required for Akt activity only toward select substrates. Evidence for this model is largely derived from studies where Ser 474 phosphorylation was prevented via ablation of its upstream kinase, mTORC2 (20 -22). However, by interfering with the upstream kinase of Ser 474 , these studies have several disadvantages, such as possible induction of longterm compensatory mechanisms and interference with other downstream actions of mTORC2 independent of Akt Ser 474 phosphorylation. MK2206-resistant W80A Akt offered a valuable tool to overcome these disadvantages and interrogate the role of Akt2 Ser 474 phosphorylation in cells through direct mutation of Ser 474 . Despite examining a cadre of Akt substrates and downstream biological processes, many of which were identical to those examined in prior studies, our data do not illustrate selective signaling in the absence of Ser 474 phosphorylation but, rather, a global decrease in Akt2 activity. We cannot exclude that these differences between our data and previous reports are due to cell-specific effects, as each study was undertaken in a different cell line, or that the role of Ser 474 phosphorylation differs for each Akt isoform. However, we speculate that these disparities are more likely reflective of other events downstream of mTORC2 conferring Akt substrate specificity rather than Ser 474 phosphorylation itself. For example, mTORC2 has been shown to phosphorylate other sites on Akt, such as Thr 450 on its turn motif (27,28), and Ser 477 /Thr 479 on its hydrophobic motif (26). This and other reported mechanisms of Akt substrate specificity (43) deserve further investigation largely because of their applicability to cancer treatments. Akt inhibitors have been tested in clinical trials; however, although they can successfully inhibit cancer growth, hyperglycemia often accompanies treatment (44,45). This is not surprising considering Akt controls glucose metabolism. To design cancer therapeutics without these metabolic toxicities, it is of interest to therapeutically target specific actions of Akt rather than using pan-Akt inhibitors.
Akt2 undergoes differential SDS-PAGE migration upon insulin stimulation as a result of changes in its phosphorylation status (Fig. S3). FLAG-W80A, FLAG-W80A-T309A, and FLAG-W80A-S474A Akt2 did not differ in their gel migration in the basal state ( Fig. 2B; indicated as Band 2 in Figs. 2E, 3A, and 4B). However, upon insulin stimulation, there was decreased migration of FLAG-W80A and FLAG-W80A-T309A Akt2 (indicated as Band 1 in Figs. 2E, 3A, and 4B) but not FLAG-W80A-S474A Akt2. These data suggest that Ser 474 phosphorylation, but not Thr 309 phosphorylation, is required for the insulin-stimulated band shift in Akt2. It is well established that some but not all phosphorylation events can result in a band shift (46). This can be attributed to the charge of the residues surrounding the phosphosite; only in the appropriate local environment does addition of a phosphate group decrease SDS binding and, consequently, migration (47). Nevertheless, this, coupled with the requirement for Ser 474 phosphorylation for the band shift, leads us to propose that the insulin-stimulated band shift in Akt2 is caused by either Ser 474 phosphorylation itself or other Ser 474 phosphorylation-dependent phosphorylation sites. For example, we speculate that phosphorylation of Akt2 at Ser 478 may be dependent on Ser 474 phosphorylation as they are closely localized, increase with similar kinetics upon insulin stimulation (38), and interdependence between the corresponding sites on Akt1 has been reported (26). Elucidating whether this is the case may help dissect why Ser 474 phosphorylation is required for maximal Akt2 kinase activity, particularly as phosphorylation events on Akt other than that at Thr 309 and Ser 474 are increasingly being shown to have substantial impact on Akt kinase activity (26,48).

Regulation of Akt2 kinase activity by Ser 474 phosphorylation
A major advantage of the W80A system used in this study is that it allowed investigation of Akt mutants in cells rather than in vitro. Our work in cells suggests that Akt can achieve half of its maximal kinase activity without Ser 474 phosphorylation, which is in striking contrast to in vitro studies showing no or very little kinase activity in the absence of Ser 474 phosphorylation (6,17,49). This difference is likely reflective of in vitro systems not reproducing complex cellular control points, comprising signal amplification, macromolecular crowding, interacting proteins, protein localization, and positive/negative feedback (36,49,50). As substantial Akt activation occurs without Ser 474 phosphorylation in the cell, our data align with studies reporting that Ser 474 phosphorylation poorly correlated with Akt activity (51); however, Ser 474 phosphorylation is used in countless studies as a proxy of Akt activity. Rather, we suggest using phosphorylation of Akt at Thr 309 or its substrates as more accurate indices of Akt activation (52).
Although we show a global defect in insulin-stimulated Akt2 activity upon loss of Ser 474 phosphorylation, a recent study by Beg et al. (30), using the same W80A system in 3T3-L1 adipocytes, reported that insulin-stimulated GLUT4 translocation and mTORC1 activation were unaffected by loss of Akt2 Ser 474 phosphorylation. There are differences between our studies that may explain these disparities. Most importantly, Beg et al. (30) used a dose of MK2206 (1 M) they and others have shown to be insufficient to completely impair substrate phosphorylation by endogenous Akt (53). Having residual endogenous Akt activity will likely alter the behavior of the ectopic W80A Akt mutant, as Akt activity is extremely sensitive to temporal changes in its activation (54 -56). Even a minute amount of residual endogenous Akt activity may skew signaling outcomes, as only a small percentage of the total Akt pool is required to achieve a maximal response for many downstream processes (36,37). In contrast, we utilized 10 M MK2206, as it was sufficient to block endogenous Akt activation, allowing specific analysis of ectopic W80A Akt2 (Figs. 1, D and E, and 2, E and F). Furthermore, to control for endogenous Akt inhibition, Beg et al. (30) used untransfected 3T3-L1 adipocytes. However, based on our studies, it is important to use adipocytes expressing FLAG-WT Akt2 to ensure effective comparison with adipocytes expressing FLAG-W80A Akt2. Nevertheless, Beg et al. (30) also reported that insulin-stimulated GLUT1 translocation requires Akt2 Ser 474 phosphorylation, which we support (Fig.  S4). These data suggest that further inquiry may reveal Akt substrates that control GLUT1 translocation and require Ser 474 -phosphorylated Akt for activation. However, an alternate explanation is that maximal Akt activity may be required to trigger insulin-stimulated GLUT1 translocation; this result may not reflect Akt substrate specificity per se but, rather, a difference in the dose-response relationship between Akt activation and various downstream processes.
The mechanism by which substantial Akt activity is possible in the cell without Ser 474 phosphorylation remains elusive. Akt crystal structures suggest that HM binding to the N-lobe of the kinase domain is required for an active conformation and that Ser 474 phosphorylation is essential for this interaction (18,19). We speculate that this interaction can occur in the absence of Ser 474 phosphorylation in cells; this weak interaction could be more favorable in cells than in vitro because of macromolecular crowding or stabilization by interacting proteins (50). Intriguingly, other AGC kinases, such as protein kinase A, lack a phosphosite within their HM, yet it retains the ability to interact with its kinase domain and induce activity (18,19,57). Thus, Ser 474 phosphorylation may increase the affinity of the HM for the kinase domain but not be a prerequisite for the interaction or Akt activation. Alternatively, it has recently been found that the HM interacts with the PH-kinase domain linker to relieve PH domain-mediated autoinhibition and increase Akt activity (58). Whether this interaction can occur without Ser 474 phosphorylation remains to be elucidated. Uncovering the mechanism by which substantial Akt kinase activity is possible without Ser 474 phosphorylation is essential, as there is interest in developing therapeutic agents that inhibit Akt by targeting its hydrophobic motif (59).

Cloning
TagRFP-T-Akt2 has been described previously (34). TagRFP-T-Akt2 with the W80A mutation was generated using site-directed mutagenesis (60). For FLAG-Akt2 constructs, Akt2 was amplified by PCR from human cDNA with addition of attB sites at either end to be compatible with gateway cloning. Akt2 cDNA was inserted into pDONR221 (Thermo Fisher Scientific) using Gateway TM BP Clonase TM II Enzyme Mix (Invitrogen) and then into the retroviral vector pMIG-t-sapphire-puromycin (pMIG-tsap-puro) using Gateway TM LR Clonase TM II Enzyme Mix (Invitrogen). A FLAG tag was then added to the N terminus of Akt2 using Gibson assembly cloning (61). Sitedirected mutagenesis (60) was used to create FLAG-tagged W80A, W80A-T309A, and W80A-S474A Akt2 plasmids. For FOXO1-mNeonGreen, the N-terminal region of FOXO1 (1-380) containing the nuclear localization and nuclear export sequences was codon-optimized and synthesized as a gene block (IDT Technologies). An mNeonGreen tag was placed at its C terminus, and this was cloned into the mammalian expression vector pcDNA3.1. The mutations S209A, H212A, and S215A were introduced to impair DNA binding and phosphorylation by STK4 as described previously (62). pHluorin-GLUT4-mRuby3 was generated from pHluorin-GLUT4-tdTomato (63) by replacing tdTomato with mammalian expression-optimized mRuby3 synthesized as a gene block (IDT Technologies). Primer and construct sequences are available upon request. All constructs were confirmed by Sanger sequencing.

Regulation of Akt2 kinase activity by Ser 474 phosphorylation
virus generated from Plat-E cells (Cell Biolabs, San Diego, CA) as described previously (64). Transduced cells were then selected using 2 g/ml puromycin. Cells were differentiated into adipocytes as described previously (65), and used for experiments 7-12 days after initiation of differentiation. To minimize differences between cells expressing each Akt2 mutant, retroviral transduction was performed simultaneously using the same population of 3T3-L1 fibroblasts. Cells were infected with high virus titers so that there was minimal cell loss upon antibiotic selection. Then, to minimize differences in genetic drift, each cell population was subject to identical culturing conditions, used for experiments at the same passage, and not passaged more than 10 times. Macroscopically, we observed no morphological differences between cell populations at any stage throughout their differentiation into adipocytes. Furthermore, we performed the central experiments comprising Figs. 3, A and B, and 4, B-D, with three independently derived cell populations. Each achieved identical results; Ser 474 phosphorylation was required for maximal Akt2 kinase activity.

Western blotting
3T3-L1 adipocytes stably expressing FLAG-Akt2 mutants were serum-starved with DMEM containing 1ϫ GlutaMAX and 0.2% BSA (w/v) for 2 h. Cells were then exposed to 10 M MK2206 (Selleckchem) or DMSO (Sigma-Aldrich) vehicle control for 5 min, followed by 1 nM insulin. We utilized 10 M MK2206 to inhibit endogenous Akt, as we have shown previously that this dose is required to completely inhibit Akt activation (53). Cells were then placed on ice, washed with cold PBS, lysed with 1% (w/v) SDS in PBS containing protease inhibitors (Roche Applied Science) and phosphatase inhibitors (2 mM Na 3 VO 4 , 1 mM Na 4 O 7 P 2 , and 10 mM NaF), and tip probesonicated. Lysates were centrifuged at 13,000 ϫ g for 15 min at 4°C. The lipid layer was removed, and protein content was quantified using the Pierce TM BCA Protein Assay Kit (Thermo Scientific). 10 g of lysate was then resolved by SDS-PAGE, transferred to PVDF membranes, and immunoblotted as described previously (39). All primary antibodies were from Cell Signaling Technology, but 14-3-3 was from Santa Cruz Biotechnology. Densitometry analysis was performed using ImageStudioLite version 5.2.5 (LI-COR). Band intensities were normalized to the loading control (14-3-3). Statistical tests were performed using GraphPad Prism version 7.0.

Live-cell TIRF microscopy
Matrigel-coated coverslips were prepared as described previously (34). To assess insulin-stimulated GLUT4 translocation to the plasma membrane, 3T3-L1 adipocytes stably expressing FLAG-Akt2 mutants were electroporated 7 days post-differentiation with 6 g of pHluorin-GLUT4-mRuby3. To assess insulin-stimulated Akt2 recruitment to the plasma membrane, 3T3-L1 adipocytes were electroporated 6 -8 days post-differentiation with 6 g of WT or W80A TagRFP-T-Akt2. Then cells were placed onto the Matrigel-coated coverslips as described previously (34). 24 -48 h after electroporation, adipocytes were serum-starved for 2 h and then incubated at 37°C with Krebs-Ringer-phosphate-HEPES (KRPH) buffer (0.6 mM Na 2 HPO 4 , 0.4 mM NaH 2 PO 4 , 120 mM NaCl, 6 mM KCl, 1 mM CaCl 2 , 1.2 mM MgSO 4 , and 12.5 mM HEPES (pH 7.4)) supplemented with 10 mM glucose, 1ϫ minimum Eagle's medium amino acids (Gibco by Life Technologies), 1ϫ GlutaMAX, and 0.2% (w/v) BSA. The cells were then treated with 10 M MK2206 and/or 1 nM insulin using a custom-made perfusion system. Images were acquired using the Nikon Ti-Lapps H-TIRF module angled to image ϳ90 nm into cells. Temperature and humidity were maintained using an Okolab cage incubator and temperature control. The change in pixel intensity over time was determined for each individual cell using Fiji (66). The average pixel intensity over time for each cell was normalized to its average intensity over the basal period.

[ 3 H]2-deoxyglucose uptake assay
3T3-L1 adipocytes stably expressing FLAG-Akt2 mutants were serum-starved for 2 h, washed with PBS, and incubated at 37°C with KRPH buffer. Cells were stimulated with 10 M MK2206 for 5 min or DMSO vehicle control, followed by 1 nM insulin. To determine background radiation, 25 M cytochalasin B (Sigma-Aldrich) as a glucose transport inhibitor was added to a well of cells following 10-min insulin stimulation. After 15 min of insulin stimulation, 50 M unlabeled 2-deoxyglucose and 0.25 Ci/ml [ 3 H]2-deoxyglucose (Perkin-Elmer Life Sciences) was added to all cells for 5 min. Glucose uptake was terminated by placing the plate on ice, washing with cold PBS, and lysing the cells with 1% (w/v) Triton X-100 in PBS. Lysates were added to a scintillation vial with 3 ml of Ultima Gold TM XR liquid scintillation mixture (Perkin-Elmer Life Sciences). The radioactivity of each sample in disintegrations per minute was determined using a Tri-Carb liquid scintillation counter (Perkin-Elmer Life Sciences). The protein content of each sample was assessed using the Pierce BCA Protein Assay Kit (Thermo Scientific). Assays were performed in duplicate, and the average of the duplicate was considered one biological replicate. The disintegrations per minute for each condition were converted to nanomoles of 2-deoxyglucose, and data were normalized to protein levels. The value for cytochalasin B-treated cells was subtracted from all other values as a background control. Statistical tests were performed using GraphPad Prism version 7.0.

Regulation of Akt2 kinase activity by Ser 474 phosphorylation
ences). The radioactivity of each sample in disintegrations per minute was determined using a Tri-Carb liquid scintillation counter (Perkin-Elmer Life Sciences). The protein content of each sample was assessed using the Pierce BCA Protein Assay Kit (Thermo Scientific). Assays were performed in triplicate, and the average of the triplicate was considered one biological replicate. The disintegrations per minute for each condition were converted to nanomoles leucine, and data were normalized to protein levels. The value for cycloheximide-treated cells was subtracted from all other values to account for background radiation. Statistical tests were performed using GraphPad Prism version 7.0.

Live-cell spinning-disk confocal microscopy
Matrigel-coated coverslips were prepared as described previously (34). To assess insulin-stimulated FOXO nuclear exclusion, 3T3-L1 adipocytes stably expressing FLAG-Akt2 mutants were electroporated 6 days post-differentiation with 6 g of FOXO1(1-380; S209A, H212A, S215A)-mNeonGreen. Then cells were placed onto the Matrigel-coated coverslips as described previously (34). 24 h after electroporation, adipocytes were serum-starved for 2 h and then stained with Hoechst 33342 for 10 min. Adipocytes were then incubated at 37°C with KRPH buffer supplemented with 10 mM glucose, 1ϫ minimum Eagle's medium amino acids, 1ϫ GlutaMAX, and 0.2% (w/v) BSA. The cells were then treated with 10 M MK2206 and 1 nM insulin using a custom-made perfusion system. Images were acquired on a Nikon Ti inverted confocal microscope with an Andor Diskovery spinning-disk system and an Andor Ixon 888 EmCCD camera (Andor). Temperature and humidity were maintained using an Okolab cage incubator and temperature control. For each individual cell, the median pixel intensity of the cytosol was divided by the median pixel intensity of the nucleus using Fiji (66).

protein phosphatase assay
3T3-L1 adipocytes were serum-starved for 2 h and then treated with 100 nM insulin for 10 min. Cells were placed on ice, washed with cold PBS, and harvested in 1% (w/v) Triton X-100 in PBS. Cells were homogenized by passing through a 22-gauge needle six times and a 27-gauge needle six times prior to centrifugation at 12,000 ϫ g for 15 min. 100 g of the supernatant was treated with 1 mM MnCl 2 (New England Biolabs), 1ϫ NEBuffer for protein metallophosphatases (New England Biolabs), and either 800 units of protein phosphatase (New England Biolabs) or phosphatase inhibitors (10 mM Na 3 VO 4 , 5 mM Na 4 O 7 P 2 , or 50 mM NaF) at 30°C for 15 min. Reactions were stopped with 2% (w/v) SDS and then resolved by SDS-PAGE.

Subcellular fractionation
3T3-L1 adipocytes stably expressing FLAG-Akt2 mutants were serum-starved for 2 h and then exposed to 10 M MK2206 for 5 min, followed by 0.5 nM insulin for 20 min. Cells were placed on ice, washed with cold PBS, and harvested in cold HES buffer (20 mM HEPES, 1 mM EDTA, and 250 mM sucrose (pH 7.4)) containing phosphatase and protease inhibitors. All subsequent steps were carried out at 4°C as described previously (67). Briefly, cells were homogenized using a Dounce tissue grinder prior to centrifugation at 500 ϫ g for 10 min. The supernatant was centrifuged at 13,550 ϫ g for 12 min to pellet the plasma membrane and mitochondria/nuclei. This pellet was resuspended in HES buffer and again centrifuged at 13,550 ϫ g for 12 min. The pellet was then resuspended in HES buffer, layered over high-sucrose HES buffer (20 mM HEPES, 1 mM EDTA, and 1.12 M sucrose (pH 7.4)), and centrifuged at 111,160 ϫ g for 60 min in a swing-out rotor. The PM fraction was collected at the interface between the sucrose layers and pelleted by centrifugation at 235,000 ϫ g for 75 min. The PM pellet was resuspended in HES buffer containing phosphatase and protease inhibitors. Protein concentrations for each fraction were determined using the Pierce BCA Protein Assay Kit (Thermo Scientific).