The Akt family of kinases are a major node in cell signaling, with regulatory roles in numerous physiologic processes, including cell growth, proliferation, differentiation, metabolism, cell migration and survival (Manning and Cantley, 2007). Perturbations in Akt signaling are linked to many diseases, from hyper-activation in cancer to blunted activity in metabolic diseases (Franke, 2008).

There are three Akt homologues: Akt1, Akt2 and Akt3 (also referred to as protein kinase B (PKB)α, -β and -γ, respectively). All three share a common domain structure consisting of an amino-terminal pleckstrin homology (PH) domain, a kinase domain, and a carboxyl-terminal regulatory region referred to as the hydrophobic motif (HM) domain (Chan et al., 1999). The canonical mode of Akt activation involves plasma membrane recruitment to sites of phosphoinositol 3-kinase (PI3K) activity by Akt PH domain binding to phosphoinositide 3,4,5 trisphosphate (PIP3), the product of growth factor activation of PI3K (Vivanco and Sawyers, 2002). Concurrent with plasma membrane recruitment, Akt is phosphorylated in its kinase domain at T308/9 (residue number for Akt1 and Akt2, respectively) by phosphoinositide-dependent protein kinase 1 (PDPK1), which is itself recruited to the plasma membrane by its own PH domain (Frech et al., 1997). Full Akt activation is associated with a second phosphorylation on S473/4 in the HM domain by mTORC2 (Sarbassov et al., 2005). The exact link between growth factor stimulation and activation of mTORC2 activity is not known (Sparks and Guertin, 2010). The kinase domain phosphorylation, T308/9, is key for activation (Alessi et al., 1996), whereas S473/4 phosphorylation of the HM domain allosterically enhances kinase activity (Alessi et al., 1997) and contributes to substrate selectivity (Jacinto et al., 2006).

A major function of Akt is to transmit insulin signaling to the control of cellular metabolism. One well characterized biological output of Akt is its role in insulin-stimulated translocation of Glut4 glucose transporter to the plasma membrane of fat and muscle cells (Jiang et al., 2003). The redistribution of Glut4 underlies enhanced glucose uptake responsible for postprandial blood glucose lowering (Abel et al., 2001; Zisman et al., 2000). Akt also mediates growth factor stimulated glucose uptake into cells other than fat and muscle (Ward and Thompson, 2012). In those cases, enhanced glucose uptake is to fulfill more local needs, such as fueling an anabolic metabolism required for cell growth and tissue repair; compared to the effect of insulin on fat and muscle cells, which is to maintain whole body glucose homeostasis. Enhanced Glut1-mediated glucose uptake by cancer cells is a prominent example of growth factor-stimulated glucose uptake untethered from regulation of whole body glucose homeostasis (Carvalho et al., 2011).

Akt is a principal mediator of growth factor action. However, technical impediments have made it challenging to define Akt isoform-specific roles as well as the precise roles of the two activating phosphorylations in signal transduction. We developed a system to study ectopically expressed Akt without interference from endogenous Akt (Kajno et al., 2015). The ectopically expressed Akt is engineered to contain a mutation in the PH domain (W80A) that confers resistance to MK2206, an allosteric Akt inhibitor (Calleja et al., 2009; Green et al., 2008). Acute inhibition of endogenous Akt’s with MK2206 allows for functional studies of the ectopically expressed Akt, while minimizing compensatory changes due to loss of Akt functions. We have previously used this system to study Akt isoform specificity in adipogenesis (Kajno et al., 2015).

Here we use this system to define the roles of Akt2 T309 and S474 phosphorylations in insulin-regulated glucose uptake by adipocytes and proliferative cells. Phosphorylation of T309 by PDPK1 is required for Glut4 translocation, whereas phosphorylation of S474 by mTORC2 is not required. However, insulin-stimulated Glut1 translocation to the plasma membrane of adipocytes and proliferative cells is dependent on both T309 and S474 phosphorylations, linking growth factor regulation of Glut1-mediated glucose uptake to mTORC2 activity.

To reveal the roles of Akt2 T309 and S474 Akt phosphorylations in insulin-stimulated Glut4 translocation, we studied Akt2-W80A, a mutant resistant to the allosteric pan Akt inhibitor MK2206. We quantitatively determined Glut4 translocation to the plasma membrane of adipocytes in studies of a HA-Glut4-GFP reporter, an established functional assay of insulin activity in fat and muscle cells (Karylowski et al., 2004; Lampson et al., 2001; Zeigerer et al., 2002; Zhao et al., 2009; Boguslavsky et al., 2012). Throughout this work, unless noted otherwise, we refer to cultured 3T3-L1 adipocytes simply as adipocytes. Although adipocytes express both Akt1 and Akt2, we focused on Akt2 since it has previously been shown to be the Akt isoform biased towards regulation of glucose metabolism (Leavens et al., 2009). Akt2-W80A and HA-Glut4-GFP plasmids were transiently co-expressed in adipocytes by electroporation (Kajno et al., 2015). The Akt2-W80A construct contains a Flag epitope tag at the amino terminus and co-expression of Akt2-W80A and HA-Glut4-GFP was confirmed by GFP fluorescence and anti-FLAG immunofluorescence. Greater than 90% of the transfected cells co-expressed both proteins. To determine the expression of the constructs relative to the native proteins, we assessed total Glut4 and Akt by quantitative immunofluorescence and compared that value between cells expressing and not expressing the constructs. HA-Glut4-GFP expression was determined by GFP fluorescence and Akt2-W80A by anti-Flag staining. The cells expressing HA-Glut4-GFP showed about 1.5 fold increase in total Glut4 staining (Figure 1—figure supplement 1), while cells expressing Akt2-W80A showed about two fold increase in total Akt (Figure 1—figure supplement 2). These data demonstrate that the constructs are expressed to about the same level as native proteins.

A 30 min incubation with 1 nM insulin promoted an approximate 10-fold increase of Glut4 in the plasma membrane of control adipocytes, and co-expression of Akt2-W80A did not affect insulin response (Figure 1C). A 60 min pre-incubation with 1 µM MK2206 abolished insulin-stimulated Glut4 translocation in control adipocytes, whereas Glut4 translocation was unaffected by MK2206 in cells expressing Akt2-W80A (Figure 1C).

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We next determined the activity of Akt2-W80A based on phosphorylation of its substrates AS160 and FoxO1. AS160 (TBC1D4), a Rab GTPase activating protein (GAP), is an Akt target required for Glut4 translocation (Mîinea et al., 2005; Sano et al., 2003; Geraghty et al., 2007; Eguez et al., 2005). Expression of Akt2-W80A fully rescued Akt phosphorylation of FoxO1 and partially rescued Akt T642 phosphorylation in cells in which native Akts are inhibited by MK2206 (Figure 1D,E,F). The Akt2-W80A partial rescue of AS160 phosphorylation, in context of its complete rescue of Glut4 translocation (Figure 1C), indicates that AS160 phosphorylation is not linearly correlated with Akt regulation of Glut4 translocation.

Akt phosphorylation of AS160 is not a reliable surrogate of physiological activity

We next performed a more detailed investigation to determine the degree to which Akt phosphorylation of AS160 is uncoupled in Akt-dependent Glut4 translocation. MK2206 in a dose-dependent manner, inhibited insulin-stimulated Glut4 translocation in control adipocytes, with an EC50 of less than 0.1 µM MK2206 (Figure 2A). Adipocytes co-expressing Akt2-W80A were resistant to a dose up to 1 µM MK2206. Thus, 1 µM MK2206 can be used to distinguish ectopically expressed Akt2-W80A activity from the activities of endogenous Akt’s. The insulin dose-response of Glut4 translocation in MK2206-treated adipocytes expressing Akt2-W80A, was similar to that in untreated control adipocytes, validating this as an experimental system to quantitatively assess Akt function downstream of insulin stimulation (Figure 2B).

Figure 2

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Although Glut4 translocation was fully blocked by 1 µM MK2206, AS160 phosphorylation on T642 was only partially inhibited (Figure 2C and D). Thus, Akt phosphorylation of AS160-T642 is not a linear readout of Akt control of Glut4 translocation. Insulin-stimulated Akt phosphorylation on T309 and S474, and phosphorylation of AS160 on T642 were unaffected by 1 µM MK2206 in adipocytes expressing Akt2-W80A, in agreement with the results of Glut4 translocation (Figure 2C and D). Based on these results, we chose 1 µM MK2206 for subsequent structure-function studies of Akt2-W80A. As was the case for the insulin-dose response measured by Glut4 translocation, MK2206 did not affect the insulin dose-response of Akt2-W80A phosphorylation on T309 and S474, confirming the system can be used to quantitatively assess Akt activation downstream of insulin-stimulation (Figure 2E and F).

T309 phosphorylation, but not S474 phosphorylation, is required for insulin-stimulated Glut4 translocation and mTORC1 activation

Activation of Akt2 involves phosphorylation of T309 and S474 (Sarbassov et al., 2005; Gonzalez and McGraw, 2009a). To probe the requirements of these phosphorylations for Akt2 signaling to Glut4, we studied the effects of alanine substitutions at these sites. Total Akt in cells expressing the mutants was about twice that in untransfected cells, demonstrating the mutants, like Akt2-W80A (Figure 1B), were expressed at about the same level as total native Akts (Figure 3A). Mutation of both T309 and S474 blocked insulin-stimulated Glut4 translocation, as did the single mutation of T309, demonstrating that T309 phosphorylation is necessary for Akt2 to signal to Glut4 (Figure 3B). Surprisingly, Glut4 translocation was fully supported by Akt2-W80A-S474A mutant, establishing that S474 phosphorylation is not required for insulin-stimulated Glut4 translocation (Figure 3B). Akt2-T309A and Akt2-S474A mutants were expressed to similar levels, demonstrating that the functional difference between these mutants was not due to difference in the expression levels (Figure 3A).

Figure 3

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Insulin-stimulated phosphorylation of S474 was unaffected by T309A mutation, demonstrating T309 phosphorylation is not a prerequisite for S474 phosphorylation (Figure 3C and D). Similarly, the S474A mutation did not affect T309 phosphorylation (Figure 3C and D). Thus, in adipocytes these phosphorylations are independent of one another.

Akt2-W80A-S474A supported insulin-stimulated phosphorylation of S6 kinase, an mTORC1 target downstream of Akt (Figure 3E and F). Thus, in addition to Glut4 translocation, Akt activation of mTORC1 does not require phosphorylation of S474. As expected, the T309A mutation blunted insulin-stimulated phosphorylation of S6 kinase, consistent with phosphorylation of T309 being required for Akt activity (Figure 3E and F).

Insulin-stimulated Glut4 translocation promoted by endogenous Akt (cells not treated with MK2206) was unaffected in adipocytes expressing either T309A or S474A mutants compared to control adipocytes (Figure 3G). These data establish that the mutants are not dominant-interfering of endogenous Akt.

Inhibition of PDPK1 and knockdown of mTORC2 phenocopy the Akt2 phosphorylation mutants

PDPK1 is responsible for phosphorylation of T309, and mTORC2 for phosphorylation of S474 (Sarbassov et al., 2005; Alessi et al., 1997; Jacinto et al., 2006). The activities of the Akt2 phosphorylation mutants predict that PDPK1 but not mTORC2 is required for insulin-stimulated Glut4 translocation. Consistent with this hypothesis, transient knockdown of mTORC2 activity by targeting the Rictor subunit had no effect on Glut4 translocation, despite reducing insulin-stimulated phosphorylation of S474 by approximately 80% (Figure 4A–D). As expected, mTORC2 knockdown did not affect phosphorylation of T309.

Figure 4

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The PDPK1 inhibitor, GSK2334470, blocked insulin-stimulated Glut4 translocation in a dose-dependent manner (Figure 4E). The inhibition of translocation correlated with reduced T309 phosphorylation (Figure 4F). GSK2334470 did not affect insulin-stimulated phosphorylation of S474. These results reinforce the conclusion that S474 phosphorylation is dispensable, whereas T309 phosphorylation is essential for Glut4 translocation in adipocytes.

Constitutive activity of Akt2 S474D mutation is dependent on T309 phosphorylation

Several studies have demonstrated that substitution of aspartate for S474 (S474D) generates a constitutively active Akt, presumably as a phosphomimetic (Yang et al., 2002a, 2002b; Hart and Vogt, 2011). In agreement with that previous work, Akt2-W80A-S474D promoted Glut4 translocation in the absence of insulin stimulation (Figure 5A). Consistent with the functional activity, T309 was constitutively phosphorylated in the S474D mutant; that is, without insulin stimulation (Figure 5B). The Akt2-W80A-S474D-induced Glut4 translocation was blocked by T309A mutation as well as by PDPK1 inhibition (Figure 5A and D). Thus, the aspartate substitution at S474 alters Akt2 in a way that enhances phosphorylation of T309 by basal activity of PDPK1, resulting in Akt2 activation and Glut4 translocation, yet phosphorylation of S474 is not required for Glut4 translocation.

Figure 5

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Complementation of HM domain deletion by membrane targeting of the HM Domain in trans

S474 phosphorylation has been linked to target selection (Jacinto et al., 2006). To determine if the HM domain, which contains S474, is required for Akt2 to support Glut4 translocation, we studied the activities of panel of Akt2 domain deletion constructs (Figure 6A). Akt2 in which the HM domain was deleted (Akt2-kinase domain construct) did not support insulin-stimulated Glut4 translocation, despite the deletion construct being expressed to the same level as full length Akt2-W80A (Figure 6B,C). T309 in Akt2-kinase domain construct was phosphorylated in insulin-stimulated cells, demonstrating the HM domain is not required for PDPK1 to phosphorylate Akt2 T309 but the HM domain is required for Glut4 translocation (Figure 6C). In addition, Akt in which the PH domain was deleted (kinase-HM domains) did not support Glut4 translocation, confirming that plasma membrane targeting is required (Figure 6C).

Figure 6

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The HM domain provided in trans did not rescue Glut4 translocation in cells expressing Akt2-kinase domain construct (Figure 6B). However, co-expression of the HM domain with a PH domain fused to its amino terminus (PH-HM domain), rescued insulin-stimulated Glut4 translocation activity to the Akt2-kinase domain construct (Figure 6B). Expression of the PH-HM domain alone had no effect on Glut4 translocation. Thus, the enhanced co-localization of the Akt2 kinase domain and the HM domain achieved when both are independently targeted to plasma membrane sites of PI3 kinase activity was required for complementation of the HM domain in trans. Despite this functional complementation, S474 of the PH-HM domain construct was not phosphorylated in insulin-stimulated adipocytes, providing further support that S474 phosphorylation is not required for Glut4 translocation.

Phospho-S474 Akt2 promotes glucose uptake by increasing GLUT1 in the plasma membrane

To confirm that phosphorylation of S474 is not required for translocation of native Glut4, we generated adipocytes stably expressing Akt2-W80A or Akt2-W80A-S474A. We established that both stably expressed Akt2-W80A and Akt2-W80A-S474A support translocation of HA-Glut4-GFP reporter. To monitor the behavior of native Glut4, we measured insulin-stimulated glucose uptake, the biological output of Glut4 translocation to the plasma membrane. Unexpectedly, insulin-stimulated glucose uptake in Akt2-W80A-S474A adipocytes was only 70% of that in cells expressing Akt2-W80A, despite Akt2-W80A-S474A supporting full translocation of Glut4 to the plasma membrane (Figure 7A and B). The difference in glucose uptake was not due to changes in expression of endogenous Glut4 between the Akt2-W80A or Akt2-W80A-S474A adipocytes (Figure 7C).

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Adipocytes also express Glut1, a widely-expressed glucose transporter whose function is not specifically linked to insulin control of blood glucose. Glut1 is used by most cells for ‘housing keeping’ glucose uptake. Nonetheless, insulin, as well as other growth factors, promote an increase of Glut1 in the plasma membrane of many cell types (Lawrence et al., 1992; Egert et al., 1999; Rett et al., 1996; Clarke et al., 1994). To explore the behavior of Glut1 in adipocytes, we quantified plasma membrane expression using an HA-tagged Glut1 construct, a reporter used previously in studies of Glut1 trafficking (Chaudhary et al., 2016; Takenouchi et al., 2007; Lee et al., 2015). In cells expressing HA-Glut1, total Glut1 was about four times that in untransfected cells, demonstrating HA-Glut1 was expressed to about three times the level of native Glut1 (Figure 7—figure supplement 1). Akt2-W80A supported a near 2-fold translocation of HA-Glut1 to the plasma membrane, whereas Akt2-W80A-S474A did not support insulin-induced Glut1 translocation. Thus, unlike Glut4, S474 phosphorylation was required for insulin to signal to Glut1 (Figure 7E). Akt2-W80A-T309A did not support Glut1 translocation, consistent with phosphorylation of T309 being required for activity (Figure 7E).

To link glucose uptake to Glut1 expression in the plasma membrane, we used Indinavir, an HIV protease inhibitor with an off-target effect of directly inhibiting Glut4-mediated glucose uptake (Murata et al., 2002; Rudich et al., 2003). Indinavir blunted insulin-stimulated glucose uptake downstream of both Akt2-W80 and Akt2-W80A-S474A, consistent with the bulk of increased glucose uptake being mediated by Glut4 (Figure 7F). Importantly, the differential in glucose uptake between adipocytes expressing Akt2-W80A and Akt2-W80A-S474A was unaffected by Indinavir, consistent with the reduced insulin-stimulated glucose uptake supported by S474A is due to reduced Glut1-mediated glucose transport. There were no differences in expression of endogenous Glut1 between adipocytes expressing Akt2-W80A or Akt2-W80A-S474A, confirming reduced glucose uptake was due a defect in translocation rather than reduced Glut1 expression (Figure 7—figure supplement 2).

Phospho-S474-Akt2-dependent increased plasma membrane Glut1 is independent of AS160 inactivation and is not due to effects on general endocytic recycling

AS160 is the principal Akt target required for insulin-stimulated Glut4 translocation (Eguez et al., 2005; Mîinea et al., 2005; Sano et al., 2003). Akt phosphorylation inhibits AS160 GAP function, resulting in Rab10-dependent Glut4 translocation (Sano et al., 2007). An AS160 mutant in which 4 of its Akt phosphorylation sites are mutated to alanine (AS160-4A) is a dominant inhibitor of insulin-stimulated Glut4 translocation (Zeigerer et al., 2004). AS160-4A did not inhibit Glut1 translocation (Figure 8A). These data demonstrate Akt2 promotes increased plasma membrane Glut1 independent of its effect on Glut4, consistent with previous studies demonstrating Glut1 is not trafficked to the plasma membrane through the same pathway used by Glut4 (Lee et al., 2015; Palmada et al., 2006; Olsen et al., 2014).

Figure 8

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Insulin promotes an increase in general membrane recycling back to the plasma membrane, revealed by increased transferrin receptor on the plasma membrane (Subtil et al., 2000; Johnson et al., 1998). This effect was not dependent on S474 phosphorylation since Akt2-W80-S474A supports insulin effects on the transferrin receptor (Figure 8B). Thus, insulin-stimulated Glut1 translocation is not an effect of Akt2 on the general endocytic recycling pathway, demonstrating that a pathway specifically downstream of Akt2 phospho-S474 targets Glut1 trafficking.

The Akt kinases are a central node in cell signaling. The two activating phosphorylations of Akt provide a mechanism by which Akt activity and signaling integrates the outputs of PDPK1 and mTORC2. The roles of the T308/9 and S473/4 for Akt activation and substrate selections have been analyzed in detail in in-vitro studies (Yang et al., 2002a; Balzano et al., 2015). Here we study their roles in intact adipocytes downstream of insulin, a natural stimulus. In adipocytes, one of the key biological output of insulin activation of Akt is the translocation of Glut4 to the plasma membrane, resulting in enhanced glucose uptake. PDPK1 phosphorylation of Akt2-T309 is required for insulin-stimulated Glut4 translocation since either T309A mutation or inhibition of PDPK1 inhibits translocation (Figure 3 and 4). These findings are in line with previous reports that T309 phosphorylation is essential for Akt activation (Alessi et al., 1997; Stokoe et al., 1997). However, Akt2 phosphorylation at S474 is not required for Glut4 translocation in adipocytes, despite the fact that this phosphorylation is robustly stimulated by insulin and therefore is often used as a sentinel of insulin-sensitivity. Neither mutation of S474 to alanine nor inactivation of mTORC2 by silencing of Rictor, affected insulin-stimulated Glut4 translocation (Figure 3 and 4). Thus, insulin-regulated glucose uptake by adipocytes, a critical feature for whole body glucose homeostasis, is independent of mTORC2 phosphorylation of Akt. S6 kinase phosphorylation, an Akt-dependent activity downstream of mTORC1, was also independent of S474 phosphorylation.

Past studies have shown that mTORC2 phosphorylation of S473/4 has a role in Akt substrate selection. For example, ablation of Rictor-mTORC2 depressed FoxO1/3 phosphorylation, whereas phosphorylation of Akt substrates TSC2 and GSK3 were not affected (Jacinto et al., 2006). Our work contributes to understanding the role of phospho-S474 Akt in substrate selection by demonstrating this phosphorylation is required for functional regulation of Glut1 but not Glut4.

The direct role of Akt S474 phosphorylation in Glut4 translocation has not been investigated in the context of native insulin regulation of Akt activity (Chen et al., 2003; Ducluzeau et al., 2002). Two groups have shown a loss of Akt S473 phosphorylation and decreased insulin-stimulated adipocyte glucose uptake in primary adipocytes from adipocyte-specific Rictor knockout mice (Tang et al., 2016; Kumar et al., 2010). However, neither study established that defective insulin-stimulated Glut4 translocation was responsible for reduced glucose uptake nor did the studies agree on a mechanism for the decreased glucose uptake. Thus, our finding that S474 phosphorylation is not required for coupling of Akt activation to Glut4 translocation is compatible with the data reported for the Rictor knockouts. Furthermore, in light of our results, it is likely that the defect in glucose uptake in the Rictor knockout mice is not due to a defect in Glut4 translocation but rather due to another effect of the Rictor knockout on glucose metabolism. mTORC2 also phosphorylates several other kinases, including protein kinase A, protein kinase G and protein kinase C (Laplante and Sabatini, 2012).

Unexpectedly, we found that Akt2 S474 phosphorylation was required for insulin-stimulated Glut1 translocation. Both translocation of Glut1 to the plasma membrane of adipocytes and its contribution to increased glucose uptake were S474 phosphorylation-dependent (Figure 7). Insulin and other growth factors stimulate an approximate 2-fold increase of Glut1 in the plasma membranes of a variety of cell types, although how this is achieved is not known. The principal Akt substrate involved in Glut4 translocation, AS160, is not required for Glut1 translocation. The dominant-inhibitory AS160 mutant, AS160-4A, which inhibits Glut4 translocation, does not affect insulin-stimulated Glut1 translocation. The S474 phosphorylation-dependent translocation of Glut1 to the plasma membrane is not a result of Akt regulation of general endocytic recycling because insulin-regulation of transferrin receptor trafficking was not dependent on S474 phosphorylation. These data establish that Akt2 specifically regulates the amount of Glut1 in the plasma membrane by a mechanism requiring S474 phosphorylation.

The control of Glut1 plasma membrane expression is not responsible for insulin regulation of glucose homeostasis. That effect of insulin is dependent on regulation of Glut4 in adipocytes and muscle, cell types that express very little Glut1 (Mitsumoto et al., 1991). Insulin stimulation of glucose uptake into those cells serves the postprandial needs of whole body metabolism rather than the intrinsic needs of the fat and muscle cells. In other cell types the effect of growth factors on Glut1 expression in the plasma membrane (and the resultant stimulated glucose uptake) fulfil cell-intrinsic needs, such as fueling an anabolic metabolism to support cell growth and tissue expansion (Olson et al., 1996). Our findings reveal a specific role for phospho-S474 Akt in cellular glucose uptake mediated by Glut1, demonstrating that mTORC2 contributes to the regulation of glucose transport into cells other than those required to maintain whole body glucose homeostasis, and it does so by signaling dependent on Akt phosphorylated at S474.

Glut1 has a role in the enhanced glucose uptake characteristic of many tumor cells. This increased Glut1 activity is achieved by increased expression as well changes in Glut1 trafficking that promote accumulation of Glut1 in the plasma membrane (Egert et al., 1999). Akt is hyper-activated in many cancers, and our data suggest that one consequence is enhanced expression of Glut1 in the plasma membrane based on phospho-S474 Akt control of Glut1 trafficking. There is an emerging appreciation of mTORC2’s role in cancer, specifically in the control of glycolytic metabolism (Masui et al., 2014). mTORC2, via phospho-S474 Akt, may promote Glut1-mediated glucose uptake and therefore contribute to enhanced glycolytic metabolism.

SIN1, a component of mTORC2 complex, is phosphorylated by PDPK1-activated Akt, which results in activation of mTORC2, and activated mTORC2 then phosphorylates Akt (Yang et al., 2015). This positive feedforward loop was demonstrated in a number of cell contexts and it may have a role to induce full Akt activation. However, this feedforward regulation is not required for all Akt regulated pathways since as we have shown here, S474A mutation did not quantitatively affect insulin-stimulated Glut4 translocation or mTORC1 activation (i.e., S6 kinase phosphorylation).

Although S474 phosphorylation, which is within the HM domain, is not required to promote Glut4 translocation, the HM domain itself is required for Akt activity. Deletion of the HM domain blocked Akt2 signaling to Glut4 translocation, despite insulin-stimulated phosphorylation of T309 and providing the HM domain in trans did not complement the defect (Figure 6). However, co-expression of the HM domain fused to a PH domain restored insulin-stimulated Glut4 translocation. The enforced co-localization, and subsequent local concentrations of the KD and HM domains, achieved by independently targeting the kinase and HM domains to plasma membrane sites of PI3 kinase activity is sufficient to reconstitute Akt function in Glut4 translocation. Although function is restored, S474 is not phosphorylated. These data reinforce the importance of the HM domain for Akt action and are consistent with the modular nature of Akt activation and substrate selection (Yang et al., 2002a).

The loss of function T309A mutation and the gain of function S474D mutation provide additional evidence that insulin activation of Akt is necessary and sufficient to induce Glut4 redistribution to the plasma membrane of adipocytes. The activity of S474D without insulin stimulation demonstrates that Akt can be activated based on the unstimulated levels of PI3 kinase and PDPK1 activities. Those findings raise the question of whether the activated levels of PI3 kinase and PDPK1 in growth factor-stimulated cells determine how quickly Akt is activated rather than PI3 kinase/PDPK1 stimulation determining the level of Akt activation achieved.

1. 1. Abel ED
   2. Peroni O
   3. Kim JK
   4. Kim YB
   5. Boss O
   6. Hadro E
   7. Minnemann T
   8. Shulman GI
   9. Kahn BB

   (2001) Adipose-selective targeting of the GLUT4 gene impairs insulin action in muscle and liver

   Nature 409:729–733.

   https://doi.org/10.1038/35055575

   • PubMed
   • Google Scholar
2. 1. Alessi DR
   2. Andjelkovic M
   3. Caudwell B
   4. Cron P
   5. Morrice N
   6. Cohen P
   7. Hemmings BA

   (1996)

   Mechanism of activation of protein kinase B by insulin and IGF-1

   The EMBO Journal 15:6541–6551.

   • PubMed
   • Google Scholar
3. 1. Alessi DR
   2. James SR
   3. Downes CP
   4. Holmes AB
   5. Gaffney PR
   6. Reese CB
   7. Cohen P

   (1997) Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase balpha

   Current Biology 7:261–269.

   https://doi.org/10.1016/S0960-9822(06)00122-9

   • PubMed
   • Google Scholar
4. 1. Balzano D
   2. Fawal MA
   3. Velázquez JV
   4. Santiveri CM
   5. Yang J
   6. Pastor J
   7. Campos-Olivas R
   8. Djouder N
   9. Lietha D

   (2015) Alternative activation mechanisms of protein kinase B trigger distinct downstream signaling responses

   Journal of Biological Chemistry 290:24975–24985.

   https://doi.org/10.1074/jbc.M115.651570

   • PubMed
   • Google Scholar
5. 1. Boguslavsky S
   2. Chiu T
   3. Foley KP
   4. Osorio-Fuentealba C
   5. Antonescu CN
   6. Bayer KU
   7. Bilan PJ
   8. Klip A

   (2012) Myo1c binding to submembrane actin mediates insulin-induced tethering of GLUT4 vesicles

   Molecular Biology of the Cell 23:4065–4078.

   https://doi.org/10.1091/mbc.E12-04-0263

   • PubMed
   • Google Scholar
6. 1. Calleja V
   2. Laguerre M
   3. Parker PJ
   4. Larijani B

   (2009) Role of a novel PH-kinase domain interface in PKB/Akt regulation: structural mechanism for allosteric inhibition

   PLoS Biology 7:e17.

   https://doi.org/10.1371/journal.pbio.1000017

   • PubMed
   • Google Scholar
7. 1. Carpten JD
   2. Faber AL
   3. Horn C
   4. Donoho GP
   5. Briggs SL
   6. Robbins CM
   7. Hostetter G
   8. Boguslawski S
   9. Moses TY
   10. Savage S
   11. Uhlik M
   12. Lin A
   13. Du J
   14. Qian YW
   15. Zeckner DJ
   16. Tucker-Kellogg G
   17. Touchman J
   18. Patel K
   19. Mousses S
   20. Bittner M
   21. Schevitz R
   22. Lai MH
   23. Blanchard KL
   24. Thomas JE

   (2007) A transforming mutation in the pleckstrin homology domain of AKT1 in Cancer

   Nature 448:439–444.

   https://doi.org/10.1038/nature05933

   • PubMed
   • Google Scholar
8. 1. Carvalho KC
   2. Cunha IW
   3. Rocha RM
   4. Ayala FR
   5. Cajaíba MM
   6. Begnami MD
   7. Vilela RS
   8. Paiva GR
   9. Andrade RG
   10. Soares FA

   (2011) GLUT1 expression in malignant tumors and its use as an immunodiagnostic marker

   Clinics 66:965–972.

   https://doi.org/10.1590/S1807-59322011000600008

   • PubMed
   • Google Scholar
9. 1. Chan DA
   2. Sutphin PD
   3. Nguyen P
   4. Turcotte S
   5. Lai EW
   6. Banh A
   7. Reynolds GE
   8. Chi JT
   9. Wu J
   10. Solow-Cordero DE
   11. Bonnet M
   12. Flanagan JU
   13. Bouley DM
   14. Graves EE
   15. Denny WA
   16. Hay MP
   17. Giaccia AJ

   (2011) Targeting GLUT1 and the Warburg effect in renal cell carcinoma by chemical synthetic lethality

   Science Translational Medicine 3:ra70.

   https://doi.org/10.1126/scitranslmed.3002394

   • PubMed
   • Google Scholar
10. 1. Chan TO
    2. Rittenhouse SE
    3. Tsichlis PN

    (1999) AKT/PKB and other D3 phosphoinositide-regulated kinases: kinase activation by phosphoinositide-dependent phosphorylation

    Annual Review of Biochemistry 68:965–1014.

    https://doi.org/10.1146/annurev.biochem.68.1.965

    • PubMed
    • Google Scholar
11. 1. Chaudhary N
    2. Gonzalez E
    3. Chang SH
    4. Geng F
    5. Rafii S
    6. Altorki NK
    7. McGraw TE

    (2016) Adenovirus protein E4-ORF1 activation of PI3 kinase reveals differential regulation of downstream effector pathways in adipocytes

    Cell Reports 17:3305–3318.

    https://doi.org/10.1016/j.celrep.2016.11.082

    • PubMed
    • Google Scholar
12. 1. Chen X
    2. Al-Hasani H
    3. Olausson T
    4. Wenthzel AM
    5. Smith U
    6. Cushman SW
    7. Activity CSW

    (2003) Activity, phosphorylation state and subcellular distribution of GLUT4-targeted Akt2 in rat adipose cells

    Journal of Cell Science 116:3511–3518.

    https://doi.org/10.1242/jcs.00675

    • PubMed
    • Google Scholar
13. 1. Choi H
    2. Sheng J
    3. Gao D
    4. Li F
    5. Durrans A
    6. Ryu S
    7. Lee SB
    8. Narula N
    9. Rafii S
    10. Elemento O
    11. Altorki NK
    12. Wong ST
    13. Mittal V

    (2015) Transcriptome analysis of individual stromal cell populations identifies stroma-tumor crosstalk in mouse lung cancer model

    Cell Reports 10:1187–1201.

    https://doi.org/10.1016/j.celrep.2015.01.040

    • PubMed
    • Google Scholar
14. 1. Clarke JF
    2. Young PW
    3. Yonezawa K
    4. Kasuga M
    5. Holman GD

    (1994) Inhibition of the translocation of GLUT1 and GLUT4 in 3T3-L1 cells by the phosphatidylinositol 3-kinase inhibitor, wortmannin

    Biochemical Journal 300 ( Pt 3:631–635.

    https://doi.org/10.1042/bj3000631

    • PubMed
    • Google Scholar
15. 1. Ducluzeau PH
    2. Fletcher LM
    3. Welsh GI
    4. Tavaré JM

    (2002)

    Functional consequence of targeting protein kinase B/Akt to GLUT4 vesicles

    Journal of Cell Science 115:2857–2866.

    • PubMed
    • Google Scholar
16. 1. Egert S
    2. Nguyen N
    3. Schwaiger M

    (1999) Myocardial glucose transporter GLUT1: translocation induced by insulin and ischemia

    Journal of Molecular and Cellular Cardiology 31:1337–1344.

    https://doi.org/10.1006/jmcc.1999.0965

    • PubMed
    • Google Scholar
17. 1. Eguez L
    2. Lee A
    3. Chavez JA
    4. Miinea CP
    5. Kane S
    6. Lienhard GE
    7. McGraw TE

    (2005) Full intracellular retention of GLUT4 requires AS160 Rab GTPase activating protein

    Cell Metabolism 2:263–272.

    https://doi.org/10.1016/j.cmet.2005.09.005

    • PubMed
    • Google Scholar
18. 1. Franke TF

    (2008) PI3K/Akt: getting it right matters

    Oncogene 27:6473–6488.

    https://doi.org/10.1038/onc.2008.313

    • PubMed
    • Google Scholar
19. 1. Frech M
    2. Andjelkovic M
    3. Ingley E
    4. Reddy KK
    5. Falck JR
    6. Hemmings BA

    (1997) High affinity binding of inositol phosphates and phosphoinositides to the pleckstrin homology domain of RAC/protein kinase B and their influence on kinase activity

    Journal of Biological Chemistry 272:8474–8481.

    https://doi.org/10.1074/jbc.272.13.8474

    • PubMed
    • Google Scholar
20. 1. Geraghty KM
    2. Chen S
    3. Harthill JE
    4. Ibrahim AF
    5. Toth R
    6. Morrice NA
    7. Vandermoere F
    8. Moorhead GB
    9. Hardie DG
    10. MacKintosh C

    (2007) Regulation of multisite phosphorylation and 14-3-3 binding of AS160 in response to IGF-1, EGF, PMA and AICAR

    Biochemical Journal 407:231–241.

    https://doi.org/10.1042/BJ20070649

    • PubMed
    • Google Scholar
21. 1. Gonzalez E
    2. McGraw TE

    (2009a) The akt kinases: isoform specificity in metabolism and Cancer

    Cell Cycle 8:2502–2508.

    https://doi.org/10.4161/cc.8.16.9335

    • PubMed
    • Google Scholar
22. 1. Gonzalez E
    2. McGraw TE

    (2009b) Insulin-modulated akt subcellular localization determines akt isoform-specific signaling

    PNAS 106:7004–7009.

    https://doi.org/10.1073/pnas.0901933106

    • PubMed
    • Google Scholar
23. 1. Green CJ
    2. Göransson O
    3. Kular GS
    4. Leslie NR
    5. Gray A
    6. Alessi DR
    7. Sakamoto K
    8. Hundal HS

    (2008) Use of Akt inhibitor and a drug-resistant mutant validates a critical role for protein kinase B/Akt in the insulin-dependent regulation of glucose and system A amino acid uptake

    Journal of Biological Chemistry 283:27653–27667.

    https://doi.org/10.1074/jbc.M802623200

    • PubMed
    • Google Scholar
24. 1. Hart JR
    2. Vogt PK

    (2011) Phosphorylation of AKT: a mutational analysis

    Oncotarget 2:467–476.

    https://doi.org/10.18632/oncotarget.293

    • PubMed
    • Google Scholar
25. 1. Jacinto E
    2. Facchinetti V
    3. Liu D
    4. Soto N
    5. Wei S
    6. Jung SY
    7. Huang Q
    8. Qin J
    9. Su B

    (2006) SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates akt phosphorylation and substrate specificity

    Cell 127:125–137.

    https://doi.org/10.1016/j.cell.2006.08.033

    • PubMed
    • Google Scholar
26. 1. Jiang ZY
    2. Zhou QL
    3. Coleman KA
    4. Chouinard M
    5. Boese Q
    6. Czech MP

    (2003) Insulin signaling through akt/protein kinase B analyzed by small interfering RNA-mediated gene silencing

    PNAS 100:7569–7574.

    https://doi.org/10.1073/pnas.1332633100

    • PubMed
    • Google Scholar
27. 1. Johnson AO
    2. Subtil A
    3. Petrush R
    4. Kobylarz K
    5. Keller SR
    6. McGraw TE

    (1998) Identification of an insulin-responsive, slow endocytic recycling mechanism in chinese hamster ovary cells

    Journal of Biological Chemistry 273:17968–17977.

    https://doi.org/10.1074/jbc.273.28.17968

    • PubMed
    • Google Scholar
28. 1. Kajno E
    2. McGraw TE
    3. Gonzalez E

    (2015) Development of a new model system to dissect isoform specific akt signalling in adipocytes

    Biochemical Journal 468:425–434.

    https://doi.org/10.1042/BJ20150191

    • PubMed
    • Google Scholar
29. 1. Karylowski O
    2. Zeigerer A
    3. Cohen A
    4. McGraw TE

    (2004) GLUT4 is retained by an intracellular cycle of vesicle formation and fusion with endosomes

    Molecular Biology of the Cell 15:870–882.

    https://doi.org/10.1091/mbc.E03-07-0517

    • PubMed
    • Google Scholar
30. 1. Kumar A
    2. Lawrence JC
    3. Jung DY
    4. Ko HJ
    5. Keller SR
    6. Kim JK
    7. Magnuson MA
    8. Harris TE

    (2010) Fat cell-specific ablation of rictor in mice impairs insulin-regulated fat cell and whole-body glucose and lipid metabolism

    Diabetes 59:1397–1406.

    https://doi.org/10.2337/db09-1061

    • PubMed
    • Google Scholar
31. 1. Lampson MA
    2. Schmoranzer J
    3. Zeigerer A
    4. Simon SM
    5. McGraw TE

    (2001) Insulin-regulated release from the endosomal recycling compartment is regulated by budding of specialized vesicles

    Molecular Biology of the Cell 12:3489–3501.

    https://doi.org/10.1091/mbc.12.11.3489

    • PubMed
    • Google Scholar
32. 1. Laplante M
    2. Sabatini DM

    (2012) mTOR signaling in growth control and disease

    Cell 149:274–293.

    https://doi.org/10.1016/j.cell.2012.03.017

    • PubMed
    • Google Scholar
33. 1. Lawrence JC
    2. Piper RC
    3. Robinson LJ
    4. James DE

    (1992) GLUT4 facilitates insulin stimulation and cAMP-mediated inhibition of glucose transport

    PNAS 89:3493–3497.

    https://doi.org/10.1073/pnas.89.8.3493

    • PubMed
    • Google Scholar
34. 1. Leavens KF
    2. Easton RM
    3. Shulman GI
    4. Previs SF
    5. Birnbaum MJ

    (2009) Akt2 is required for hepatic lipid accumulation in models of insulin resistance

    Cell Metabolism 10:405–418.

    https://doi.org/10.1016/j.cmet.2009.10.004

    • PubMed
    • Google Scholar
35. 1. Lee EE
    2. Ma J
    3. Sacharidou A
    4. Mi W
    5. Salato VK
    6. Nguyen N
    7. Jiang Y
    8. Pascual JM
    9. North PE
    10. Shaul PW
    11. Mettlen M
    12. Wang RC

    (2015) A protein kinase C phosphorylation motif in GLUT1 affects glucose transport and is mutated in GLUT1 deficiency syndrome

    Molecular Cell 58:845–853.

    https://doi.org/10.1016/j.molcel.2015.04.015

    • PubMed
    • Google Scholar
36. 1. Manning BD
    2. Cantley LC

    (2007) AKT/PKB signaling: navigating Downstream

    Cell 129:1261–1274.

    https://doi.org/10.1016/j.cell.2007.06.009

    • PubMed
    • Google Scholar
37. 1. Masui K
    2. Cavenee WK
    3. Mischel PS

    (2014) mTORC2 in the center of Cancer metabolic reprogramming

    Trends in Endocrinology & Metabolism 25:364–373.

    https://doi.org/10.1016/j.tem.2014.04.002

    • PubMed
    • Google Scholar
38. 1. Mitsumoto Y
    2. Burdett E
    3. Grant A
    4. Klip A

    (1991) Differential expression of the GLUT1 and GLUT4 glucose transporters during differentiation of L6 muscle cells

    Biochemical and Biophysical Research Communications 175:652–659.

    https://doi.org/10.1016/0006-291X(91)91615-J

    • PubMed
    • Google Scholar
39. 1. Murata H
    2. Hruz PW
    3. Mueckler M

    (2002) Indinavir inhibits the glucose transporter isoform Glut4 at physiologic concentrations

    AIDS 16:859–863.

    https://doi.org/10.1097/00002030-200204120-00005

    • PubMed
    • Google Scholar
40. 1. Mîinea CP
    2. Sano H
    3. Kane S
    4. Sano E
    5. Fukuda M
    6. Peränen J
    7. Lane WS
    8. Lienhard GE

    (2005) AS160, the Akt substrate regulating GLUT4 translocation, has a Functional Rab GTPase-activating protein domain

    Biochemical Journal 391:87–93.

    https://doi.org/10.1042/BJ20050887

    • PubMed
    • Google Scholar
41. 1. Olsen JM
    2. Sato M
    3. Dallner OS
    4. Sandström AL
    5. Pisani DF
    6. Chambard JC
    7. Amri EZ
    8. Hutchinson DS
    9. Bengtsson T

    (2014) Glucose uptake in brown fat cells is dependent on mTOR complex 2-promoted GLUT1 translocation

    The Journal of Cell Biology 207:365–374.

    https://doi.org/10.1083/jcb.201403080

    • PubMed
    • Google Scholar
42. 1. Olson AL
    2. Pessin JE
    3. Structure PJE

    (1996) Structure, function, and regulation of the mammalian facilitative glucose transporter gene family

    Annual Review of Nutrition 16:235–256.

    https://doi.org/10.1146/annurev.nu.16.070196.001315

    • PubMed
    • Google Scholar
43. 1. Palmada M
    2. Boehmer C
    3. Akel A
    4. Rajamanickam J
    5. Jeyaraj S
    6. Keller K
    7. Lang F

    (2006) SGK1 kinase upregulates GLUT1 activity and plasma membrane expression

    Diabetes 55:421–427.

    https://doi.org/10.2337/diabetes.55.02.06.db05-0720

    • PubMed
    • Google Scholar
44. 1. Rett K
    2. Wicklmayr M
    3. Dietze GJ
    4. Häring HU

    (1996) Insulin-induced glucose transporter (GLUT1 and GLUT4) translocation in cardiac muscle tissue is mimicked by bradykinin

    Diabetes 45 Suppl 1:S66–S69.

    https://doi.org/10.2337/diab.45.1.S66

    • PubMed
    • Google Scholar
45. 1. Rudich A
    2. Konrad D
    3. Török D
    4. Ben-Romano R
    5. Huang C
    6. Niu W
    7. Garg RR
    8. Wijesekara N
    9. Germinario RJ
    10. Bilan PJ
    11. Klip A

    (2003) Indinavir uncovers different contributions of GLUT4 and GLUT1 towards glucose uptake in muscle and fat cells and tissues

    Diabetologia 46:649–658.

    https://doi.org/10.1007/s00125-003-1080-1

    • PubMed
    • Google Scholar
46. 1. Sano H
    2. Eguez L
    3. Teruel MN
    4. Fukuda M
    5. Chuang TD
    6. Chavez JA
    7. Lienhard GE
    8. McGraw TE

    (2007) Rab10, a target of the AS160 Rab GAP, is required for insulin-stimulated translocation of GLUT4 to the adipocyte plasma membrane

    Cell Metabolism 5:293–303.

    https://doi.org/10.1016/j.cmet.2007.03.001

    • PubMed
    • Google Scholar
47. 1. Sano H
    2. Kane S
    3. Sano E
    4. Mîinea CP
    5. Asara JM
    6. Lane WS
    7. Garner CW
    8. Lienhard GE

    (2003) Insulin-stimulated phosphorylation of a Rab GTPase-activating protein regulates GLUT4 translocation

    Journal of Biological Chemistry 278:14599–14602.

    https://doi.org/10.1074/jbc.C300063200

    • PubMed
    • Google Scholar
48. 1. Sarbassov DD
    2. Guertin DA
    3. Ali SM
    4. Sabatini DM

    (2005) Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex

    Science 307:1098–1101.

    https://doi.org/10.1126/science.1106148

    • PubMed
    • Google Scholar
49. 1. Sparks CA
    2. Guertin DA

    (2010) Targeting mTOR: prospects for mTOR complex 2 inhibitors in Cancer therapy

    Oncogene 29:3733–3744.

    https://doi.org/10.1038/onc.2010.139

    • PubMed
    • Google Scholar
50. 1. Stokoe D
    2. Stephens LR
    3. Copeland T
    4. Gaffney PR
    5. Reese CB
    6. Painter GF
    7. Holmes AB
    8. McCormick F
    9. Hawkins PT

    (1997) Dual role of phosphatidylinositol-3,4,5-trisphosphate in the activation of protein kinase B

    Science 277:567–570.

    https://doi.org/10.1126/science.277.5325.567

    • PubMed
    • Google Scholar
51. 1. Subtil A
    2. Lampson MA
    3. Keller SR
    4. McGraw TE

    (2000) Characterization of the insulin-regulated endocytic recycling mechanism in 3T3-L1 adipocytes using a novel reporter molecule

    Journal of Biological Chemistry 275:4787–4795.

    https://doi.org/10.1074/jbc.275.7.4787

    • PubMed
    • Google Scholar
52. 1. Takenouchi N
    2. Jones KS
    3. Lisinski I
    4. Fugo K
    5. Yao K
    6. Cushman SW
    7. Ruscetti FW
    8. Jacobson S

    (2007) GLUT1 is not the primary binding receptor but is associated with cell-to-cell transmission of human T-cell leukemia virus type 1

    Journal of Virology 81:1506–1510.

    https://doi.org/10.1128/JVI.01522-06

    • PubMed
    • Google Scholar
53. 1. Tang Y
    2. Wallace M
    3. Sanchez-Gurmaches J
    4. Hsiao WY
    5. Li H
    6. Lee PL
    7. Vernia S
    8. Metallo CM
    9. Guertin DA

    (2016) Adipose tissue mTORC2 regulates ChREBP-driven de novo lipogenesis and hepatic glucose metabolism

    Nature Communications 7:11365.

    https://doi.org/10.1038/ncomms11365

    • PubMed
    • Google Scholar
54. 1. Vazirani RP
    2. Verma A
    3. Sadacca LA
    4. Buckman MS
    5. Picatoste B
    6. Beg M
    7. Torsitano C
    8. Bruno JH
    9. Patel RT
    10. Simonyte K
    11. Camporez JP
    12. Moreira G
    13. Falcone DJ
    14. Accili D
    15. Elemento O
    16. Shulman GI
    17. Kahn BB
    18. McGraw TE

    (2016) Disruption of adipose Rab10-Dependent insulin signaling causes hepatic insulin resistance

    Diabetes 65:1577–1589.

    https://doi.org/10.2337/db15-1128

    • PubMed
    • Google Scholar
55. 1. Vivanco I
    2. Sawyers CL

    (2002) The phosphatidylinositol 3-Kinase AKT pathway in human Cancer

    Nature Reviews Cancer 2:489–501.

    https://doi.org/10.1038/nrc839

    • PubMed
    • Google Scholar
56. 1. Ward PS
    2. Thompson CB

    (2012) Signaling in control of cell growth and metabolism

    Cold Spring Harbor Perspectives in Biology 4:a006783.

    https://doi.org/10.1101/cshperspect.a006783

    • PubMed
    • Google Scholar
57. 1. Yang G
    2. Murashige DS
    3. Humphrey SJ
    4. James DE

    (2015) A positive feedback Loop between Akt and mTORC2 via SIN1 phosphorylation

    Cell Reports 12:937–943.

    https://doi.org/10.1016/j.celrep.2015.07.016

    • PubMed
    • Google Scholar
58. 1. Yang J
    2. Cron P
    3. Good VM
    4. Thompson V
    5. Hemmings BA
    6. Barford D

    (2002a) Crystal structure of an activated akt/protein kinase B ternary complex with GSK3-peptide and AMP-PNP

    Nature Structural Biology 9:940–944.

    https://doi.org/10.1038/nsb870

    • PubMed
    • Google Scholar
59. 1. Yang J
    2. Cron P
    3. Thompson V
    4. Good VM
    5. Hess D
    6. Hemmings BA
    7. Barford D

    (2002b) Molecular mechanism for the regulation of protein kinase B/Akt by hydrophobic motif phosphorylation

    Molecular Cell 9:1227–1240.

    https://doi.org/10.1016/S1097-2765(02)00550-6

    • PubMed
    • Google Scholar
60. 1. Zeigerer A
    2. Lampson MA
    3. Karylowski O
    4. Sabatini DD
    5. Adesnik M
    6. Ren M
    7. McGraw TE

    (2002) GLUT4 retention in adipocytes requires two intracellular insulin-regulated transport steps

    Molecular Biology of the Cell 13:2421–2435.

    https://doi.org/10.1091/mbc.E02-02-0071

    • PubMed
    • Google Scholar
61. 1. Zeigerer A
    2. McBrayer MK
    3. McGraw TE

    (2004) Insulin stimulation of GLUT4 exocytosis, but not its inhibition of endocytosis, is dependent on RabGAP AS160

    Molecular Biology of the Cell 15:4406–4415.

    https://doi.org/10.1091/mbc.E04-04-0333

    • PubMed
    • Google Scholar
62. 1. Zhao P
    2. Yang L
    3. Lopez JA
    4. Fan J
    5. Burchfield JG
    6. Bai L
    7. Hong W
    8. Xu T
    9. James DE

    (2009) Variations in the requirement for v-SNAREs in GLUT4 trafficking in adipocytes

    Journal of Cell Science 122:3472–3480.

    https://doi.org/10.1242/jcs.047449

    • PubMed
    • Google Scholar
63. 1. Zisman A
    2. Peroni OD
    3. Abel ED
    4. Michael MD
    5. Mauvais-Jarvis F
    6. Lowell BB
    7. Wojtaszewski JF
    8. Hirshman MF
    9. Virkamaki A
    10. Goodyear LJ
    11. Kahn CR
    12. Kahn BB

    (2000) Targeted disruption of the glucose transporter 4 selectively in muscle causes insulin resistance and glucose intolerance

    Nature Medicine 6:924–928.

    https://doi.org/10.1038/78693

    • PubMed
    • Google Scholar

Author details

1. Muheeb Beg

   Department of Biochemistry, Weill Cornell Medicine, New York, United States

   Contribution

   MB, Conceptualization, Data curation, Formal analysis, Writing—original draft

   Contributed equally with

   Nazish Abdullah

   Competing interests

   The authors declare that no competing interests exist.

2. Nazish Abdullah

   Department of Biochemistry, Weill Cornell Medicine, New York, United States

   Contribution

   NA, Conceptualization, Formal analysis, Investigation

   Contributed equally with

   Muheeb Beg

   Competing interests

   The authors declare that no competing interests exist.

3. Fathima Shazna Thowfeik

   1. Department of Cardiothoracic Surgery, Weill Cornell Medicine, New York, United States
   2. Lung Cancer Program, Meyer Cancer Center, Weill Cornell Medicine, New York, United States

   Contribution

   FST, Investigation, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

4. Nasser K Altorki

   1. Department of Cardiothoracic Surgery, Weill Cornell Medicine, New York, United States
   2. Lung Cancer Program, Meyer Cancer Center, Weill Cornell Medicine, New York, United States

   Contribution

   NKA, Conceptualization, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

5. Timothy E McGraw

   1. Department of Biochemistry, Weill Cornell Medicine, New York, United States
   2. Department of Cardiothoracic Surgery, Weill Cornell Medicine, New York, United States
   3. Lung Cancer Program, Meyer Cancer Center, Weill Cornell Medicine, New York, United States

   Contribution

   TEM, Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   temcgraw@med.cornell.edu

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0001-9748-263X

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