A herpesvirus kinase that masquerades as Akt: You don't have to look like Akt,to act like it

The cellular protein synthesis machinery is tightly regulated and capable of rapid reaction to a variety of physiological inputs critical in stress-response, cell cycle control, cancer biology and virus infection. One important strategy for stimulating protein synthesis involves the ser/thr kinase Akt, which subsequently triggers inactivation of the cap-dependent translational repressor 4E-BP1 by an mTOR-containing protein complex (mTORC1). A recent paper demonstrated that herpes simplex virus utilizes a remarkable tactic to activate mTOR in infected cells. Instead of using the cellular Akt, the virus produces a ser/thr kinase called Us3 that doesn''t look like Akt, but masquerades as Akt. By making the Akt-like protein unrecognizable, this disguise allows it to bypass the strict limits normally imposed on the real cellular Akt. Importantly, preventing the virus Akt-imposter from triggering mTORC1 inhibited viral growth, suggesting a new way to block herpes simplex virus. This study also raises the possibility that other Akt-impersonators may lurk hidden in our own genomes, possibly contributing to diseases ranging from diabetes to cancer.Key words: Akt signaling, translational control, mTORC1 activation, virus replication, viral kinaseBy manipulating activity of the translation repressor 4E-BP1, Akt signaling through mTORC1 controls a critical step regulating the initiation of protein synthesis in eukaryotes.¹ 4E-BP1 represses translation by binding to the cellular cap-binding protein, eukaryotic translation initiation factor 4E (eIF4E), preventing its incorporation into the eIF4F multi-subunit complex required to initiate translation.² Hyperphosphorylation of 4E-BP1 by mTORC1 inactivates the repressor, allowing eIF4E to associate with the large molecular scaffold eIF4G and assemble eIF4F (Fig. 1).¹ Subsequently, hyperphosphorylated 4E-BP1 can be degraded by the proteasome.^3–5 Regulating assembly of eIF4F is a major point through which 40S ribosome subunits are recruited and loaded onto the mRNA 5′ end. Indeed, numerous biological regulatory processes where differential control of translation plays a fundamental role often involve 4E-BP1 hyperphosphorylation by mTORC1.⁶Open in a separate windowFigure 1Repression of eIF4F assembly and cap-dependent mRNA translation by 4E-BP1 phosphorylation. The cellular cap-binding protein (4E) is depicted bound to the translational repressor eIF4E-binding protein 1 (4E-BP1) and is unable to assemble into an eIF4F complex with the other translation initiation factors, eIF4G and eIF4A. Activation of the kinase mTOR in response to a variety of cues such as HSV-1 infection, growth factor signaling, alterations to the nutrient pool or changes in cellular energy reserves results in phosphorylation of the translational repressor protein 4E-BP1 and the release of eIF4E from 4E-BP1. Binding of eIF4E to eIF4G and eIF4A results in assembly of the multisubunit initiation factor eIF4F complex, which in turn recognizes the 7-methyl guanine cap (m⁷) at the mRNA 5′ end. The 40S ribosome is recruited through its association with eIF3.Diverse inputs including nutrient, energy and growth factor availability are integrated by the tuberous sclerosis heterodimer complex (TSC1/2), which controls mTORC1 activation.¹ TSC is a GTPase activating protein (GAP) for the small G-protein rheb (ras-homolog enriched in brain). Whereas rheb•GTP activates mTORC1, rheb•GDP cannot. Thus, TSC GAP activation results in rheb•GDP accumulation and inactive mTORC1, while TSC GAP inhibition results in rheb•GTP accumulation and stimulates mTORC1 (Fig. 2). TSC activity is controlled by phosphorylation of the TSC2 subunit by different cellular kinases. One of these is Akt. In response to PI3-kinase activation, recruitment of PDK1 and Akt to the plasma membrane results in Akt phosphorylation at T308. Fully active Akt results after a second mTOR-containing complex, mTORC2, phosphorylates S473. Phosphorylation of TSC2 by Akt on S939 and T1462 inhibits TSC Gap activity and thereby activates mTORC1. Although mTORC1 can be regulated by inputs from other signaling pathways including Erk/RSK, which also can phosphorylate TSC2 to inhibit its GAP function and Rag-GTPases, which are required for mTORC1 activation in response to amino acids, Akt signaling is required for growth factors and hormone-responsive mTORC1 activation and the resulting stimulation of protein synthesis.⁷ In addition to TSC2 phosphorylation, Akt can also phosphorylate and inactivate PRAS40, an mTORC1 inhibitory subunit.⁸ Phosphorylation of the mTORC1 substrates ATG1, ribosomal protein S6 kinase and 4E-BP1 regulate autophagy, cell size, cell proliferation and protein synthesis.^9,10 In addition, inhibition of mTORC1 with rapamycin decelerates cellular senescence.¹¹ These basic processes are important in a variety of pathophysiological settings, including response to stress, cell cycle control, age-related diseases, cancer biology and virus infection.Open in a separate windowFigure 2Phosphorylation of TSC2 by Akt or Us3 activates mTORC1. Growth factor-mediated activation of mTORC1 is illustrated. IRS1 is recruited to the cytoplasmic face of activated growth factor receptors and stimulates PI3-kinase signaling. PDK1 and Akt are both localized to the plasma membrane via a lipid-binding plekstrin homology domain. Upon activation by PI3-kinase, PDK1 phosphorylates Akt on T308, thereby contributing to Akt activation. Full Akt activation also requires S473 phosphorylation by mTORC2. Akt inhibits TSC rheb-GAP activity by phosphorylating TSC2 on T1462/S939, promoting rheb•GTP-mediated mTORC1 activation and subsequent 4E-BP1 hyperphosphorylation and activation of p70 S6K (S6K). Inactivation of the translational repressor 4E-BP1 promotes binding of eIF4E to eIF4G, stimulating cap-dependent translation. An intrinsic feedback control circuit whereby inactivation of IR S1 and mTORC2 by activated p70 S6K limits Akt activation. Even though it is unrelated to Akt at the primary sequence level, the HSV1 ser/thr kinase Us3 phosphorylates TSC2 on the same residues targeted by Akt (T1462, S939). By targeting TSC2, this strategy allows Us3 to bypass the intrinsic feedback controls designed to limit Akt activation, allowing HSV1 to constitutively inactivate 4E-BP1 and maintain high levels of viral mRNA translation in infected cells.Following lytic infection of a host cell or reactivation from latency, herpesviruses stimulate the cap-dependent translation machinery of their hosts by promoting eIF4F assembly.^3,12–15 This is often a critical step in the virus replication cycle, as viruses are completely dependent on the translation machinery resident in their cellular hosts to produce viral proteins required for their productive growth. Similar to uninfected cells, herpesvirus-induced eIF4F assembly was sensitive to recently-developed mTOR active site inhibitors and impaired by expression of a dominant 4E-BP1 repressor allele with T→A substitutions at the key T37 and T46 sites.^16–18 However, in a major departure from findings in uninfected cells, 4E-BP1 was constitutively hyperphosphorylated in HSV-1-infected cells in the presence of allosteric Akt inhibitors.¹⁷ Precisely how HSV-1 was able to stimulate mTORC1 in the absence of Akt signaling was not known.Recently, we established that the HSV-1 ser/thr kinase encoded by the Us3 gene is required to activate mTORC1, inactivate the 4E-BP1 translational repressor and stimulate eIF4F assembly. Surprisingly, Us3 displays no sequence homology with the cellular kinase Akt, yet directly phosphorylates tuberous sclerosis complex 2 (TSC2) on S939 and T1462, the same sites targeted by Akt to inhibit TSC activity and activate mTORC1 in uninfected cells.¹⁷ While it is not unusual for virus infection to stimulate Akt signaling, this typically involves PI3-kinase activation by a virus-encoded gene product, such as Influenza virus A NS1, HSV1 VP11/12, KSHV-encoded GPCR, HCMV IE1/2, EBV LMP2A and Adenovirus E4 orf1 or a less well-understood protein phosphatase 2A-dependent process involving HPV E7 or Adenovirus E4 orf4 that may prevent dephosphorylation of mTORC1 substrates.^19–22 Akt activated in this manner is potentially limited by intrinsic feedback circuitry built into this important pathway, prohibiting sustained Akt activation (Fig. 2).²³ Indeed, transient Akt activation early in the replication cycle is observed in primary human fibroblasts infected with wild-type HSV-1 or HCMV (C. McKinney, IM, in preparation).²⁴ Failure to continuously stimulate Akt could limit mTORC1 activation, imposing substantial constraints on viruses that require the host cap-dependent translation machinery and seek to inactivate the 4E-BP1 translational repressor. Some viruses encode multiple functions capable of activating Akt or downstream targets, further illustrating the importance of this task in the virus lifecycle.²² Significantly, TSC-inactivation by Us3 allows HSV1 to activate mTORC1 even when Akt activity is low or undetectable, as may be the case in non-proliferating cells. Furthermore, by acting at the level of TSC, mTORC1 activation by Us3 is not responsive to p70 S6K-mediated cellular feedback controls in place to limit both receptor-mediated activation of the PI3K/Akt/mTORC1 signaling axis and mTORC2-mediated Akt activation (Fig. 2).^23,25 Other viruses likewise activate mTORC1 via TSC, albeit via different mechanism, illustrating the potential advantages of targeting TSC2 to stimulate mTORC1 in infected cells. The related herpesvirus HCMV encodes a TSC2-binding protein (UL38) that inhibits TSC activity, while HPV E6 binds TSC2 and targets it for proteasome degradation.^26,27 None of these strategies, however, involve direct phosphorylation of TSC2 by viral enzymes.Disabling TSC allows viruses to overcome a natural antiviral checkpoint. Interestingly, Us3-deficient virus replication is impaired relative to wild-type in normal primary human fibroblasts and replication is significantly restored upon siRNA-mediated TSC2-depletion. WT virus replication, however, is not impacted by TSC2-depletion, consistent with the observation that TSC is already inactivated in cells infected with WT HSV-1.¹⁷ Thus, TSC comprises an antiviral checkpoint for viruses that require the host cap-dependent translation machinery to produce virus-encoded polypeptides. Replication of viruses that are not equipped to counteract TSC-mediated mTORC1 repression will be limited, unless they bypass this requirement by using an alternative, cap-independent mode of translation initiation that does not require eIF4E.Not only is TSC regulated by Akt, it is also a critical juncture integrating signaling inputs from other pathways. In contrast to Akt, differential TSC2 phosphorylation by AMP-activated protein kinase (AMPK), which is responsive to elevated AMP levels resulting from energy deprivation, stimulates TSC Rheb-GAP activity and prevents mTORC1 activation.²⁸ Induction of the p53-responsive sestrins 1 and 2 in response to genotoxic stress likewise activate AMPK and stimulate TSC Rheb-GAP.²⁹ TSC2 phosphorylation by glycogen synthase kinase 3 cooperates with AMPK-mediated phosphorylation to inhibit mTORC1, linking bioenergetic state with Wnt-signaling responsive GSK3.³⁰ Finally, hypoxia-induced REDD1 activates TSC by interfering with phosphorylation-dependent association of TSC with 14-3-3 proteins.³¹ Binding of phosphorylated TSC2 to 14-3-3 has been proposed to account for Akt-mediated inhibition of TSC. Thus, REDD1-mediated displacement of 14-3-3 from phospho-TSC2 prevents mTORC1 activation even though Akt is constitutively active. In HSV1 infected primary human fibroblasts, Erk activation, which can also phosphorylate TSC2 and inhibit TSC rheb-GAP, is suppressed, making it unlikely that Erk/Rsk signaling plays a role in mTORC1 activation.³ However, how other TSC regulators respond to HSV-1 infection and the potential for Us3 to repress TSC Rheb-GAP activity in response to different stress inputs remains largely unexplored.Given the lack of primary sequence homology between Akt and Us3, it is amazing that Us3 stimulates phosphorylation of Akt substrates other than TSC2 in an Akt-independent manner. Both FOXO1 and GSK3 were among the Akt substrates phosphorylated by Us3 on the same residues targeted by Akt.¹⁷ Thus, Us3 appears to be an Akt surrogate with overlapping substrate specificity that activates mTORC1, stimulating translation and virus replication. As a unique viral kinase unrelated to any single cellular kinase, Us3 is a potential drug development target. Small molecule Us3 inhibitors could prevent HSV1-induced mTORC1 activation and effectively suppress replication without the immune suppressive side-effects associated with targeting mTOR itself.³² The benefits of such a strategy may not be confined to HSV1, given the prevalence of different virus-encoded TSC-inhibitory functions all focused on activating mTORC1 in virus-infected cells.Viruses are masters at encoding multifunctional proteins like Us3, extracting maximum functionality from limited coding regions. Nevertheless, all known Us3 functions are impaired by mutations that eliminate its kinase activity. In addition to the cellular targets TSC2, GSK3 and FOXO1, Us3 has anti-apoptotic activity that likely involves phosphorylation of yet another Akt substrate BAD.^33,34 Us3 also phosphorylates viral proteins some of which stimulate nuclear lamina disassembly and egress of newly assembled progeny virions.^35,36 While not essential for replication, Us3-deficient viruses exhibit cell type-specific replication defects in culture and are severely impaired in mouse pathogenesis models.^37–39 A recently developed cultured rat neuron model of HSV latency/reactivation dependent upon PI-3K/Akt signaling provides an exciting opportunity to probe the role of Us3 in reactivation.⁴⁰ Indeed, by allowing Us3 access to a diverse palate of substrates, a multitude of host and virus-specific tasks can be subverted through the catalytic actions of a single virus enzyme. Given the sheer breadth of these diverse processes involving cellular and viral substrates, Us3 substrate specificity may not be restricted to those of a single cellular kinase like Akt. This raises some important questions regarding the limits of Us3 substrate targeting. For example, are all Akt substrates targeted by Us3, or are some Akt substrates effectively excluded, as their phosphorylation by Us3 may interfere somehow with viral replication? By mimicking Akt activity downstream of the cellular kinase, Us3 could effectively cherry pick which Akt substrates will be phosphorylated and which will be left untouched. Alternatively, the substrate specificity of Us3 could conceivably be so broad that some substrates make little or no detectable contribution to infected cell physiology, representing biological “noise” tolerated but neither advantageous nor detrimental to virus biology. Ultimately, understanding the role of each Us3 substrate in the virus lifecycle will require detailed genetic and biochemical analysis.How Us3 recognizes its full spectrum of substrates, including those that overlap with Akt, remains unknown. Since Us3 bears little resemblance to any specific Ser/Thr kinase, target site prediction is problematic.¹⁷ Studies with synthetic peptide substrates in vitro have not yielded clear-cut results. Although capable of phosphorylating PKA and PKC peptide substrates, Us3 appeared to have a distinct specificity.⁴¹ Sequence recognition motifs, however, are not likely to provide the required in vivo specificity, since (1) many kinases share in vitro recognition motifs (i.e., Arg residues N-terminal to phospho-acceptor site for p90 RSK, PKA and Akt), (2) recognition motifs may not be physiologically phosphorylated, (3) peptides may not mimic intact protein phosphorylation kinetics and (4) not all kinases have a clear consensus motif in peptide substrates.⁴² Instead, interactions beyond the active site that tether the kinase to physiological substrates are often responsible for biological specificity.^43,44 Despite the lack of significant primary sequence homology between Akt and Us3, their functional motifs governing substrate recognition may in fact be related structurally. Such structural similarity in the presence of only limited primary sequence homology has been observed previously among other proteins including globin, lysozyme and thioredoxin family members.^45–47 Finally, the existence of an Akt-like kinase such as Us3 that shares Akt substrates, but not primary sequence homology has potential consequences for the biology of its human host. Is encoding an Akt mimic unrelated at the primary sequence level confined to virus biology, defining an effective strategy to escape from normal constraints that limit Akt activation? Or are there other ways to make kinases with Akt-like substrate specificity lurking in our own genomes? Their unrelatedness at the primary sequence level would render them invisible to present day functional genomic-based identification methods other than pinning them as kinases. Should their identity ever be unmasked, they are likely to play important roles given the critical contributions of Akt signaling to human health and disease.     

Mammalian Target of Rapamycin Complex 1 (mTORC1) Activity Is Associated with Phosphorylation of Raptor by mTOR

mTORC1 contains multiple proteins and plays a central role in cell growth and metabolism. Raptor (regulatory-associated protein of mammalian target of rapamycin (mTOR)), a constitutively binding protein of mTORC1, is essential for mTORC1 activity and critical for the regulation of mTORC1 activity in response to insulin signaling and nutrient and energy sufficiency. Herein we demonstrate that mTOR phosphorylates raptor in vitro and in vivo. The phosphorylated residues were identified by using phosphopeptide mapping and mutagenesis. The phosphorylation of raptor is stimulated by insulin and inhibited by rapamycin. Importantly, the site-directed mutation of raptor at one phosphorylation site, Ser⁸⁶³, reduced mTORC1 activity both in vitro and in vivo. Moreover, the Ser⁸⁶³ mutant prevented small GTP-binding protein Rheb from enhancing the phosphorylation of S6 kinase (S6K) in cells. Therefore, our findings indicate that mTOR-mediated raptor phosphorylation plays an important role on activation of mTORC1.Mammalian target of rapamycin (mTOR)² has been shown to function as a critical controller in cellular growth, survival, metabolism, and development (1). mTOR, a highly conserved Ser-Thr phosphatidylinositol 3-kinase-related protein kinase, structurally forms two distinct complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), each of which catalyzes the phosphorylation of different substrates (1). The best characterized substrates for mTORC1 are eIF4E-binding protein (4E-BP, also known as PHAS) and p70 S6 kinase (S6K) (1), whereas mTORC2 phosphorylates the hydrophobic and turn motifs of protein kinase B (Akt/protein kinase B) (2) and protein kinase C (3, 4). mTORC1 constitutively consists of mTOR, raptor, and mLst8/GβL (1), whereas the proline-rich Akt substrate of 40 kDa (PRAS40) is a regulatory component of mTORC1 that disassociates after growth factor stimulation (5, 6). Raptor is essential for mTORC1 activity by providing a substrate binding function (7) but also plays a regulatory role on mTORC1 with stimuli of growth factors and nutrients (8). In response to insulin, raptor binding to substrates is elevated through the release of the competitive inhibitor PRAS40 from mTORC1 (9, 10) because PRAS40 and the substrates of mTORC1 (4E-BP and S6K) appear to bind raptor through a consensus sequence, the TOR signaling (TOS) motif (10–14). In response to amino acid sufficiency, raptor directly interacts with a heterodimer of Rag GTPases and promotes mTORC1 localization to the Rheb-containing vesicular compartment (15).mTORC1 integrates signaling pathways from growth factors, nutrients, energy, and stress, all of which generally converge on the tuberous sclerosis complex (TSC1-TSC2) through the phosphorylation of TSC2 (1). Growth factors inhibit the GTPase-activating protein activity of TSC2 toward the small GTPase Rheb via the PI3K/Akt pathway (16, 17), whereas energy depletion activates TSC2 GTPase-activating protein activity by stimulating AMP-activated protein kinase (AMPK) (18). Rheb binds directly to mTOR, albeit with very low affinity (19), and upon charging with GTP, Rheb functions as an mTORC1 activator (6). mTORC1 complexes isolated from growth factor-stimulated cells show increased kinase activity yet do not contain detectable levels of associated Rheb. Therefore, how Rheb-GTP binding to mTOR leads to an increase in mTORC1 activity toward substrates, and what the role of raptor is in this activation is currently unknown. More recently, the AMPK and p90 ribosomal S6 kinase (RSK) have been reported to directly phosphorylate raptor and regulate mTORC1 activity. The phosphorylation of raptor directly by AMPK reduced mTORC1 activity, suggesting an alternative regulation mechanism independent of TSC2 in response to energy supply (20). RSK-mediated raptor phosphorylation enhances mTORC1 activity and provides a mechanism whereby stress may activate mTORC1 independent of the PI3K/Akt pathway (21). Therefore, the phosphorylation status of raptor can be critical for the regulation of mTORC1 activity.In this study, we investigated phosphorylation sites in raptor catalyzed by mTOR. Using two-dimensional phosphopeptide mapping, we found that Ser⁸⁶³ and Ser⁸⁵⁹ in raptor were phosphorylated by mTOR both in vivo and in vitro. mTORC1 activity in vitro and in vivo is associated with the phosphorylation of Ser⁸⁶³ in raptor.     

S6K1 and 4E-BP1 are independent regulated and control cellular growth in bladder cancer

Aberrant activation and mutation status of proteins in the phosphatidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) and the mitogen activated protein kinase (MAPK) signaling pathways have been linked to tumorigenesis in various tumors including urothelial carcinoma (UC). However, anti-tumor therapy with small molecule inhibitors against mTOR turned out to be less successful than expected. We characterized the molecular mechanism of this pathway in urothelial carcinoma by interfering with different molecular components using small chemical inhibitors and siRNA technology and analyzed effects on the molecular activation status, cell growth, proliferation and apoptosis. In a majority of tested cell lines constitutive activation of the PI3K was observed. Manipulation of mTOR or Akt expression or activity only regulated phosphorylation of S6K1 but not 4E-BP1. Instead, we provide evidence for an alternative mTOR independent but PI3K dependent regulation of 4E-BP1. Only the simultaneous inhibition of both S6K1 and 4E-BP1 suppressed cell growth efficiently. Crosstalk between PI3K and the MAPK signaling pathway is mediated via PI3K and indirect by S6K1 activity. Inhibition of MEK1/2 results in activation of Akt but not mTOR/S6K1 or 4E-BP1. Our data suggest that 4E-BP1 is a potential new target molecule and stratification marker for anti cancer therapy in UC and support the consideration of a multi-targeting approach against PI3K, mTORC1/2 and MAPK.     

Targeting mTOR to Overcome Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Resistance in Non-Small Cell Lung Cancer Cells

Aims

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have shown dramatic clinical benefits in advanced non-small cell lung cancer (NSCLC); however, resistance remains a serious problem in clinical practice. The present study analyzed mTOR-associated signaling-pathway differences between the EGFR TKI-sensitive and -resistant NSCLC cell lines and investigated the feasibility of targeting mTOR with specific mTOR inhibitor in EGFR TKI resistant NSCLC cells.

Methods

We selected four different types of EGFR TKI-sensitive and -resistant NSCLC cells: PC9, PC9GR, H1650 and H1975 cells as models to detect mTOR-associated signaling-pathway differences by western blot and Immunoprecipitation and evaluated the antiproliferative effect and cell cycle arrest of ku-0063794 by MTT method and flow cytometry.

Results

In the present study, we observed that mTORC2-associated Akt ser473-FOXO1 signaling pathway in a basal state was highly activated in resistant cells. In vitro mTORC1 and mTORC2 kinase activities assays showed that EGFR TKI-resistant NSCLC cell lines had higher mTORC2 kinase activity, whereas sensitive cells had higher mTORC1 kinase activity in the basal state. The ATP-competitive mTOR inhibitor ku-0063794 showed dramatic antiproliferative effects and G1-cell cycle arrest in both sensitive and resistant cells. Ku-0063794 at the IC₅₀ concentration effectively inhibited both mTOR and p70S6K phosphorylation levels; the latter is an mTORC1 substrate and did not upregulate Akt ser473 phosphorylation which would be induced by rapamycin and resulted in partial inhibition of FOXO1 phosphorylation. We also observed that EGFR TKI-sensitive and -resistant clinical NSCLC tumor specimens had higher total and phosphorylated p70S6K expression levels.

Conclusion

Our results indicate mTORC2-associated signaling-pathway was hyperactivated in EGFR TKI-resistant cells and targeting mTOR with specific mTOR inhibitors is likely a good strategy for patients with EGFR mutant NSCLC who develop EGFR TKI resistance; the potential specific roles of mTORC2 in EGFR TKI-resistant NSCLC cells were still unknown and should be further investigated.     

Simultaneous inhibition of mTOR-containing complex 1 (mTORC1) and MNK induces apoptosis of cutaneous T-cell lymphoma (CTCL) cells

Background

mTOR kinase forms the mTORC1 complex by associating with raptor and other proteins and affects a number of key cell functions. mTORC1 activates p70S6kinase 1 (p70S6K1) and inhibits 4E-binding protein 1 (4E-BP1). In turn, p70S6K1 phosphorylates a S6 protein of the 40S ribosomal subunit (S6rp) and 4E-BP1, with the latter negatively regulating eukaryotic initiation factor 4E (eIF-4E). MNK1 and MNK2 kinases phosphorylate and augment activity of eIF4E. Rapamycin and its analogs are highly specific, potent, and relatively non-toxic inhibitors of mTORC1. Although mTORC1 activation is present in many types of malignancies, rapamycin-type inhibitors shows relatively limited clinical efficacy as single agents. Initially usually indolent, CTCL displays a tendency to progress to the aggressive forms with limited response to therapy and poor prognosis. Our previous study (M. Marzec et al. 2008) has demonstrated that CTCL cells display mTORC1 activation and short-term treatment of CTCL-derived cells with rapamycin suppressed their proliferation and had little effect on the cell survival.

Methods

Cells derived from CTCL were treated with mTORC1 inhibitor rapamycin and MNK inhibitor and evaluated for inhibition of the mTORC1 signaling pathway and cell growth and survival.

Results

Whereas the treatment with rapamycin persistently inhibited mTORC1 signaling, it suppressed only partially the cell growth. MNK kinase mediated the eIF4E phosphorylation and inhibition or depletion of MNK markedly suppressed proliferation of the CTCL cells when combined with the rapamycin-mediated inhibition of mTORC1. While MNK inhibition alone mildly suppressed the CTCL cell growth, the combined MNK and mTORC1 inhibition totally abrogated the growth. Similarly, MNK inhibitor alone displayed a minimal pro-apoptotic effect; in combination with rapamycin it triggered profound cell apoptosis.

Conclusions

These findings indicate that the combined inhibition of mTORC1 and MNK may prove beneficial in the treatment of CTCL and other malignancies.     

Regulation of mTOR Complex 1 (mTORC1) by Raptor Ser863 and Multisite Phosphorylation

The rapamycin-sensitive mTOR complex 1 (mTORC1) promotes protein synthesis, cell growth, and cell proliferation in response to growth factors and nutritional cues. To elucidate the poorly defined mechanisms underlying mTORC1 regulation, we have studied the phosphorylation of raptor, an mTOR-interacting partner. We have identified six raptor phosphorylation sites that lie in two centrally localized clusters (cluster 1, Ser⁶⁹⁶/Thr⁷⁰⁶ and cluster 2, Ser⁸⁵⁵/Ser⁸⁵⁹/Ser⁸⁶³/Ser⁸⁷⁷) using tandem mass spectrometry and generated phosphospecific antibodies for each of these sites. Here we focus primarily although not exclusively on raptor Ser⁸⁶³ phosphorylation. We report that insulin promotes mTORC1-associated phosphorylation of raptor Ser⁸⁶³ via the canonical PI3K/TSC/Rheb pathway in a rapamycin-sensitive manner. mTORC1 activation by other stimuli (e.g. amino acids, epidermal growth factor/MAPK signaling, and cellular energy) also promote raptor Ser⁸⁶³ phosphorylation. Rheb overexpression increases phosphorylation on raptor Ser⁸⁶³ as well as on the five other identified sites (e.g. Ser⁸⁵⁹, Ser⁸⁵⁵, Ser⁸⁷⁷, Ser⁶⁹⁶, and Thr⁷⁰⁶). Strikingly, raptor Ser⁸⁶³ phosphorylation is absolutely required for raptor Ser⁸⁵⁹ and Ser⁸⁵⁵ phosphorylation. These data suggest that mTORC1 activation leads to raptor multisite phosphorylation and that raptor Ser⁸⁶³ phosphorylation functions as a master biochemical switch that modulates hierarchical raptor phosphorylation (e.g. on Ser⁸⁵⁹ and Ser⁸⁵⁵). Importantly, mTORC1 containing phosphorylation site-defective raptor exhibits reduced in vitro kinase activity toward the substrate 4EBP1, with a multisite raptor 6A mutant more strongly defective that single-site raptor S863A. Taken together, these data suggest that complex raptor phosphorylation functions as a biochemical rheostat that modulates mTORC1 signaling in accordance with environmental cues.     

The class I PI3K/Akt pathway is critical for cancer cell survival in dogs and offers an opportunity for therapeutic intervention

ABSTRACT: BACKGROUND: Using novel small-molecular inhibitors, we explored the feasibility of the class I PI3K/Akt/mTORC1 signaling pathway as a therapeutic target in canine oncology either by using pathway inhibitors alone, in combination or combined with conventional chemotherapeutic drugs in vitro. RESULTS: We demonstrate that growth and survival of the cell lines tested are predominantly dependent on class I PI3K/Akt signaling rather than mTORC1 signaling. In addition, the newly developed inhibitors ZSTK474 and KP372-1 which selectively target pan-class I PI3K and Akt, respectively, and Rapamycin which has been well-established as highly specific mTOR inhibitor, decrease viability of canine cancer cell lines. All inhibitors demonstrated inhibition of phosphorylation of pathway members. Annexin V staining demonstrated that KP372-1 is a potent inducer of apoptosis whereas ZSTK474 and Rapamycin are weaker inducers of apoptosis. Simultaneous inhibition of class I PI3K and mTORC1 by ZSTK474 combined with Rapamycin additively or synergistically reduced cell viability whereas responses to the PI3K pathway inhibitors in combination with conventional drug Doxorubicin were cell linedependent. CONCLUSION: This study highlighted the importance of class I PI3K/Akt axis signaling in canine tumour cells and identifies it as a promising therapeutic target.     

Hyperforin inhibits Akt1 kinase activity and promotes caspase-mediated apoptosis involving Bad and Noxa activation in human myeloid tumor cells

Background

The natural phloroglucinol hyperforin HF displays anti-inflammatory and anti-tumoral properties of potential pharmacological interest. Acute myeloid leukemia (AML) cells abnormally proliferate and escape apoptosis. Herein, the effects and mechanisms of purified HF on AML cell dysfunction were investigated in AML cell lines defining distinct AML subfamilies and primary AML cells cultured ex vivo.

Methodology and Results

HF inhibited in a time- and concentration-dependent manner the growth of AML cell lines (U937, OCI-AML3, NB4, HL-60) by inducing apoptosis as evidenced by accumulation of sub-G1 population, phosphatidylserine externalization and DNA fragmentation. HF also induced apoptosis in primary AML blasts, whereas normal blood cells were not affected. The apoptotic process in U937 cells was accompanied by downregulation of anti-apoptotic Bcl-2, upregulation of pro-apoptotic Noxa, mitochondrial membrane depolarization, activation of procaspases and cleavage of the caspase substrate PARP-1. The general caspase inhibitor Z-VAD-fmk and the caspase-9- and -3-specific inhibitors, but not caspase-8 inhibitor, significantly attenuated apoptosis. HF-mediated apoptosis was associated with dephosphorylation of active Akt1 (at Ser⁴⁷³) and Akt1 substrate Bad (at Ser¹³⁶) which activates Bad pro-apoptotic function. HF supppressed the kinase activity of Akt1, and combined treatment with the allosteric Akt1 inhibitor Akt-I-VIII significantly enhanced apoptosis of U937 cells.

Significance

Our data provide new evidence that HF''s pro-apoptotic effect in AML cells involved inhibition of Akt1 signaling, mitochondria and Bcl-2 members dysfunctions, and activation of procaspases -9/-3. Combined interruption of mitochondrial and Akt1 pathways by HF may have implications for AML treatment.     

New Hierarchical Phosphorylation Pathway of the Translational Repressor eIF4E-binding Protein 1 (4E-BP1) in Ischemia-Reperfusion Stress

Eukaryotic initiation factor (eIF) 4E-binding protein 1 (4E-BP1) is a translational repressor that is characterized by its capacity to bind specifically to eIF4E and inhibit its interaction with eIF4G. Phosphorylation of 4E-BP1 regulates eIF4E availability, and therefore, cap-dependent translation, in cell stress. This study reports a physiological study of 4E-BP1 regulation by phosphorylation using control conditions and a stress-induced translational repression condition, ischemia-reperfusion (IR) stress, in brain tissue. In control conditions, 4E-BP1 was found in four phosphorylation states that were detected by two-dimensional gel electrophoresis and Western blotting, which corresponded to Thr⁶⁹-phosphorylated alone, Thr⁶⁹- and Thr³⁶/Thr⁴⁵-phosphorylated, all these plus Ser⁶⁴ phosphorylation, and dephosphorylation of the sites analyzed. In control or IR conditions, no Thr³⁶/Thr⁴⁵ phosphorylation alone was detected without Thr⁶⁹ phosphorylation, and neither was Ser⁶⁴ phosphorylation without Thr³⁶/Thr⁴⁵/Thr⁶⁹ phosphorylation detected. Ischemic stress induced 4E-BP1 dephosphorylation at Thr⁶⁹, Thr³⁶/Thr⁴⁵, and Ser⁶⁴ residues, with 4E-BP1 remaining phosphorylated at Thr⁶⁹ alone or dephosphorylated. In the subsequent reperfusion, 4E-BP1 phosphorylation was induced at Thr³⁶/Thr⁴⁵ and Ser⁶⁴, in addition to Thr⁶⁹. Changes in 4E-BP1 phosphorylation after IR were according to those found for Akt and mammalian target of rapamycin (mTOR) kinases. These results demonstrate a new hierarchical phosphorylation for 4E-BP1 regulation in which Thr⁶⁹ is phosphorylated first followed by Thr³⁶/Thr⁴⁵ phosphorylation, and Ser⁶⁴ is phosphorylated last. Thr⁶⁹ phosphorylation alone allows binding to eIF4E, and subsequent Thr³⁶/Thr⁴⁵ phosphorylation was sufficient to dissociate 4E-BP1 from eIF4E, which led to eIF4E-4G interaction. These data help to elucidate the physiological role of 4E-BP1 phosphorylation in controlling protein synthesis.     

Hydrogen peroxide impairs insulin-stimulated assembly of mTORC1

Oxidants are well recognized for their capacity to reduce the phosphorylation of the mammalian target of rapamycin (mTOR) substrates, eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and p70 S6 kinase 1 (S6K1), thereby hindering mRNA translation at the level of initiation. mTOR functions to regulate mRNA translation by forming the signaling complex mTORC1 (mTOR, raptor, GβL). Insulin signaling to mTORC1 is dependent upon phosphorylation of Akt/PKB and the inhibition of the tuberous sclerosis complex (TSC1/2), thereby enhancing the phosphorylation of 4E-BP1 and S6K1. In this study we report the effect of H₂O₂ on insulin-stimulated mTORC1 activity and assembly using A549 and bovine aortic smooth muscle cells. We show that insulin stimulated the phosphorylation of TSC2 leading to a reduction in raptor–mTOR binding and in the quantity of proline-rich Akt substrate 40 (PRAS40) precipitating with mTOR. Insulin also increased 4E-BP1 coprecipitating with mTOR and the phosphorylation of the mTORC1 substrates 4E-BP1 and S6K1. H₂O₂, on the other hand, opposed the effects of insulin by increasing raptor–mTOR binding and the ratio of PRAS40/raptor derived from the mTOR immunoprecipitates in both cell types. These effects occurred in conjunction with a reduction in 4E-BP1 phosphorylation and the 4E-BP1/raptor ratio. siRNA-mediated knockdown of PRAS40 in A549 cells partially reversed the effect of H₂O₂ on 4E-BP1 phosphorylation but not on S6K1. These findings are consistent with PRAS40 functioning as a negative regulator of insulin-stimulated mTORC1 activity during oxidant stress.     

Effect of Testosterone on Insulin Stimulated IRS1 Ser Phosphorylation in Primary Rat Myotubes—A Potential Model for PCOS-Related Insulin Resistance

Background

Polycystic ovary syndrome (PCOS) is characterized by a hyperandrogenic state and frequently develops skeletal muscle insulin resistance. We determined whether testosterone adversely affects insulin action by increasing serine phosphorylation of IRS-1^636/639 in differentiated rat skeletal muscle myotubes. The phosphorylation of Akt, mTOR, and S6K, downstream targets of the PI3-kinase-IRS-1 complex were also studied.

Methods

Primary differentiated rat skeletal muscle myotubes were subjected to insulin for 30 min after 16-hour pre-exposure to either low (20 ng/ml) or high (200 ng/ml) doses of testosterone. Protein phosphorylation of IRS-1 Ser^636/639, Akt Ser⁴⁷³, mTOR-Ser²⁴⁴⁸, and S6K-Thr³⁸⁹ were measured by Western blot with signal intensity measured by immunofluorescence.

Results

Cells exposed to 100 nM of insulin had increased IRS-1 Ser^636/639 and Akt Ser⁴⁷³ phosphorylation. Cells pre-exposed to low-dose testosterone had significantly increased insulin-induced mTOR-Ser²⁴⁴⁸ and S6K-Thr³⁸⁹ phosphorylation (p<0.05), and further increased insulin-induced IRS-1 Ser^636/639 phosphorylation (p = 0.042) compared to control cells. High-dose testosterone pre-exposure attenuated the insulin-induced mTOR-Ser²⁴⁴⁸ and S6K-Thr³⁸⁹ phosphorylation.

Conclusions

The data demonstrated an interaction between testosterone and insulin on phosphorylation of intracellular signaling proteins, and suggests a link between a hyperandrogenic, hyperinsulinemic environment and the development of insulin resistance involving serine phosphorylation of IRS-1 Ser^636/639. These results may guide further investigations of potential mechanisms of PCOS-related insulin resistance.     

Akt1 Intramitochondrial Cycling Is a Crucial Step in the Redox Modulation of Cell Cycle Progression

Akt is a serine/threonine kinase involved in cell proliferation, apoptosis, and glucose metabolism. Akt is differentially activated by growth factors and oxidative stress by sequential phosphorylation of Ser⁴⁷³ by mTORC2 and Thr³⁰⁸ by PDK1. On these bases, we investigated the mechanistic connection of H₂O₂ yield, mitochondrial activation of Akt1 and cell cycle progression in NIH/3T3 cell line with confocal microscopy, in vivo imaging, and directed mutagenesis. We demonstrate that modulation by H₂O₂ entails the entrance of cytosolic P-Akt1 Ser⁴⁷³ to mitochondria, where it is further phosphorylated at Thr³⁰⁸ by constitutive PDK1. Phosphorylation of Thr³⁰⁸ in mitochondria determines Akt1 passage to nuclei and triggers genomic post-translational mechanisms for cell proliferation. At high H₂O₂, Akt1-PDK1 association is disrupted and P-Akt1 Ser⁴⁷³ accumulates in mitochondria in detriment to nuclear translocation; accordingly, Akt1 T308A is retained in mitochondria. Low Akt1 activity increases cytochrome c release to cytosol leading to apoptosis. As assessed by mass spectra, differential H₂O₂ effects on Akt1-PDK interaction depend on the selective oxidation of Cys³¹⁰ to sulfenic or cysteic acids. These results indicate that Akt1 intramitochondrial-cycling is central for redox modulation of cell fate.     

Akt1 inhibition promotes breast cancer metastasis through EGFR-mediated β-catenin nuclear accumulation

Background

Knockdown of Akt1 promotes Epithelial-to-Mesenchymal Transition in breast cancer cells. However, the mechanisms are not completely understood.

Methods

Western blotting, immunofluorescence, luciferase assay, real time PCR, ELISA and Matrigel invasion assay were used to investigate how Akt1 inhibition promotes breast cancer cell invasion in vitro. Mouse model of lung metastasis was used to measure in vivo efficacy of Akt inhibitor MK2206 and its combination with Gefitinib.

Results

Knockdown of Akt1 stimulated β-catenin nuclear accumulation, resulting in breast cancer cell invasion. β-catenin nuclear accumulation induced by Akt1 inhibition depended on the prolonged activation of EGFR signaling pathway in breast cancer cells. Mechanistic experiments documented that knockdown of Akt1 inactivates PIKfyve via dephosphorylating of PIKfyve at Ser³¹⁸ site, resulting in a decreased degradation of EGFR signaling pathway. Inhibition of Akt1 using MK2206 could induce an increase in the expression of EGFR and β-catenin in breast cancer cells. In addition, MK2206 at a low dosage enhance breast cancer metastasis in a mouse model of lung metastasis, while an inhibitor of EGFR tyrosine kinase Gefitinib could potentially suppress breast cancer metastasis induced by Akt1 inhibition.

Conclusion

EGFR-mediated β-catenin nuclear accumulation is critical for Akt1 inhibition-induced breast cancer metastasis.

     

Autophagy induction by tetrahydrobiopterin deficiency

Tetrahydrobiopterin (BH₄) deficiency is a genetic disorder associated with a variety of metabolic syndromes such as phenylketonuria (PKU). In this article, the signaling pathway by which BH₄ deficiency inactivates mTORC1 leading to the activation of the autophagic pathway was studied utilizing BH₄-deficient Spr^-/- mice generated by the knockout of the gene encoding sepiapterin reductase (SR) catalyzing BH₄ synthesis. We found that mTORC1 signaling was inactivated and autophagic pathway was activated in tissues from Spr^-/- mice. This study demonstrates that tyrosine deficiency causes mTORC1 inactivation and subsequent activation of autophagic pathway in Spr^-/- mice. Therapeutic tyrosine diet completely rescued dwarfism and mTORC1 inhibition but inactivated autophagic pathway in Spr^-/- mice. Tyrosine-dependent inactivation of mTORC1 was further supported by mTORC1 inactivation in Pah^enu2 mouse model lacking phenylalanine hydroxylase (Pah). NIH3T3 cells grown under the condition of tyrosine restriction exhibited autophagy induction. However, mTORC1 activation by RhebQ64L, a positive regulator of mTORC1, inactivated autophagic pathway in NIH3T3 cells under tyrosine-deficient conditions. In addition, this study first documents mTORC1 inactivation and autophagy induction in PKU patients with BH₄ deficiency.Key words: tetrahydrobiopterin, autophagy, mTORC1, tyrosine, phenylalanine, phenylketonuria, Akt, AMPK     

High-dose rapamycin induces apoptosis in human cancer cells by dissociating mTOR complex 1 and suppressing phosphorylation of 4E-BP1

mTOR, the mammalian target of rapamycin, has been widely implicated in signals that promote cell cycle progression and survival in cancer cells. Rapamycin, which inhibits mTOR with high specificity, has consequently attracted much attention as an anticancer therapeutic. Rapamycin suppresses phosphorylation of S6 kinase at nanomolar concentrations; however, at higher micro-molar doses, rapamycin induces apoptosis in several human cancer cell lines. While much is known about the effect of low-dose rapamycin treatment, the mechanistic basis for the apoptotic effects of high-dose rapamycin treatment is not understood. We report here that the apoptotic effects of high-dose rapamycin treatment correlate with suppressing phosphorylation of the mTOR complex 1 substrate, eukaryotic initiation factor 4E (eIF4E) binding protein-1 (4E-BP1). Consistent with this observation, ablation of eIF4E also resulted in apoptorsis in MDA-MB 231 breast cancer cells. We also provide evidence that the differential dose effects of rapamycin are correlated with partial and complete dissociation of Raptor from mTORC1 at low and high doses, respectively. In contrast with MDA-MB-231 cells, MCF-7 breast cancer cells survived rapamycin-induced suppression of 4E-BP1 phosphorylation. We show that survival correlated with a hyperphosphorylation of Akt at S473 at high rapamycin doses, the suppression of which conferred rapamycin sensitivity. This study reveals that the apoptotic effect of rapamycin requires doses that completely dissociate Raptor from mTORC1 and suppress that phosphorylation of 4E-BP1 and inhibit eIF4E.Key words: rapamycin, mTOR, 4E-BP1, eIF4E, Akt, apoptosis     

Different Patterns of Akt and ERK Feedback Activation in Response to Rapamycin,Active-Site mTOR Inhibitors and Metformin in Pancreatic Cancer Cells

The mTOR pathway is aberrantly stimulated in many cancer cells, including pancreatic ductal adenocarcinoma (PDAC), and thus it is a potential target for therapy. However, the mTORC1/S6K axis also mediates negative feedback loops that attenuate signaling via insulin/IGF receptor and other tyrosine kinase receptors. Suppression of these feed-back loops unleashes over-activation of upstream pathways that potentially counterbalance the antiproliferative effects of mTOR inhibitors. Here, we demonstrate that treatment of PANC-1 or MiaPaCa-2 pancreatic cancer cells with either rapamycin or active-site mTOR inhibitors suppressed S6K and S6 phosphorylation induced by insulin and the GPCR agonist neurotensin. Rapamycin caused a striking increase in Akt phosphorylation at Ser⁴⁷³ while the active-site inhibitors of mTOR (KU63794 and PP242) completely abrogated Akt phosphorylation at this site. Conversely, active-site inhibitors of mTOR cause a marked increase in ERK activation whereas rapamycin did not have any stimulatory effect on ERK activation. The results imply that first and second generation of mTOR inhibitors promote over-activation of different pro-oncogenic pathways in PDAC cells, suggesting that suppression of feed-back loops should be a major consideration in the use of these inhibitors for PDAC therapy. In contrast, metformin abolished mTORC1 activation without over-stimulating Akt phosphorylation on Ser⁴⁷³ and prevented mitogen-stimulated ERK activation in PDAC cells. Metformin induced a more pronounced inhibition of proliferation than either KU63794 or rapamycin while, the active-site mTOR inhibitor was more effective than rapamycin. Thus, the effects of metformin on Akt and ERK activation are strikingly different from allosteric or active-site mTOR inhibitors in PDAC cells, though all these agents potently inhibited the mTORC1/S6K axis.     

Anti-Myeloma Activity of Akt Inhibition Is Linked to the Activation Status of PI3K/Akt and MEK/ERK Pathway

The PI3K/Akt/mTOR signal transduction pathway plays a central role in multiple myeloma (MM) disease progression and development of therapeutic resistance. mTORC1 inhibitors have shown limited efficacy in the clinic, largely attributed to the reactivation of Akt due to rapamycin induced mTORC2 activity. Here, we present promising anti-myeloma activity of MK-2206, a novel allosteric pan-Akt inhibitor, in MM cell lines and patient cells. MK-2206 was able to induce cytotoxicity and inhibit proliferation in all MM cell lines tested, albeit with significant heterogeneity that was highly dependent on basal pAkt levels. MK-2206 was able to inhibit proliferation of MM cells even when cultured with marrow stromal cells or tumor promoting cytokines. The induction of cytotoxicity was due to apoptosis, which at least partially was mediated by caspases. MK-2206 inhibited pAkt and its down-stream targets and up-regulated pErk in MM cells. Using MK-2206 in combination with rapamycin (mTORC1 inhibitor), {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (PI3K inhibitor), or U0126 (MEK1/2 inhibitor), we show that Erk- mediated downstream activation of PI3K/Akt pathway results in resistance to Akt inhibition. These provide the basis for clinical evaluation of MK-2206 alone or in combination in MM and potential use of baseline pAkt and pErk as biomarkers for patient selection.     

Role of Inositol Trisphosphate Receptors in Autophagy in DT40 Cells

Previous studies have shown that small interfering RNA knockdown and pharmacological inhibition of inositol 1,4,5-trisphosphate receptors (IP₃Rs) stimulate autophagy. We have investigated autophagy in chicken DT40 cell lines containing targeted deletions of all three IP₃R isoforms (triple knock-out (TKO) cells). Using gel shifts of microtubule-associated protein 1 light chain 3 as a marker of autophagy, we find that TKO cells have enhanced basal autophagic flux even under nutrient-replete conditions. Stable DT40 cell lines derived from TKO cells containing the functionally inactive D2550A IP₃R mutant did not suppress autophagy in the same manner as wild-type receptors. This suggests that the channel function of the receptor is important in its regulatory role in autophagy. There were no marked differences in the phosphorylation state of AMP-activated protein kinase, Akt, or mammalian target of rapamycin between wild-type and TKO cells. The amount of immunoprecipitated complexes of Bcl-2-Beclin-1 and Beclin-1-Vps34 were also not different between the two cell lines. The major difference noted was a substantially decreased mTORC1 kinase activity in TKO cells based on decreased phosphorylation of S6 kinase and 4E-BP1. The discharge of intracellular stores with thapsigargin stimulated mTORC1 activity (measured as S6 kinase phosphorylation) to a greater extent in wild-type than in TKO cells. We suggest that basal autophagic flux may be negatively regulated by IP₃R-dependent Ca²⁺ signals acting to maintain an elevated mTORC1 activity in wild-type cells and that Ca²⁺ regulation of this enzyme is defective in TKO cells. The protective effect of a higher autophagic flux in cells lacking IP₃Rs may play a role in the delayed apoptotic response observed in these cells.     

Capillary Isoelectric Focusing of Akt Isoforms Identifies Highly Dynamic Phosphorylation in Neuronal Cells and Brain Tissue

The PI3K/PTEN/Akt pathway has been established as a core signaling pathway that is crucial for the integration of neurons into neuronal circuits and the maintenance of the architecture and function of neurons in the adult brain. Akt1–3 kinases are specifically activated by two phosphorylation events on residues Thr³⁰⁸ and Ser⁴⁷³ upon growth factor signaling, which subsequently phosphorylate a vast cohort of downstream targets. However, we still lack a clear understanding of the complexity and regulation of isoform specificity within the PI3K/PTEN/Akt pathway. We utilized a capillary-based isoelectric focusing method to study dynamics of Akt phosphorylation in neuronal cells and the developing brain and identify previously undescribed features of Akt phosphorylation and activation. First, we show that the accumulation of multiple phosphorylation events on Akt forms occur concurrently with Ser⁴⁷³ and Thr³⁰⁸ phosphorylation upon acute PI3K activation and provide evidence for uncoupling of Ser⁴⁷³ and Thr³⁰⁸ phosphorylation, as well as differential sensitivities of Akt1 forms upon PI3K inhibition. Second, we detect a transient shift in Akt isoform phosphorylation and activation pattern during early postnatal brain development, at stages corresponding to synapse development and maturation. Third, we show differential sensitivities of Ser⁴⁷³-Akt species to PTEN deletion in mature neurons, which suggests inherent differences in the Akt pools that are accessible to growth factors as compared with the pools that are controlled by PTEN. Our study demonstrates the presence of complex phosphorylation events of Akt in a time- and signal-dependent manner in neurons.