Phosphoinositide-dependent kinase 1: Biological Overview | References
Gene name - Phosphoinositide-dependent kinase 1
Cytological map position - 61B1-61B1
Keywords - growth response, insulin receptor signaling pathway, synapse, neuromuscular junction, CNS, activation of protein kinase B by phosphorylation, phosphoinositide 3-kinase-dependent signaling pathway
Symbol - Pdk1
FlyBase ID: FBgn0020386
Genetic map position - chr3L:129450-144642
Cellular location - cytoplasmic
|Recent literature||Genc, O., An, J. Y., Fetter, R. D., Kulik, Y., Zunino, G., Sanders, S. J. and Davis, G. W. (2020). Homeostatic plasticity fails at the intersection of autism-gene mutations and a novel class of common genetic modifiers. Elife 9. PubMed ID: 32609087
This study identified a set of common phenotypic modifiers that interact with five independent autism gene orthologs [RIMS1 (Rim), CHD8 (Kismet), CHD2 (Chd1), WDFY3 (Blue cheese), ASH1L (ASH1)] causing a common failure of presynaptic homeostatic plasticity (PHP) in Drosophila. Heterozygous null mutations in each autism gene are demonstrated to have normal baseline neurotransmission and PHP. However, PHP is sensitized and rendered prone to failure. A subsequent electrophysiology-based genetic screen identifies the first known heterozygous mutations that commonly genetically interact with multiple ASD gene orthologs, causing PHP to fail. Two phenotypic modifiers identified in the screen, PDPK1 and PPP2R5D, are characterized. Finally, transcriptomic, ultrastructural and electrophysiological analyses define one mechanism by which PHP fails; an unexpected, maladaptive up-regulation of CREG, a conserved, neuronally expressed, stress response gene and a novel repressor of PHP. Thus, we define a novel genetic landscape by which diverse, unrelated autism risk genes may converge to commonly affect the robustness of synaptic transmission.
The dimensions of neuronal dendrites, axons, and synaptic terminals are reproducibly specified for each neuron type, yet it remains unknown how these structures acquire their precise dimensions of length and diameter. Similarly, it remains unknown how active zone number and synaptic strength are specified relative the precise dimensions of presynaptic boutons. This paper demonstrates that S6 kinase (S6K) localizes to the presynaptic active zone. Specifically, S6K colocalizes with the presynaptic protein Bruchpilot (Brp) and requires Brp for active zone localization. Evidence is provided that S6K functions downstream of presynaptic PDK1 to control synaptic bouton size, active zone number, and synaptic function without influencing presynaptic bouton number. It was further demonstrated that PDK1 is also a presynaptic protein, though it is distributed more broadly. A model is presented in which synaptic S6K responds to local extracellular nutrient and growth factor signaling at the synapse to modulate developmental size specification, including cell size, bouton size, active zone number, and neurotransmitter release (Cheng, 2011).
Dendrite diameter, the size of a dendritic field, and the area of the presynaptic nerve terminal are all reproducibly specified for a given cell type. The size and complexity of a neuron far exceeds that of any other cell type, suggesting that there may be unique solutions to the challenge of controlling and coordinating the growth of the many different and distinct features of neuronal architecture. Specification of neuronal dimension is further complicated because it is intimately associated with the electrochemical function of the neuron. Indeed, a specific form of neuronal growth, isoelectronic growth, has been observed in both invertebrates and vertebrates in which dendrite diameter grows precisely as the square of dendrite length, thereby maintaining the cable properties of individual dendrites. This type of growth is fundamentally different from most types of neuronal growth that are characterized experimentally, including axon extension, dendrite branching, spine formation, and synapse expansion. It remains unknown how the dimensions of individual neuronal compartments can be precisely specified and how these growth-related parameters are coordinated with neuronal function. Mechanistically, very few studies report the identification of genes that specifically control neuronal dimensions without otherwise perturbing the ability of these structures to form properly (Cheng, 2011).
Candidates for the control of neuronal dimension are genes associated with the regulation of cell size, including mammalian target of rapamycin, PDK1, and S6 kinase (S6K). S6K is necessary for long-term facilitation (Weatherill, 2010), the early phase of long-term potentiation, learning, and activity-dependent neuronal sprouting during epilepsy. S6K also influences the growth of dendritic arbors in cultured hippocampal neurons (Jaworski, 2005). However, there are conflicting studies regarding whether S6K influences bouton number at the Drosophila melanogaster neuromuscular junction (Cheng, 2011).
The formation of complex neural circuitry is largely determined by the sequential processes of axon guidance, target recognition, and the activity-dependent refinement of synaptic connectivity. However, the dimensions of individual neuronal compartments, including the length and diameter of dendrites, axons, and presynaptic boutons are also reproducibly specified for each cell type, and these parameters strongly influence neuronal function. It was previously established that PDK1 and S6K control cell size (Montagne, 1999; Rintelen, 2001), but it has remained unclear how these potent signaling molecules influence synaptic growth. This study provides evidence that signaling through PDK1 and S6K is specifically required to control synaptic bouton size without strongly influencing the proliferation of bouton number or the length of the nerve terminal. Many genes have been identified that perturb synapse morphology at the Drosophila NMJ when mutated. In most cases, however, the gross morphology of the synapse is perturbed, indicating that the cellular mechanics of nerve terminal extension are altered. In contrast, signaling via PDK1 and S6K primarily control the dimensions of individual synaptic boutons without otherwise altering the appearance of the presynaptic terminal (Cheng, 2011).
How are changes in compartment dimensions (cell diameter, axon diameter, and bouton diameter) mechanistically executed downstream of PDK1 and S6K? It is particularly interesting that both PDK1 and S6K are distributed throughout the cell, being present in axons, presynaptic terminals, and in the case of S6K, at the active zone. This distribution suggests that PDK1 and S6K could exert local effects that contribute to the specification of cell shape. Indeed, there is a correlation between impaired S6K localization at the active zone in the brp mutant and decreased bouton size as well as decreased axon size. Future experiments will be necessary to determine whether the changes in compartment size are a direct consequence of S6K mislocalization or whether size is influenced by altered synaptic transmission in the brp mutant. It is interesting to speculate about the cell biological processes that might function downstream of S6K to influence cellular dimensions. A previous study has provided evidence that the submembranous skeleton composed of spectrin and ankyrin provides structural integrity and modulates the shape of axons and presynaptic terminals, in part through organization of the underlying microtubule cytoskeleton (Pielage, 2008). However, a connection between PDK1, S6K, and the submembranous spectrin/ankyrin skeleton remains to be identified. In other systems, S6K has been shown to interact with spinophillin/neurabin, which is an F-actin- and protein phosphatase 1-binding protein linked to the control of dendritic spine size in vertebrates and active zone integrity in Caenorhabditis elegans (Burnett, 1998; Oliver, 2002; Ryan, 2005; Sieburth, 2005; Terry-Lorenzo, 2005). A link between S6K and neurabin/spinophillin could provide a mechanism to link local nutrient detection to the modulation of the neuronal cytoskeleton and cell shape (Cheng, 2011).
It remains unknown how active zone number is specified for a given cell type. The neuromuscular synapse, the Calyx of Held, and other large, powerful synapses harbor a robust and reproducibly large number of active zones. In contrast, other neuronal cell types make synaptic connections composed of fewer active zones, many forming a single release site within a synaptic bouton. Genes have been identified that negatively regulate active zone assembly. Other genes have been identified that are necessary for correct placement of active zones, active zone assembly, and active zone dimension. In contrast, the specification of total active zone number has remained less well defined. Target-derived growth factors clearly influence the growth and development of the presynaptic nerve terminal and have been shown to influence total active zone number. These signaling systems will activate downstream intracellular signaling cascades, such as MAPK signaling, recently implicated in the specification of active zone number at the Drosophila NMJ (Wairkar, 2009). However, it also seems likely that the control of active zone number during development will be more complex than simple specification based upon the quantity of a target-derived trophic signal. It is speculated that there will exist cell type-specific programs that interface with growth factor signaling to determine characteristic active zone densities. Cellular metabolic signaling might be one such cell type-specific parameter, including the actions of S6K and PDK1 (Cheng, 2011).
It is particularly remarkable that presynaptic overexpression of S6K and PDK1 are sufficient to increase active zone number at the NMJ. These data indicate that synapse assembly during neuromuscular development is a process that can be driven by signaling that originates within the motoneuron. It is speculated that the localization of S6K at the active zone may be critical in this regard. In some respects, it seems counterintuitive that a signaling system coupled to the metabolism of the motoneuron (PDK1-S6K) would be able to determine active zone number and, therefore, the level of postsynaptic excitation. Therefore, in keeping with well-established trophic mechanisms, it is predicted that S6K and PDK1 normally function downstream of muscle-derived factors, including nutrients and growth factors, that couple the needs of the muscle to the insertion of active zones by the motoneuron (Cheng, 2011).
There is increasing evidence that local protein synthesis plays a prominent role within postsynaptic dendrites. In contrast, the evidence for presynaptic protein translation is less abundant, including an apparent absence of polyribosomes within axons and nerve terminals. The most convincing evidence for local presynaptic protein translation is observed in Aplysia, in which serotonin-dependent long-term facilitation can be induced in synaptic compartments separated from the soma. Interestingly, two recent studies implicate S6K in presynaptic, translation-dependent long-term facilitation in Aplysia (Khan, 2001; Weatherill, 2010). The localization of S6K to the presynaptic active zone, where additional RNA-interacting signaling molecules have recently been identified, is intriguing. In Drosophila, the translational repressors nanos and pumillio have been implicated in the regulation of neuromuscular growth, membrane excitability, and postsynaptic glutamate receptor abundance. Nanos is present presynaptically, and recent data demonstrate that loss of nanos leads to an increase in total bouton number and an increase in the total active zone number. In a separate study, the IGF-II RNA-binding protein (Imp-GFP) was observed to traffic to the presynaptic terminal, and loss of Imp caused a decrease in bouton number. Although none of these data provide direct evidence for local presynaptic protein translation being important for presynaptic development or function, there is an accumulation of data suggesting that this may be a realistic possibility. Perhaps local protein translation, downstream of nutrient and growth factor signaling, could help independently shape the dimensions of each neuronal compartment (soma, axon, dendrite, and nerve terminal) and, thereby, fine tune the input-output properties of neurons during development. In this regard, it is particularly interesting that the synaptic localization of S6K by Brp seems to be important for size regulation at the synapse and perhaps throughout the cell (Cheng, 2011).
3-Phosphoinositide-dependent protein kinase 1 (PDK1) operates in cells in response to phosphoinositide 3-kinase activation and phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3] production by activating a number of AGC kinases [named after the protein kinase A, G, and C families (PKA, PKC, PKG)], including protein kinase B (PKB)/Akt. Both PDK1 and PKB contain pleckstrin homology (PH) domains that interact with the PtdIns(3,4,5)P3 second messenger. Disrupting the interaction of the PDK1 PH domain with phosphoinositides by expressing the PDK1 K465E knock-in mutation resulted in mice with reduced PKB activation. This study explored the physiological consequences of this biochemical lesion in the central nervous system. The PDK1 knock-in mice displayed a reduced brain size due to a reduction in neuronal cell size rather than cell number. Reduced BDNF-induced phosphorylation of PKB at Thr308, the PDK1 site, was observed in the mutant neurons, which was not rate limiting for the phosphorylation of those PKB substrates governing neuronal survival and apoptosis, such as FOXO1 or glycogen synthase kinase 3 (GSK3). Accordingly, the integrity of the PDK1 PH domain was not essential to support the survival of different embryonic neuronal populations analyzed. In contrast, PKB-mediated phosphorylation of PRAS40 and TSC2, allowing optimal mTORC1 activation and brain-specific kinase (BRSK) protein synthesis, was markedly reduced in the mutant mice, leading to impaired neuronal growth and differentiation (Zurashvili, 2013).
During the development of the nervous system, among all the neuronal precursors initially produced during the neurogenesis stage, only those encountering the appropriate set of neurotrophic factors along with a complex set of extracellular positional signals will be further selected to survive and differentiate. The phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB) axis is one of the critical intracellular signaling pathways that promotes neuronal survival by inhibiting the apoptotic cell death machinery in response to a number of extracellular stimuli. Thus, pharmacological inhibition of PI3K catalytic activity causes neuronal cell death, while forced expression of constitutively active forms of the PKB/Akt kinase promotes the survival of many neuronal cell type. PI3K also plays fundamental roles in regulating neuronal differentiation by defining the axon-dendrite axis through the activation of PKB. PKB promotes axon specification by inhibiting glycogen synthase kinase 3β (GSK3β). PKB also inhibits the TSC1-TSC2 complex, which antagonizes axon formation by inhibiting mTORC1 and in this way restricting the expression of the brain-specific kinase (BRSK)/SAD kinases, which are known to play fundamental roles in neuronal polarization in vivo (Zurashvili, 2013).
However, mice lacking the neuronal Akt3/PKBγ isoform are viable and do not exhibit any overt phenotype, although they display a reduced brain size, with neurons more sensitive to apoptotic insults. Therefore, the contribution of kinases activated downstream of the PI3K cascade besides PKB cannot be overlooked. In this regard, a role for the closely related kinase serum- and glucocorticoid-induced kinase (SGK) or p90 ribosomal S6 kinase (RSK) in promoting neuronal survival, and for RSK in promoting neurite outgrowth, has also been proposed (Zurashvili, 2013).
3-Phosphoinositide-dependent protein kinase 1 (PDK1) elicits cellular responses to growth factors, hormones, and many other agonists that signal through PI3K activation and phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3] production by directly activating as many as 23 protein kinases of the AGC family. These protein kinases include PKB/Akt, p70 ribosomal S6 kinase (S6K), SGK, RSK, and protein kinase C (PKC) isoforms, which in turn regulate cell growth, proliferation, survival, as well as metabolism. All these AGC kinases share structural homology and a common mechanism of activation based on the dual phosphorylation of two residues lying within two highly conserved motifs, namely, the T loop (Thr308 residue for PKBα) and the hydrophobic motif (Ser473 residue for PKBα). PDK1 acts as the master upstream kinase activating this set of AGC kinases by phosphorylating their T-loop sites (Pearce, 2010). The hydrophobic motif kinase is different among the different AGC family members, although a prominent role for mTOR complexes has emerged. Thus, the mTORC1 complex phosphorylates the hydrophobic motif of S6K isoforms and novel PKC isoforms , while the mTORC2 complex is the hydrophobic motif kinase for PKB (Sarbassov, 2005), PKCα, and SGK isoforms (Zurashvili, 2013).
PDK1 is expressed in cells as a constitutively active enzyme which is not modulated by any stimuli. Regulation of this intricate signaling network relies instead on the ability of PDK1 to specifically recognize and interact with its substrates (Biondi, 2004). The interaction of PDK1 with most AGC kinases needs the previous phosphorylation of their hydrophobic motifs, which in this manner become a substrate docking site for PDK1 binding (Biondi, 2001). Activation of PKB/Akt isoforms represents an exception to this general mechanism. Among all the PDK1-activated kinases, PKB isoforms are the only ones possessing pleckstrin homology domains, a phosphoinositide binding domain that is also present in the PDK1 protein. The specific binding of the pleckstrin homology domain of PKB with PtdIns(3,4,5)P3 becomes rate limiting for the translocation of PKB to the plasma membrane and colocalization with PDK1, where PDK1 can then efficiently phosphorylate PKB at Thr308, while mTORC2 phosphorylates the Ser473 site in the hydrophobic motif, resulting in maximal activation of the enzyme. The significance of the interaction of the PDK1 PH domain with phosphoinositides in the activation of PKB has been evaluated in vivo using PDK1K465E/K465E knock-in mice, which express a rationally designed point mutant form of PDK1 that retains catalytic activity but is incapable of phosphoinositide binding. In tissues derived from these mice, PKB is still activated by growth factors albeit to a reduced level, whereas the activation of the rest of the PDK1 substrates proceeds normally. As a consequence, these mice are smaller, prone to diabetes, and protected from PTEN-induced tumorigenesis. The PDK1K465E/K465E mouse is a genuine model in which PKB activation is only moderately reduced, which might originate from the ability of PDK1 to recognize the PKB Ser473 phospho-docking site in the absence of phosphoinositide binding. This genetic model has proven instrumental in dissecting PDK1 signaling (Bayascas, 2008) and has revealed that in T cells a PKB/Akt signaling threshold depending on PDK1-phosphoinositide interactions dictates specific cellular responses such as cell migration but not cell proliferation (Zurashvili, 2013).
The present study has employed PDK1K465E/K465E knock-in mice to explore the role that the interaction of the PDK1 PH domain with phosphoinositides plays in neuronal tissues. It was found that the brain of homozygous PDK1K465E/K465E knock-in mice was reduced in size due to a reduction in cell size rather than cell number. In agreement with the maintenance of the number of cells, both the sensitivity of the mutant neurons to apoptosis induced by serum withdrawal as well as the ability of different growth factors to support neuronal survival in the absence of serum were preserved in the mutant neurons. The deficient activation of PKB and incomplete phosphorylation and inactivation of PRAS40 and TSC2 observed in the mutant neurons caused decreased mTORC1 activation, leading to reduced BRSK protein synthesis and deficient neuronal differentiation (Zurashvili, 2013).
Deregulation of Akt/protein kinase B (PKB) is implicated in the pathogenesis of cancer and diabetes. Akt/PKB activation requires the phosphorylation of Thr308 in the activation loop by the phosphoinositide-dependent kinase 1 (PDK1) and Ser473 within the carboxyl-terminal hydrophobic motif by an unknown kinase. This study shows in Drosophila and human cells the target of rapamycin (TOR) kinase and its associated protein Rictor are necessary for Ser473 phosphorylation and that a reduction in rictor or mammalian TOR (mTOR) expression inhibits an Akt/PKB effector. The rictor-mTOR complex directly phosphorylated Akt/PKB on Ser473 in vitro and facilitated Thr308 phosphorylation by PDK1. Rictor-mTOR may serve as a drug target in tumors that have lost the expression of PTEN, a tumor suppressor that opposes Akt/PKB activation (Sarbassov, 2005).
The Akt/PKB kinase is a well-characterized effector of phosphoinositide 3-kinase (PI3K), and its deregulation plays important roles in the pathogenesis of human cancers. PI3K is necessary for the activation of Akt/PKB, and current models suggest that phosphatidylinositol-3,4,5-triphosphates produced upon growth factor stimulation recruit Akt/PKB to the plasma membrane by binding to its N-terminal pleckstrin homology (PH) domain. At the membrane, Akt/PKB is phosphorylated on two key residues: Thr308 (T308) of the activation loop by PDK1 (Alessi, 1997a, Stephens, 1998) and Ser473 (S473) in the hydrophobic motif of the C-terminal tail by a kinase whose identity has been elusive. The role of S473 phosphorylation is controversial, but there is an emerging view that it precedes the phosphorylation of T308 and is important for the recognition and activation of Akt/PKB by PDK1 (Alessi, 1996; Schied, 2002; Sarbassov, 2005 and references therein).
The molecular identity of the S473 kinase (S473K), at times referred to as 'PDK2' or the 'hydrophobic motif (HM) kinase,' has been hotly debated for many years. Several candidate S473Ks have been proposed, including PDK1, integrin-linked kinase (ILK), Akt/PKB itself, and, most recently, DNA-PKcs. Many lines of evidence argue that neither PDK1, ILK, nor Akt/PKB is the physiological S473K, and for several reasons, DNA-PKcs is also unlikely to have this function. There is no Drosophila ortholog of DNA-PKcs, and, thus, if DNA-PKcs is a physiological S473K in mammals, a distinct kinase must play that role in flies even though all other core components of the pathway (e.g., PI3K, Akt/PKB, PDK1, and PTEN) are well conserved. Moreover, it has not been shown that DNA-PKcs phosphorylates full-length Akt/PKB, and DNA-PKcs null mice do not suffer the growth retardation or insulin signaling defects associated with Akt1/PKB1 or Akt2/PKB2 (Sarbassov, 2005).
Mammalian TOR (mTOR) is a large protein kinase that exists in two distinct complexes within cells: one that contains mTOR, GβL, and raptor and another containing mTOR, GβL, and rictor. The raptor-containing complex is sensitive to the drug rapamycin and regulates cell growth, in part by phosphorylating the hydrophobic motif of S6K1, a member of the same family of kinases to which Akt/PKB belongs. The rictor-containing complex does not appear to be rapamycin-sensitive, and its cellular function is just beginning to be understood. Despite its structural similarity to S6K1, Akt/PKB phosphorylation is not sensitive to acute rapamycin treatment, and thus mTOR has not previously been considered as the S473K (Sarbassov, 2005).
This study used RNA interference (RNAi) in cultured Drosophila cells to determine the role of TOR pathway components in the phosphorylation of the hydrophobic motif sites of Drosophila Akt/PKB (dAKT/dPKB) and S6K (dS6K). In mammals and Drosophila, S6K suppresses signaling through the PI3K/Akt pathway so that inhibition of S6K boosts Akt/PKB phosphorylation. Knockdown of dS6K or Drosophila Raptor expression with double-stranded RNAs (dsRNAs) inhibited the phosphorylation and activity of dS6K and increased the phosphorylation of dAkt/dPKB. Despite reducing dS6K phosphorylation to the same extent as did dRaptor dsRNA, the dTOR dsRNA failed to increase dAkt/dPKB phosphorylation and, surprisingly, decreased it by a small amount. The contrasting effects on dAkt/dPKB phosphorylation by the dTOR and dRaptor dsRNAs suggest that dTOR has an unexpected positive role in dAkt/dPKB signaling that is not shared with dRaptor and that dTOR is required for the increase in dAkt/dPKB phosphorylation caused by dS6K inhibition. Consistent with the dRaptor-independent role for dTOR in dAkt/dPKB phosphorylation, a knockdown of dRictor reduced dAkt/dPKB phosphorylation (Sarbassov, 2005).
Because basal dAkt/dPKB phosphorylation is low in Drosophila Kc167 cells, the roles of dRictor and dTOR were verified in cells in which dAkt/dPKB phosphorylation was enhanced by decreasing the expression of dPTEN, the negative regulator of the PI3K/Akt pathway. Knockdown of dS6K or dRaptor expression in dPTEN-depleted cells further boosted dAkt/dPKB phosphorylation. In contrast, knockdown of dRictor expression almost completely prevented the dramatic increase in dAkt/dPKB phosphorylation caused by a dPTEN knockdown, whereas the knockdown of dTOR expression caused a slightly smaller suppression. Also, dRictor and dTOR were required for the increase in phosphorylation of dAkt/dPKB caused by a knockdown in the expression of dRaptor (Sarbassov, 2005).
The results in Drosophila cells suggest that dTOR and dRictor have a shared positive role in the phosphorylation of the hydrophobic motif site of dAkt/dPKB. This finding was unexpected, because previously no decrease was observed in the phosphorylation of the hydrophobic motif site of Akt/PKB after reducing mTOR expression in human cells with small interfering RNAs (siRNAs). In retrospect, however, these experiments were undertaken when RNAi-mediated knockdowns of expression in mammalian cells were relatively inefficient. In this study, with the use of a lentiviral short hairpin RNA (shRNA) expression system that robustly suppresses gene expression, results in human cell lines were obtained analogous to those in Drosophila cells. In human HT-29 colon and A549 lung cancer cells, knockdown of rictor or mTOR expression using two different sets of shRNAs decreased phosphorylation of both S473 and T308 of Akt/PKB. Mammalian cells may try to compensate for the effects of the rictor and mTOR knockdowns by boosting Akt/PKB expression. The decrease in T308 phosphorylation is consistent with the importance of S473 phosphorylation for T308 phosphorylation and with the fact that the Ser473 --> Asp473 mutant of Akt/PKB is a better substrate than the wild-type protein for T308 phosphorylation by PDK1. Knockdown of raptor expression increased the phosphorylation of both S473 and T308 despite reducing Akt/PKB expression. Knockdown of rictor or mTOR expression also decreased S473 phosphorylation in HeLa and HEK-293T cells, two human cell lines that, like A549 and HT-29 cells, contain wild-type PTEN. In addition, the knockdowns also decreased S473 phosphorylation in the PTEN-null PC-3 prostate cancer cell line, a result reminiscent of that in Drosophila cells with reduced dPTEN expression. Furthermore, the knockdowns decreased S473 phosphorylation in M059J glioblastoma cells that are null for DNA-PKcs, a proposed S473K candidate. Thus, in six distinct human cell lines, rictor and mTOR but not raptor are necessary for the phosphorylation of the hydrophobic motif of Akt/PKB (Sarbassov, 2005).
Because the rictor and mTOR knockdowns inhibit phosphorylation events critical for Akt/PKB activity, they should affect Akt/PKB-regulated effectors. In HeLa cells, a reduction in the expression of rictor or mTOR but not raptor decreased phosphorylation of AFX (Foxo4a), a forkhead family transcription factor that is a direct substrate of Akt/PKB. Because the raptor-mTOR complex directly phosphorylates the hydrophobic motif site of S6K1, whether rictor-mTOR has an analogous function for Akt/PKB was determined. Rictor-mTOR complexes isolated from HEK-293T or HeLa phosphorylated S473 but not T308 of full-length, wild-type Akt/PKB in vitro. Immunoprecipitates of raptor, the ataxia telagiectasia mutated (ATM) protein, or protein kinase C α (PKCα) did not phosphorylate either site, and Akt/PKB did not autophosphorylate S473. Importantly, the raptor immunoprecipitates also contain mTOR but did not phosphorylate Akt/PKB, suggesting that for mTOR to phosphorylate Akt/PKB, it must be bound to rictor and that raptor cannot substitute. This lack of phosphorylation holds even in the raptor immunoprecipitates isolated from HEK-293T cells that contain as much mTOR as the rictor immunoprecipitates. Consistent with a key role for rictor, mTOR immunoprecipitates prepared from the rictor knockdown cells did not phosphorylate Akt/PKB despite containing a similar amount of mTOR as the controls. To verify that mTOR is the S473K in the rictor immunoprecipitates, immunoprecipitates were prepared from control cells and from two different lines of mTOR knockdown cells. Although rictor levels were equivalent in all the immunoprecipitates, only those prepared from cells expressing mTOR phosphorylated Akt/PKB in vitro. Both the LY294002 and wortmannin mTOR kinase inhibitors blocked the in vitro phosphorylation of Akt/PKB by rictor-mTOR, and LY294002 acted at concentrations that inhibit S473 phosphorylation in cells. Staurosporine, an inhibitor of Akt/PKB kinase activity, did not decrease the phosphorylation of Akt/PKB by rictor-mTOR. Thus, in vitro the rictor-mTOR complex phosphorylates S473 of Akt/PKB in a rictor- and mTOR-dependent fashion and with a drug sensitivity profile consistent with mTOR being the phosphorylating kinase (Sarbassov, 2005).
To determine whether the phosphorylation of Akt/PKB on S473 by rictor-mTOR activates Akt/PKB activity, rictor-mTOR was used to phosphorylate Akt/PKB on S473, and then PDK1 was added to the assay to phosphorylate T308. Prior phosphorylation of Akt/PKB on S473 boosted subsequent phosphorylation by PDK1 of T308, consistent with the importance of S473 phosphorylation for T308 phosphorylation and with the inhibitory effects of the rictor and mTOR knockdowns on T308 phosphorylation. After phosphorylation with rictor-mTOR and PDK1, Akt1/PKB1 had about four- to fivefold more activity than after phosphorylation with PDK1 alone, confirming the important role of S473 in fully activating Akt/PKB. Because growth factors control the phosphorylation of Akt/PKB on S473, it was determined whether the concentration of serum in the cell media regulated the in vitro kinase activity of rictor-mTOR toward Akt/PKB. Rictor-mTOR had decreased activity in HeLa cells deprived of serum and was reactivated by serum stimulation for 30 min, indicating that modulation of the intrinsic kinase activity of rictor-mTOR may be a mechanism for regulating S473 phosphorylation (Sarbassov, 2005).
These results indicate that the rictor-mTOR complex is a hydrophobic motif kinase for Akt/PKB. Rictor-TOR has essential roles in Akt/PKB hydrophobic motif site phosphorylation in Drosophila and human cells and in vitro phosphorylates full-length, wild-type Akt/PKB in a serum-sensitive fashion. No other proposed hydrophobic motif kinase has been shown to fulfill all these criteria. With hindsight, clues are seen in the literature to the important role of mTOR in Akt/PKB activation. Prolonged but not acute treatment of certain human cells with rapamycin partially inhibits Akt/PKB phosphorylation, and the current findings provide a possible rationale to explain these results. Although rapamycin does not bind to a preformed rictor-mTOR complex, during long-term rapamycin treatment the drug should eventually sequester many of the newly synthesized mTOR molecules within cells. Thus, as the rictor-mTOR complex turns over, rapamycin may interfere with its reassembly or over time become part of the new complexes. It is reasonable to expect then that prolonged rapamycin treatment may partially inhibit rictor-mTOR activity, which would explain why rapamycin is particularly effective at suppressing the proliferation of tumor cells with hyperactive Akt/PKB. The PI3K/Akt pathway is frequently deregulated in human cancers that have lost the expression of the PTEN tumor suppressor gene, and the current findings suggest that direct inhibitors of mTOR-rictor should strongly suppress Akt/PKB activity. Thus, the rictor-mTOR complex, like its raptor-mTOR sibling, may be a valuable drug target (Sarbassov, 2005).
Genetic studies in Drosophila underscore the importance of the insulin-signaling pathway in controlling cell, organ and animal size. Effectors of this pathway include Chico (the insulin receptor substrate homolog), PI(3)K, PKB, PTEN, and S6k. Mutations in any of these components have a striking effect on cell size and number, with the exception of S6k. Mutants in S6k affect cell size but not cell number, seemingly consistent with arguments that S6k is a distal effector in the signaling pathway, directly controlled by Target of rapamycin (Tor), a downstream effector of PI(3)K and PKB. Unexpectedly, recent studies showed that S6k activity is unimpaired in chico-deficient larvae, suggesting that S6k activation may be mediated through the PI(3)K docking sites of the Drosophila insulin receptor. It has been shown genetically, pharmacologically and biochemically that S6k resides on an insulin signaling pathway distinct from that of PKB, and surprisingly also from that of PI(3)K. More striking, despite PKB-PI(3)K-independence, S6k activity is dependent on the Drosophila homolog of the phosphoinositide-dependent protein kinase 1, PDK1, demonstrating that both PDK1 (as well as Tor) mediated S6K activation is phosphatidylinositide-3,4,5-trisphosphate (PIP3)-independent (Radimerski, 2002).
The protein kinase B (PKB)/Akt family of serine kinases is rapidly activated following agonist- induced stimulation of phosphoinositide 3-kinase (PI3K). To probe the molecular events important for the activation process, two distinct models of posttranslational inducible activation and membrane recruitment were employed. PKB induction requires phosphorylation of two critical residues, threonine 308 in the activation loop and serine 473 near the carboxyl terminus. Membrane localization of PKB was found to be a primary determinant of serine 473 phosphorylation. PI3K activity was equally important for promoting phosphorylation of serine 473, but this was separable from membrane localization. PDK1 phosphorylation of threonine 308 was primarily dependent upon prior serine 473 phosphorylation and, to a lesser extent, localization to the plasma membrane. Mutation of serine 473 to alanine or aspartic acid modulated the degree of threonine 308 phosphorylation in both models, while a point mutation in the substrate-binding region of PDK1 (L155E) rendered PDK1 incapable of phosphorylating PKB. Together, these results suggest a mechanism in which 3' phosphoinositide lipid-dependent translocation of PKB to the plasma membrane promotes serine 473 phosphorylation, which is, in turn, necessary for PDK1-mediated phosphorylation of threonine 308 and, consequentially, full PKB activation (Scheid, 2002).
The insulin/insulin-like growth factor-1 signaling pathway promotes growth in invertebrates and vertebrates by increasing the levels of phosphatidylinositol 3,4,5-triphosphate through the activation of p110 phosphatidylinositol 3-kinase. Two key effectors of this pathway are the phosphoinositide-dependent protein kinase 1 (PDK1) and Akt/PKB. Although genetic analysis in C. elegans has implicated Akt as the only relevant PDK1 substrate, cell culture studies have suggested that PDK1 has additional targets. In Drosophila, PDK1 controls cellular and organism growth by activating Akt1 and S6 kinase, dS6K (FlyBase name: RPS6-p70-protein kinase). Furthermore, dPDK1 genetically interacts with dRSK but not with dPKN (FlyBase name: Protein kinase related to protein kinase N), encoding two substrates of PDK1 in vitro. Thus, the results suggest that dPDK1 is required for dRSK but not dPKN activation and that it regulates insulin-mediated growth through two main effector branches, dAkt and dS6K (Rintelen, 2001).
To analyze the function of dPDK1 in Drosophila, gain- and loss-of-function alleles of the kinase were generated. Drosophila contains a single gene that encodes a kinase that is highly homologous to PDK1 in its primary sequence and its domain structure. Initially, two EP transposable elements in the 5' region of the endogenous Drosophila PDK1 gene dPDK1 were identified. These EP elements drive expression of dPDK1 under the control of the Gal4 system, allowing a test whether dPDK1 and dAkt cooperate in promoting growth in Drosophila. Overexpression of either kinase in the eye imaginal disc during the last cell division cycle and subsequent differentiation shows little effect on the size or the structure of the eye. Co-overexpression of dAkt and dPDK1, however, leads to a significant increase in eye size. Furthermore, analysis of clones of cells in the eye overexpressing dPDK1 and/or dAkt reveals that the observed effect on cell size is strictly autonomous. These results indicate that overexpression of dPDK1 does not interfere with the normal differentiation of eye disc cells and that it promotes local growth through dAkt activation (Rintelen, 2001).
To generate loss-of-function alleles of dPDK1, the dominant eye size phenotype caused by co-overexpression of dPDK1 and dAkt was reverted by using EMS mutagenesis, leading to three partial or complete loss-of-function mutations. dPDK13 causes a G(352) to S substitution in the conserved DFG motif in the kinase subdomain VII. The D residue in this motif is essential for kinase activity by orienting the ATP-Mg2+ complex for phosphotransfer. dPDK14 causes a P(441) to L substitution in a conserved residue in kinase subdomain VIII. In the dPDK15 allele, a Q codon at position 437 in kinase subdomain VIII is mutated to a STOP codon. Because this latter mutation results in the formation of a truncated dPDK1 protein lacking part of the kinase domain and the Pleckstrin-homology domain, dPDK15 is likely to be a null mutation. A fourth allele EP(3)3091 (dPDK11), from the Berkeley Drosophila Genome Project, has an EP element located in the third intron of dPDK1 and is homozygous lethal. It failed to complement dPDK15, and the lethality was reversed by EP element excision (Rintelen, 2001).
Combinations of loss-of-function alleles provide mutants of varying strengths. Larvae homozygous for the dPDK15 null allele or larvae of the dPDK11/5 heteroallelic combination die during the second instar stage. A less severe reduction in dPDK1 function (dPDK14/5) permits development of viable dPDK1 mutant flies that are delayed 1 day in development and smaller than their heterozygous siblings, having an 18% reduction in body weight. By measuring the cell density in the wing, the reduction in size and weight apparently is primarily caused by a decrease in cell size, because cell number is only slightly affected. The lethality associated with the dPDK1 null allele and the size defect of dPDK1 hypomorphs was rescued by ubiquitous expression of a wild-type dPDK1 transgene with armadillo (arm)-Gal4 as a driver. dPDK14/5 male flies are almost completely sterile, although they show no obvious defect in sperm morphology and motility and in mating behavior. That loss of zygotic dPDK1 function results in larval lethality is in contrast to a recent analysis of two dPDK1 mutations caused by the EP insertion EP(3)3091 (dPDK11) or a 10-kb deletion (dPDK12), which are homozygous embryonic lethal. It is possible that the embryonic lethality observed by Cho (2001) is not caused by loss of dPDK1 function but by a linked lethal mutation on the same chromosome, because no rescue was attempted, and the phenotype was only analyzed in homozygotes. Consistent with this observation, larvae homozygous for a dPDK11 mutant chromosome, which has been cleaned from second hits by recombination, die during the second instar stage. Although it is very likely that dPDK1 functions during embryogenesis, like dAkt, maternal transcripts may be sufficient to support embryonic development (Rintelen, 2001).
To determine whether the effects of loss of dPDK1 function on cell growth and organ development are autonomous events, loss of dPDK1 was analyzed in clones of cells by using the FRT mitotic recombination system. In contrast to organism lethality, clones of cells homozygous for the dPDK1 null allele dPDK15 survive to adulthood. These cells show no defect in their ability to differentiate into photoreceptor cells or accessory cells, but mutant photoreceptor cells are ~30% smaller than the heterozygous cells outside the clone, a strictly cell autonomous effect. To test whether an entire body part could develop in the absence of dPDK1 function, dPDK1 was selectively removed in much of the head primordium by using the ey-Flp system. Heads homozygous mutant for anyone of the three alleles, dPDK13, dPDK14, and dPDK15, are reduced in size, which indicates that entire organs differentiate and develop in the absence of dPDK1 function, but that the final size of these organs autonomously depends on the amount of dPDK1 activity. The reduction in head size was most severe with dPDK15 followed by dPDK14 and dPDK13, with the complete removal of dPDK1 function similar to that observed for loss-of-function mutations in the Drosophila insulin receptor (dInr), Dp110/PI(3)K, and dAkt (Rintelen, 2001).
The pronounced effect of loss of dPDK1 function on head size suggests that it is a dominant constituent in the dInr pathway. To test this possibility, the ability of complete and partial loss-of-function alleles of dPDK1 to reverse phenotypes caused by either overexpression of dInr or by mutations in dPTEN, the 3-phosphatidylinositide phosphatase, was evaluated. Overexpression of a wild-type dInr cDNA under the control of GMR-Gal4 leads to a marked increase in eye size and a slightly rough eye surface, an effect dominantly suppressed by removing one copy of dPDK1. Further reduction of dPDK1 function by the dPDK11/4 heteroallelic combination reduces the eye to almost wild-type size, suggesting that the amount of dPDK1 protein is rate-limiting for the dInr overgrowth phenotype. Null mutations in dPTEN cause lethality, and removal of dPTEN function in clones stimulates cell autonomous growth, suggesting that increased levels of PIP3 promote growth and are the likely cause of lethality. Thus, if dPDK1 is an essential target of PIP3, mutations in dPDK1 may suppress the dPTEN phenotype. Surprisingly, some dPTEN/dPDK1 double mutant flies survive to adulthood, indicating that the presumed PIP3-induced lethality is primarily caused by the hyperactivation of dPDK1 or of one of its targets (Rintelen, 2001).
The fact that the growth phenotypes of dPDK1 mutations are similar to those caused by mutations in genes coding for dS6K, and dAkt, and that S6K1 is a mammalian PDK1 substrate, raises the possibility that dPDK1 may independently control growth through dS6K. This possibility was tested in the wing, which is composed of a dorsal and a ventral epithelial sheet that are tightly attached to each other through extracellular matrix. Selective overexpression of a wild-type dS6K cDNA in the dorsal wing epithelium with the apterous (ap)-Gal4 driver leads to a bending down of the wing blade, probably because of a cell-size increase in the dorsal surface. This phenotype is suppressed by a reduction of dPDK1 function. Although ap-Gal4 induced overexpression of wild-type dPDK1 alone had little effect on wing morphology, overexpression of a dPDK1A467V variant is sufficient to cause a bent-wing phenotype. The corresponding amino acid substitution in the C. elegans PDK1 is thought to cause a hyperactivation of the kinase. The dPDK1A467V-induced bent wing phenotype depends on normal levels of dS6K and dAkt, because null mutations in either of the corresponding genes dominantly suppress the phenotype. Together with the biochemical evidence in both cultured cells and in vivo, that dPDK1 controls the activity of dAkt and dS6K, these results provide functional evidence that dPDK1 is a key regulator in the control of growth and cell size by regulating the activity of two AGC kinases, dAkt and dS6K (Rintelen, 2001).
The effects of dPDK1 on dS6K raised the possibility that dPDK1 controls the activity of other AGC kinases in vivo, such as dRSK and dPKN, which have been implicated as mammalian PDK1 substrates. Because the developing eye depends on endogenous levels of dPDK1, whether lowering the dose of dPDK1 is sufficient to suppress dominantly the rough eye phenotype caused by overexpression of dRSK and dPKN under GMR-Gal4 control was tested. Reduction of dPDK1 activity in a viable dPDK1 mutant combination is sufficient to suppress the rough eye phenotype of dRSK but not of dPKN overexpression. These results suggest that at least in this in vivo assay, dRSK activity critically depends on dPDK1 function, whereas dPKN activity is not changed by a reduction in dPDK1 levels. This idea is in line with the recent finding that in PDK1-/- embryonic stem cells the protein kinase C-related protein kinase PRK2 (CG2049), which shares extensive homology with PKN, is still partially phosphorylated at its T loop residue, indicating that PDK1-independent mechanisms may exist for the phosphorylation of the T loop of certain AGC kinases including dPKN (Rintelen, 2001).
These results show that dPDK1 is an essential component in the insulin signaling pathway in the control of cell growth and body size through its two substrates, dAkt and dS6K. These results are distinct from the genetic evidence in C. elegans where Akt is the primary target of PDK1 in dauer formation. Because mutations in the insulin signaling pathway do not show an autonomous alteration of cell size in C. elegans, the regulation of the rate of protein synthesis through S6K does not seem to be a primary target of this pathway. However, the fact that dPDK1 may yet have additional substrates is suggested by the genetic interaction with dRSK gain-of-function mutations and because viable dPDK1 males are almost completely sterile. Although mutations in components of the insulin signaling pathway such as dInr, chico, Dp110/PI(3)K, and dAkt cause female sterility, male sterility is not observed. Further genetic dissection of dPDK1 function is required to determine the role of dPDK1 in male fertility. These findings in Drosophila are consistent with the absence of insulin growth factor-1-induced activation of S6K, Akt, and RSK in mammalian PDK1-/- embryonic stem cells, and therefore provide evidence for the functional conservation of branch points in kinase networks during evolution (Rintelen, 2001).
Phosphoinositide-dependent kinase-1 (PDK-1) is a central mediator of the cell signaling between phosphoinositide 3-kinase (PI3K) and various intracellular serine/threonine kinases including Akt/protein kinase B (PKB), p70 S6 kinases, and protein kinase C. Recent studies with cell transfection experiments have implied that PDK-1 may be involved in various cell functions including cell growth and apoptosis. However, despite its pivotal role in cellular signalings, the in vivo functions of PDK-1 in a multicellular system have rarely been investigated. Drosophila PDK-1 (dPDK-1) mutants have been isolated and the in vivo roles of their kinases have been characterized. Drosophila deficient in the dPDK-1 gene exhibit lethality and an apoptotic phenotype in the embryonic stage. Conversely, overexpression of dPDK-1 increases cell and organ size in a Drosophila PI3K-dependent manner. dPDK-1 not only can activate Drosophila Akt/PKB (Dakt1), but also substitutes the in vivo functions of its mammalian ortholog to activate Akt/PKB. This functional interaction between dPDK-1 and Dakt1 was further confirmed through genetic analyses in Drosophila. However, cAMP-dependent protein kinase, which has been proposed as a possible target of dPDK-1, did not interact with dPDK-1. In conclusion, these findings provide direct evidence that dPDK-1 regulates cell growth and apoptosis during Drosophila development via the PI3K-dependent signaling pathway and demonstrate this Drosophila system to be a powerful tool for elucidating the in vivo functions and targets of PDK-1 (Cho, 2001).
PDK-1 originally was identified as an upstream regulatory kinase of Akt/protein kinase B (PKB). Consequently, the in vivo roles of PDK-1 have been inferred mainly from those of Akt/PKB. Akt/PKB is a growth factor-regulated serine/threonine kinase that contains a pleckstrin homology domain, as does PDK-1. It acts downstream of phosphoinositide 3-kinase (PI3K) to regulate various cellular activities, including glucose metabolism, transcription, and protein translation. Akt/PKB also negatively regulates apoptosis in various ways. To exert its antiapoptotic effects, Akt/PKB either inhibits the activities of proapoptotic proteins, such as BAD and caspase-9, or induces antiapoptotic signals via the NF-kappaB- and forkhead transcription factor-dependent pathways. Recent transgenic studies in Drosophila have revealed an unexpected function of Akt/PKB and the PI3K signaling pathway: the pathway plays an essential role in the control of cell size. When the activities of one or multiple components of the pathway, including PI3K, Drosophila akt1 (Dakt1), and Drosophila p70 S6 kinase, are down-modulated, cell size as well as body size are dramatically reduced in a cell-autonomous manner (Cho, 2001 and references therein).
Recent studies also suggest that PDK-1 is involved in the activation of members of the AGC superfamily of serine/threonine protein kinases, through phosphorylation of their activation loop in response to extracellular stimulations induced by peptide growth factors and hormones. A number of important kinases in this family, including Akt/PKB, p70 S6 kinase, various protein kinase Cs, protein kinase C-related kinases, and cAMP-dependent protein kinase (PKA), have been proposed as either in vivo or in vitro targets of PDK-1. These results implicate that PDK-1 may play the role of a 'master kinase' in regulating these downstream kinases. However, further investigation is required to determine the actual in vivo targets of PDK-1, as it has been revealed that some of the AGC family kinases are not directly phosphorylated by PDK-1 in vivo, despite possessing a putative PDK-1 phosphorylation site at the activation loop and being phosphorylated by PDK-1 in vitro. In addition, although PDK-1 is regarded as a regulator of at least some of these important kinases, the physiological role of the kinase in a multicellular system has not yet been defined at all (Cho, 2001 and references therein).
The PDK-1 Drosophila homolog, dPDK-1 is 54% identical to its human counterpart in the catalytic domain and is also highly homologous in the noncatalytic carboxyl terminus. Flies containing mutations in the dPDK-1 locus were isolated for genetic analyses. Three P-element insertion mutants, EP(3)837, EP(3)3553, and EP(3)3091, have been found containing P-element insertions in either the 5' or intron region of the dPDK-1 gene. In detail, the inserted positions of the P-element in EP(3)837 and EP(3)3553, which have been determined by inverse PCR, are located at 179 bp and 144 bp upstream of the dPDK-1 transcription start site, respectively. The insertion sites and directions of the P-elements are oriented to induce gene expression and imply that these mutants can be used to study the gain of function of dPDK-1. Another EP line, EP(3)3091, has a P-element in the fourth intron of dPDK-1. The insertion site of EP(3)3091 predicts that the transcription of dPDK-1 is disrupted by the insertion of the transposon. Indeed, EP(3)3091 displays a complete lethal phenotype. In addition, another PDK-1-deficient lethal line, DeltadPDK5, has been generated by an imprecise excision of P-element in EP(3)837. This line contains about a 10-kb deletion that includes the first exon of dPDK-1. This mutant fails to complement the lethality of EP(3)3091, suggesting that both lines are alleles of dPDK-1 mutants. Thus, EP(3)3091 and DeltadPDK5 are hereafter referred to as dPDK-11 and dPDK-12, respectively (Cho, 2001).
None of the homozygous dPDK-11 and dPDK-12 flies emerge as larva, and both display an embryonic lethality. To isolate dPDK-11 homozygous individuals, a GFP balancer chromosome was used. The GFP-negative embryos were selected as dPDK-1 homozygotes. All of the hatched larvae from dPDK-11 or dPDK-12/TM3, GFP, Ser females show GFP expression. dPDK-11 homozygous embryos produce no ventral cuticles, and they do not develop into the larval stage. These results are similar to those seen in the mutation of Dakt1, whose mammalian homologs are well-known targets of PDK-1. Briefly, absence of maternal and zygotic Dakt1 activity also results in an embryonic lethality, along with defective cuticle formation (Cho, 2001).
Whether dPDK-1 also is involved in the cell survival-signaling pathway was tested. TUNEL assays were performed with dPDK-11 homozygous embryos to examine the involvement of the kinase in apoptosis. Apoptotic activity is dramatically induced in the dPDK-1 zygotic loss-of-function mutant. The induced apoptosis in dPDK-11 mutant embryos is extensively suppressed by expression of Dakt1 using the hs-GAL4-UAS system. Collectively, these results strongly suggest that PDK-1 plays an important role in Drosophila embryonic development and apoptosis (Cho, 2001).
A series of components in the PI3K pathway including Dakt1 and Drosophila p70 S6 kinase modulate cell size in a cell-autonomous manner. Thus, whether overexpression of dPDK-1 affects cell size was examined using the GAL4-UAS system. dPDK-1 was overexpressed under the control of gmr-GAL4, which directs expression of the gene in the developing eye. This ectopic overexpression of dPDK-1 causes an increase in ommatidia size, ~1.33-fold bigger than controls. In addition, the effect of overexpression of dPDK-1 was examined in a specific compartment of the wing disk. The wing disk is composed of two compartments (dorsal and ventral), which fold and generate the flattened wing blade. When dPDK-1 is ectopically overexpressed in the dorsal compartment with ap-GAL4 driver, the wing of EP(3)837 is convex toward the dorsal side. This is likely the result of an increase in the size of the cells in the dorsal compartment. Indeed, a similar situation is observed in the UAS-Drosophila p70 S6 kinase flies. These results suggest that dPDK-1 regulates cell and organ size (Cho, 2001).
Despite the fact that there is no clear evidence on how the intrinsic kinase activity of PDK-1 is regulated, the kinase has been found to act downstream of PI3K. Thus, whether dPDK-1 and PI3K can genetically interact was examined in fly lines in which dPDK-1 was coexpressed with PI3K or a dominant negative Dp110 (PI3KDN). Overexpression of the PI3K catalytic subunit, Dp110, increases cell size, whereas overexpression of a PI3KDN results in the opposite phenotype. This change in cell size results in the change of organ and body size. Overexpression of PI3KDN under ptc-GAL4 (the driver induces GAL4 expression throughout the anterior compartment with a stripe of maximal intensity along the border of anterior/posterior compartment extending into the posterior compartment) results in reduction of the distance between L3 and L4 veins. However, this phenotype is strongly suppressed by coexpression of dPDK-1 with PI3KDN, suggesting that dPDK-1 acts as a vital downstream effector of PI3K in cell and compartment size control. Conversely, overexpression of the PI3K wild-type causes an increase in the distance between L3 and L4 veins, and coexpression of dPDK-1 and PI3K further increases the distance in a synergistic manner. These results provide strong in vivo evidence that dPDK-1 functions downstream of Drosophila PI3K in the control of cell and compartment size (Cho, 2001).
To determine whether dPDK-1 functions in a manner similar to its mammalian counterpart, myc-tagged dPDK-1, myc-tagged human PDK-1 (hPDK-1), and/or HA-tagged human Akt/PKB were transiently expressed in COS cells. As expected, dPDK-1 strongly induces human Akt/PKB activity, to levels comparable to those induced by hPDK-1. Conversely, coexpression of a dominant negative hPDK-1 or a dominant negative dPDK-1 strongly inhibits the epidermal growth factor-induced activation of human Akt/PKB. These results indicate that the Drosophila ortholog of PDK-1 can properly function and substitute its mammalian counterpart to relay the growth factor-induced activation signals to a mammalian Akt/PKB (Cho, 2001).
Whether dPDK-1 can activate Dakt1 in Drosophila was examined. To test this, dPDK-1 and HA-tagged Dakt1 were coexpressed in the Drosophila eye using the gmr-GAL4 driver, and the phosphotransferase activities of Dakt1 were measured from the head extracts of gmr-GAL4, gmr-GAL4; UAS-HA-Dakt1, or gmr-GAL4; UAS-HA-Dakt1/EP(3)837. Dakt1 activity is strongly increased in the flies coexpressing dPDK-1. Consistent with this increased activity, an electrophoretically retarded Akt/PKB band, corresponding to a highly phosphorylated and activated form, is observed. This biochemical evidence strongly supports that Dakt1 is indeed a physiological target of dPDK-1 (Cho, 2001).
To further confirm the in vivo roles of dPDK-1, genetic interactions between Dakt1 and dPDK-1 were examined in flies. Overexpression of Dakt1 in the Drosophila eye increases eye size and generates a bulging eye with enlarged ommatidia. In addition to this change in size, the ommatidia array becomes irregular, and eye bristles are enlarged with a frequent loss of number. When dPDK-1 is coexpressed with Dakt1 in the eye, it displays a severely crumpled morphology. The eye bristles are enlarged even more severely, and the boundaries of all ommatidia and photoreceptor cells disappear. These dPDK-1/Dakt1 phenotypes are further enhanced by an increased dose of gmr-GAL4 driver. These findings, taken together, clearly demonstrate the functional and genetic interactions between dPDK-1 and Dakt1 (Cho, 2001).
The genetic interactions between dPDK-1 and Drosophila PKA were examined. Although PKA has been proposed to be a putative substrate of PDK-1, the in vivo relevance of this has not been clearly determined. When the catalytic subunit of Drosophila PKA (dPKAc) is overexpressed in the developing eye of Drosophila, the eye is discolored and swells up with wrinkles. Scanning electron microscopic views of the eye show that the boundaries of all ommatidia and photoreceptor cells disappear. However, unlike Akt/PKB, coexpression of dPDK-1 does not affect these phenotypes of dPKAc. Furthermore, the regulatory subunit of Drosophila PKA (dPKAr) also does not interact with dPDK-1. These results support that PKA is not regulated by PDK-1 in Drosophila, which is highly consistent with recent results that PKA is phosphorylated and activated normally in a PDK-1-deficient cell line. These results strongly support that the Drosophila system is a physiologically relevant tool for determining the actual in vivo targets of PDK-1 (Cho, 2001).
Protein kinase B (PKB), also known as c-Akt, is activated rapidly when mammalian cells are stimulated with insulin and growth factors, and much of the current interest in this enzyme stems from the observation that it lies 'downstream' of phosphoinositide 3-kinase on intracellular signalling pathways. Insulin or insulin-like growth factor 1 induce the phosphorylation of PKB at two residues, Thr308 and Ser473. The phosphorylation of both residues is required for maximal activation of PKB. The kinases that phosphorylate PKB are, however, unknown. This study purified 500,000-fold from rabbit skeletal muscle extracts a protein kinase which phosphorylates PKBalpha at Thr308 and increases its activity over 30-fold. The kinase was tested in the presence of several inositol phospholipids, and only low micromolar concentrations of the D enantiomers of either phosphatidylinositol 3,4,5-triphosphate (PtdIns(3,4,5)P3) or PtdIns(3,4)P2 were found to be effective in potently activating the kinase, which has been named PtdIns(3,4,5)P3-dependent protein kinase-1 (PDK1). None of the inositol phospholipids tested activated or inhibited PKBalpha or induced its phosphorylation under the conditions used. PDK1 activity was not affected by wortmannin, indicating that it is not likely to be a member of the phosphoinositide 3-kinase family. It is concluded PDK1 is likely to be one of the protein kinases that mediate the activation of PKB by insulin and growth factors. PDK1 may, therefore, play a key role in mediating many of the actions of the second messenger(s) PtdIns(3,4, 5)P3 and/or PtdIns(3,4)P2 (Alessi, 1997a).
The activation of protein kinase B (PKB, also known as c-Akt) is stimulated by insulin or growth factors and results from its phosphorylation at Thr308 and Ser473. A protein kinase, termed PDK1, has been identified that phosphorylates PKB at Thr308 only in the presence of lipid vesicles containing phosphatidylinositol 3,4,5-trisphosphate [Ptdlns(3,4,5)P3] or phosphatidylinositol 3,4-bisphosphate [Ptdlns(3,4)P2]. Human PDK1 has been cloned and sequenced. The 556-residue monomeric enzyme comprises a catalytic domain that is most similar to the PKA, PKB and PKC subfamily of protein kinases and a carboxy-terminal pleckstrin homology (PH) domain. The PDK1 gene is located on human chromosome 16p13.3 and is expressed ubiquitously in human tissues. Human PDK1 is homologous to the Drosophila protein kinase DSTPK61, which has been implicated in the regulation of sex differentiation, oogenesis and spermatogenesis. Expressed PDK1 and DSTPK61 phosphorylated Thr308 of PKB alpha only in the presence of Ptdlns(3,4,5)P3 or Ptdlns(3,4)P2. Overexpression of PDK1 in 293 cells activated PKB alpha and potentiated the IGF1-induced phosphorylation of PKB alpha at Thr308. Experiments in which the PH domains of either PDK1 or PKB alpha were deleted indicated that the binding of Ptdlns(3,4,5)P3 or Ptdlns(3,4)P2 to PKB alpha is required for phosphorylation and activation by PDK1. IGF1 stimulation of 293 cells did not affect the activity or phosphorylation of PDK1. It is concluded PDK1 is likely to mediate the activation of PKB by insulin or growth factors. DSTPK61 is a Drosophila homologue of PDK1. The effect of Ptdlns(3,4,5)P3/Ptdlns(3,4)P2 in the activation of PKB alpha is at least partly substrate directed (Alessi, 1997b).
Search PubMed for articles about Drosophila Pdk1
Alessi, D. R., Andjelkovic, M., Caudwell, B., Cron, P., Morrice, N., Cohen, P. and Hemmings, B. A. (1996). Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J 15: 6541-6551. PubMed ID: 8978681
Alessi, D. R., James, S. R., Downes, C. P., Holmes, A. B., Gaffney, P. R., Reese, C. B. and Cohen, P. (1997a). Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Balpha. Curr Biol 7: 261-269. PubMed ID: 9094314
Alessi, D. R., Deak, M., Casamayor, A., Caudwell, F. B., Morrice, N., Norman, D. G., Gaffney, P., Reese, C. B., MacDougall, C. N., Harbison, D., Ashworth, A. and Bownes, M. (1997b). 3-Phosphoinositide-dependent protein kinase-1 (PDK1): structural and functional homology with the Drosophila DSTPK61 kinase. Curr Biol 7: 776-789. PubMed ID: 9368760
Bayascas, J. R. (2008). Dissecting the role of the 3-phosphoinositide-dependent protein kinase-1 (PDK1) signalling pathways. Cell Cycle 7: 2978-2982. PubMed ID: 18802401
Biondi, R. M. (2004). Phosphoinositide-dependent protein kinase 1, a sensor of protein conformation. Trends Biochem Sci 29: 136-142. PubMed ID: 15003271
Biondi, R. M., Kieloch, A., Currie, R. A., Deak, M. and Alessi, D. R. (2001). The PIF-binding pocket in PDK1 is essential for activation of S6K and SGK, but not PKB. EMBO J 20: 4380-4390. PubMed ID: 11500365
Burnett, P. E., Blackshaw, S., Lai, M. M., Qureshi, I. A., Burnett, A. F., Sabatini, D. M. and Snyder, S. H. (1998). Neurabin is a synaptic protein linking p70 S6 kinase and the neuronal cytoskeleton. Proc Natl Acad Sci U S A 95: 8351-8356. PubMed ID: 9653190
Cheng, L., Locke, C. and Davis, G. W. (2011). S6 kinase localizes to the presynaptic active zone and functions with PDK1 to control synapse development. J Cell Biol 194: 921-935. PubMed ID: 21930778
Cho, K. S., et al. (2001). Drosophila phosphoinositide-dependent kinase-1 regulates apoptosis and growth via the phosphoinositide 3-kinase-dependent signaling pathway. Proc. Natl. Acad. Sci. 98: 6144-6149. 11344272
Jaworski, J., Spangler, S., Seeburg, D. P., Hoogenraad, C. C. and Sheng, M. (2005). Control of dendritic arborization by the phosphoinositide-3'-kinase-Akt-mammalian target of rapamycin pathway. J Neurosci 25: 11300-11312. PubMed ID: 16339025
Khan, A., Pepio, A. M. and Sossin, W. S. (2001). Serotonin activates S6 kinase in a rapamycin-sensitive manner in Aplysia synaptosomes. J Neurosci 21: 382-391. PubMed ID: 11160419
Montagne, J., Stewart, M. J., Stocker, H., Hafen, E., Kozma, S. C. and Thomas, G. (1999). Drosophila S6 kinase: a regulator of cell size. Science 285: 2126-2129. PubMed ID: 10497130
Oliver, C. J., Terry-Lorenzo, R. T., Elliott, E., Bloomer, W. A., Li, S., Brautigan, D. L., Colbran, R. J. and Shenolikar, S. (2002). Targeting protein phosphatase 1 (PP1) to the actin cytoskeleton: the neurabin I/PP1 complex regulates cell morphology. Mol Cell Biol 22: 4690-4701. PubMed ID: 12052877
Pearce, L. R., Komander, D. and Alessi, D. R. (2010). The nuts and bolts of AGC protein kinases. Nat Rev Mol Cell Biol 11: 9-22. PubMed ID: 20027184
Pielage, J., Cheng, L., Fetter, R. D., Carlton, P. M., Sedat, J. W. and Davis, G. W. (2008). A presynaptic giant ankyrin stabilizes the NMJ through regulation of presynaptic microtubules and transsynaptic cell adhesion. Neuron 58: 195-209. PubMed ID: 18439405
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date revised: 25 April 2013
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