Mutation of the PDK1 PH Domain Inhibits Protein Kinase B/Akt, Leading to Small Size and Insulin Resistance

Abstract

PDK1 activates a group of kinases, including protein kinase B (PKB)/Akt, p70 ribosomal S6 kinase (S6K), and serum and glucocorticoid-induced protein kinase (SGK), that mediate many of the effects of insulin as well as other agonists. PDK1 interacts with phosphoinositides through a pleckstrin homology (PH) domain. To study the role of this interaction, we generated knock-in mice expressing a mutant of PDK1 incapable of binding phosphoinositides. The knock-in mice are significantly small, insulin resistant, and hyperinsulinemic. Activation of PKB is markedly reduced in knock-in mice as a result of lower phosphorylation of PKB at Thr308, the residue phosphorylated by PDK1. This results in the inhibition of the downstream mTOR complex 1 and S6K1 signaling pathways. In contrast, activation of SGK1 or p90 ribosomal S6 kinase or stimulation of S6K1 induced by feeding is unaffected by the PDK1 PH domain mutation. These observations establish the importance of the PDK1-phosphoinositide interaction in enabling PKB to be efficiently activated with an animal model. Our findings reveal how reduced activation of PKB isoforms impinges on downstream signaling pathways, causing diminution of size as well as insulin resistance.The 3-phosphoinositide-dependent protein kinase 1 (PDK1) functions as an upstream activator of a group of AGC family protein kinases that are stimulated by insulin, growth factors, and numerous other agonists (42). These include isoforms of protein kinase B (PKB), also known as Akt (23), p70 ribosomal S6 kinase (S6K) (17), serum and glucocorticoid-induced protein kinase (SGK) (32), and p90 ribosomal S6 kinase (RSK) (27). Activation of PKB and other AGC kinases plays crucial roles in regulating diverse effects of extracellular agonists on cells by phosphorylating regulatory proteins that control metabolism, growth, proliferation, and survival (21). Many if not all of the cellular effects of insulin are mediated through activation of PKB (18, 58). PKB also stimulates the activation of S6K1, which plays an important role in regulating protein synthesis and cell growth (17). Genetic analysis of the PDK1-signaling pathway in Drosophila melanogaster and mice also suggests that this pathway plays an important role in regulating organism size. For example, Drosophila organisms with reduced levels of PDK1 (51), PKB (56), or S6K (40) are all small, possessing cells with reduced volume. Similarly, mice with decreased levels of PDK1 (33) and mice lacking PKB␣ (18) or S6K isoforms (48) also display small-organism and -cell phenotypes.PDK1 activates at least 23 AGC kinases by phosphorylating a specific Thr or Ser residue located within the T-loop of the kinase domain (42). Maximal activation also necessitates phosphorylation of a Ser/Thr residue located C-terminal to the catalytic domain within a region known as the hydrophobic motif. Recent work has established that the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) and mTORC2 phosphorylate the hydrophobic motif of S6K1 and PKB (52,61). In the case of RSK, a se...

Interaction of PDK1 with Phosphoinositides Is Essential for Neuronal Differentiation but Dispensable for Neuronal Survival

“…The specific binding of the pleckstrin homology domain of PKB with PtdIns(3,4,5)P 3 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 (28,29), while mTORC2 phosphorylates the Ser473 site in the hydrophobic motif (21), 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 PDK1 K465E/K465E knock-in mice (30), which express a rationally designed point mutant form of PDK1 that retains catalytic activity but is incapable of phosphoinositide binding (27). In tissues derived from these mice, PKB is still activated by growth factors albeit to a reduced level (30)(31)(32), whereas the activation of the rest of the PDK1 substrates proceeds normally.…”

“…The significance of the interaction of the PDK1 PH domain with phosphoinositides in the activation of PKB has been evaluated in vivo using PDK1 K465E/K465E knock-in mice (30), which express a rationally designed point mutant form of PDK1 that retains catalytic activity but is incapable of phosphoinositide binding (27). In tissues derived from these mice, PKB is still activated by growth factors albeit to a reduced level (30)(31)(32), whereas the activation of the rest of the PDK1 substrates proceeds normally. As a consequence, these mice are smaller, prone to diabetes (30), and protected from PTEN-induced tumorigenesis (32).…”

Hepatic Gluconeogenesis Is Enhanced by Phosphatidic Acid Which Remains Uninhibited by Insulin in Lipodystrophic Agpat2−/− Mice

“…Normally PIP 3 , the second messenger of PI3K, recruits Akt and PDK1 to the plasma membrane via their PH domains. The current study suggests that Akt or PDK1 or both fail to be recruited onto the plasma membrane and thus Akt is inefficiently phosphorylated (30). Prior studies have shown that when a mutation was created in the PH domain of PDK1 at serine 241 to alanine, it resulted in a 50-fold decrease in the phosphorylation and activation of Akt (31).…”

AMIGO2, a novel membrane anchor of PDK1, controls cell survival and angiogenesis via Akt activation

“…Platelet-specific PDK1-deficient mice exhibit thrombocytopenia and inhibition of platelet aggregation and Akt activation (Chen et al, 2013). Moreover, the disruption of the PH domain in PDK1 knockin embryoid bodies resulted in impairment of Akt activation, vessel formation, and EC migration (Primo et al, 2007;Bayascas et al, 2008). Likewise, depletion of AMI GO2 using siRNA resulted in inhibition of PDK1 localization to the plasma membrane as well as activation and caused defective EC survival, It is well established that the PI3K-mediated PDK1-Akt signaling pathway plays a crucial role in regulating tumorigenesis and angiogenesis through VEGF and several other growth factors (Jiang and Liu, 2008;Meuillet et al, 2010).…”

“…5 D). Because the PH domain of PDK1 binds to PIP3 and activates Akt signaling (Bayascas et al, 2008), we examined the direct binding of the AMI GO2 CD (AMI GO2 CD ) and the PDK1 PH domain (PDK1 PH ). As anticipated, His-PDK1 PH proteins, but not His-PDK1 kinase , could bind to GST-AMI GO2 CD under cellfree conditions (Fig.…”