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

Abstract

b 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)P 3 ] production by activating a number of AGC kinases, including protein kinase B (PKB)/Akt. Both PDK1 and PKB contain pleckstrin homology (PH) domains that interact with the PtdIns(3,4,5)P 3 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. We 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.

Mutation of the 3-Phosphoinositide-Dependent Protein Kinase 1 (PDK1) Substrate-Docking Site in the Developing Brain Causes Microcephaly with Abnormal Brain Morphogenesis Independently of Akt, Leading to Impaired Cognition and Disruptive Behaviors

“…This is consistent with the fact that the PKB/Akt kinase, which is the only PDK1 substrate that is not affected by the PDK1 L155E mutation, is thought to be the critical downstream effector of this pathway in promoting survival by antagonizing programmed cell death (47). However, in PDK1 K465E/K465E knock-in mice, reduced activation of Akt isoforms was also sufficient to dictate neuronal survival (22). We recently demonstrated how the inhibition of mTORC2, the Akt and SGK hydrophobic-motif kinase, modestly compromised the viability of PDK1 wild-type neurons at doses that did not affect Akt activation, and this was further aggravated in the PDK1 K465E/K465E mutant neurons (43).…”

“…We and others recently described how, in the absence of PtdIns(3,4,5)P 3 binding, PDK1 can still activate Akt by binding to the phosphorylated-Akt hydrophobic motif (42,43). In agreement with that, in the PDK1 K465E/K465E knock-in mice expressing a mutant form of PDK1 incapable of phosphoinositide binding, activation of Akt was selectively affected but not fully abolished (22). In contrast, in the PDK1 fl/fl CRE ϩ neurons and tissues, Akt was normally activated, demonstrating that under physiological conditions, Akt activation by PDK1 relies mostly on the phosphoinositide-mediated colocalization of both kinases at PtdIns(3,4,5)P 3 -rich cellular membranes rather than on the docking-site interaction.…”

“…It is well known than the PI3K/PDK1/Akt signaling pathway plays critical roles in mediating the responses to survival signals that antagonize the intrinsic programmed cell death in the developing nervous system (6, 37). We recently showed how decreasing the efficiency of PKB/Akt activation by expressing the mutant form of PDK1 K465E, which is incapable of phosphoinositide binding, had no consequences for neuronal survival (22). From that, it was postulated that either the reduced levels of Akt activity attained in the PDK1 K465E/K465E mice were sufficient to preserve normal neuronal viability or that PDK1-activated kinases other than Akt were responsible for transducing the survival program elicited by extracellular signals.…”

“…The PI3K signaling pathway controls several aspects of neuronal morphogenesis, including neuronal polarization, axonal outgrowth, and dendrite arborization (38). In PDK1 K465E/K465E mice, the Akt/ mTORC1 axis regulated PI3K-mediated neuronal polarization and axon outgrowth (22). Since S6K is a major effector of mTORC1 in controlling the translation of proteins that are relevant for neuronal differentiation, we reasoned that the PDK1 L155E mouse model, with normal levels of Akt activity but in which S6K cannot be activated by PDK1, could be instrumental in defining the contribution of S6K to neuronal differentiation.…”

“…For the survival studies, cortical neurons obtained at E15.5 were cultured in complete neurobasal medium with the B27 supplement for 6 days in vitro, washed twice with Dulbecco's modified Eagle's medium (DMEM) without serum, and then either reincubated in conditioned medium or trophic factor deprived for 24 h in serum-free neurobasal medium in the absence or presence of 50 ng/ml of brain-derived neurotrophic factor (BDNF) (Alomone). Cell viability was determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction assay, whereas the percentage of apoptosis was determined after Hoechst staining by scoring the number of cells with fragmented or condensed nuclei, as described previously (22). For the proliferation and apoptosis studies, E15.5 cortical neurons were cultured in complete neurobasal medium supplemented with B27 for the indicated number of days in vitro and then processed for immunocytochemistry with antibodies recognizing the Ki67 proliferation marker and the active caspase 3 apoptotic marker.…”