The role of the PDK1/PKB kinases in regulating neuronal survival and differentiation: characterization of the PDK1 K465E knock-in mice
Year: 2015
Type: dissertation
Abstract: Neuronal cell death programmes are counteracted by survival signals during development in \norder to maintain the tissue homeostasis. Neuronal differentiation is a mechanism generating \nfunctionally integrated neuronal cells from their progenitors. These processes appear to be \nmediated via activation of the Ras/Raf/MAPK and the PI3K/PDK1/PKB signaling pathways \nand are associated with a selective increase in protein translation. Protein kinase B (PKB/Akt) \nis a serine/threonine protein kinase which is claimed to be the critical transducer for several \nextracellular signals provided by different neurotransmitters, growth factors and hormones \nthat promote phosphoinositide 3-kinase (PI3K) activation. PI3K is a lipid kinase characterized \nby its ability to phosphorylate the 3-OH group in the inositol ring of phospholipids at the inner \nside of the plasma membrane to generate phosphatidylinositol-3,4,5-trisphosphate \n(PtdIns(3,4,5)P3 or PIP3), which is a potent second messenger. PKB regulation by its \nactivator PDK1 precisely relies on a PtdIns(3,4,5)P3 binding domain, named Pleckstrin \nHomology domain (PH-domain). Both PDK1 and PKB are protein kinases of the AGC family \ncontaining PH-domains which mediate their recruitment to the plasma membrane, where PKB \nis activated by phosphorylation at two regulatory residues, namely Thr308 at the T-loop by \nPDK1, and Ser473 at the hydrophobic motif by mTORC2. In fact, PDK1 was shown to be a \nmaster kinase also playing an essential role in the activation of a number of AGC family \nmembers by phosphorylating their T-loops by means of a PH domain-independent \nmechanism. Activated PKB modulates the function of numerous substrates involved in the \nregulation of cell metabolism, survival, proliferation and growth, which deregulation has \nconsequences in pathologies such as diabetes, cancer and neurodegeneration. \nThe crystal structure high resolution of the PDK1 PH domain revealed that the positivelycharged \nlateral chain of Lysine at position 465 within the PH domain crystal establishes \nfundamental interactions with the negatively-charged phosphate groups of PIP3. Targeted \nmutagenesis of Lysine 465 to the negatively-charged aminoacid Glutamic acid abolished \nbinding of PDK1 to PIP3 by disrupting the phosphoinositide binding pocket. Therefore, it was \nthought that this mutation could be instrumental in ablating this part of the PDK1 signaling \npathway. In order to analyze the role of the PDK1-PIP3 interaction in vivo, PDK1K465E/K465E \nknock-in mice were generated which physiologically express from the endogenous locus a \nmutant form of PDK1 incapable of phosphoinositide binding. This knock-in mice model was \nshown to be a good tool to analyse the contributory role of the PKB signaling pathway to glucose metabolism. The PDK1K465E/K465E mice were shown to be viable but smaller, with a \nmodest reduction in PKB activity compared with the wild type littermates, and prone to \ndiabetes. The importance of this pathway in tumourogenesis has been highlighted by \nintroducing the PDK1 PH domain knock-in mutation into cancer-prone PTEN+/- mice, which \nresulted in the delayed tumour onset, suggesting that even moderate reduction of PKB activity \ncan significantly delay tumour initiation and development. This makes the PDK1K465E/K465E \nmutant mouse model an excellent tool to explore the contribution of PKB to different human \npathologies and to identify downstream substrates that could provide targets for therapeutic \nintervention. In particular, I aimed to use this genetic model to unravel the role of PKB on \ndifferent aspects of brain development and function. \nStereological analysis of embryonic brain sections showed that the PDK1K465E/K465E mice \ndisplayed reduced brain size due to a reduction in neuronal cell size rather than cell number, \nsince the number of cortical and hippocampal neuronal populations between PDK1K465E/K465E \nand PDK1+/+ mice was not significantly different, whereas the volume of the mutant neuronal \nsoma was approximately 80% of the volume of the wild type neuronal soma. \nStimulation of cortical neurons with BDNF induced a robust phosphorylation of Trk receptors \nfollowed by the phosphorylation of PKB at Thr308 in the PDK1+/+ cells, which is blunted in \nthe PDK1K465E/K465E neurons, whereas PKB phosphorylation at the mTORC2 site (Ser473) \nproceeded normally in both type of cells. The moderate reduction of PKB activation was not \nrate-limiting for the phosphorylation of those PKB substrates governing neuronal survival and \napoptosis such as FOXO and GSK3. Then, it was questioned whether such mutation could \naffect survival responses in primary neuronal cultures. The findings from this study illustrate \nthat the integrity of the PDK1 PH domain is not essential to support the survival of different \nembryonic neuronal populations analyzed. Cell viability is compromised after trophic factor \ndeprivation, whilst BDNF treatment rescues cells from death to the same extent in both \nPDK1+/+ and PDK1K465E/K465E neurons. \nIn contrast, the moderate reduction of PKB activity in the PDK1K465E/K465E neurons markedly \nreduced phosphorylation of the PRAS40 and TSC2 substrates, leading to decreased \nmTORC1/S6K activation and also reduced BRSK protein synthesis. The PDK1K465E/K465E \nneurons in culture showed reduced neurite outgrowth, delayed polarization and deficient \naxonogenesis. To establish the possible causal relation between the PKB pathway defects and \naxon formation, the impact of specific pharmacological treatments with PKB and mTORC1 \ninhibitors on neuronal differentiation were assessed, which provided strong evidence that the differentiation defects were due to reduced PKB activity and inefficient activation of the \nmTORC1 signaling. Moreover, the overexpression of BRSK isoforms rescued the \naxonogenesis defects of the PDK1K465E/K465E hippocampal cells. Altogether, these findings \nillustrate how the binding of PDK1 to PIP3 creates a PKB signaling threshold which is \nsufficient to support survival, but not differentiation of neuronal cells. In this regard, there is \nincreasing evidence that PI3K/PDK1 dependent, PKB independent pathways might be \nresponsible for the control of essential cellular processes, for example cell survival, which rely \non other members of the AGC family activated by PDK1. \nThese other PDK1-regulated members of the AGC family include SGK1, S6K and RSK. The \nactivation of these kinases is not dependent on PDK1 binding to PIP3 and therefore they \nshould be normally phosphorylated in the PDK1K465E/K465E knock-in mice neurons. However, I \nobserved decreased phosphorylation of the SGK substrate NDRG1. This study clearly states \nfor the first time, that NDRG1 is regulated by PKB, at least in neurons. Activation of S6K was \nfound also incomplete in the PDK1K465E/K465E neurons due to reduced mTORC1 PKBdependent \nactivation, which could be overcome by nutrients. In fact, the only PDK1 substrate \nanalyzed that appears to not to be affected by the PDK1 K465E mutation is RSK, which \nserves as a control of the specificity of this knock-in mutation. \nIn summary, the data allow to conclude that full activation of PKB is not essential in \ncontrolling neuronal survival. In marked contrast, reduced PKB-mediated, mTORC1- \ndependent, BRSK expression resulting from lack of PDK1-phosphoinositide binding prevents \nneuronal differentiation.
Author: Tinatin Zurashvili
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W1605465619