Akt-PDK1 complex mediates epidermal growth factor-induced membrane protrusion through Ral activation.

We studied the spatiotemporal regulation of Akt (also called protein kinase B), phosphatidylinositol-3,4-bisphosphate [PtdIns(3,4)P2], and phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3] by using probes based on the principle of fluorescence resonance energy transfer. On epidermal growth factor (EGF) stimulation, the amount of PtdIns(3,4,5)P3 was increased diffusely in the plasma membrane, whereas that of PtdIns(3,4)P2 was increased more in the nascent lamellipodia than in the plasma membrane of the central region. The distribution and time course of Akt activation were similar to that of increased PtdIns(3,4)P2 levels, which were most prominent in the nascent lamellipodia. Moreover, we found that upon EGF stimulation 3-phosphoinositide-dependent protein kinase-1 (PDK1) was also recruited to nascent lamellipodia in an Akt-dependent manner. Because PDK1 is known to activate Ral GTPase and because Ral is required for EGF-induced lamellipodial protrusion, we speculated that the PDK1-Akt complex may be indispensable for the induction of lamellipodia. In agreement with this idea, EGF-induced lamellipodia formation was promoted by the overexpression of Akt and inhibited by an Akt inhibitor or a Ral-binding domain of Sec5. These results identified the Akt-PDK1 complex as an upstream positive regulator of Ral GTPase in the induction of lamellipodial protrusion.

[1]  M. White,et al.  RalB Mobilizes the Exocyst To Drive Cell Migration , 2006, Molecular and Cellular Biology.

[2]  Brian A. Hemmings,et al.  Protein kinase B/Akt at a glance , 2005, Journal of Cell Science.

[3]  Jin Zhang,et al.  Signal propagation from membrane messengers to nuclear effectors revealed by reporters of phosphoinositide dynamics and Akt activity. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[4]  D. Theodorescu,et al.  RalA and RalB: antagonistic relatives in cancer cell migration. , 2005, Cancer research.

[5]  Michiyuki Matsuda,et al.  A FRET-based probe for epidermal growth factor receptor bound non-covalently to a pair of synthetic amphipathic helixes. , 2005, Experimental cell research.

[6]  B. Dahlbäck,et al.  C1‐TEN is a negative regulator of the Akt/PKB signal transduction pathway and inhibits cell survival, proliferation, and migration , 2005, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[7]  M. White,et al.  Ral GTPases: corrupting the exocyst in cancer cells. , 2005, Trends in cell biology.

[8]  K. Aoki,et al.  Local phosphatidylinositol 3,4,5-trisphosphate accumulation recruits Vav2 and Vav3 to activate Rac1/Cdc42 and initiate neurite outgrowth in nerve growth factor-stimulated PC12 cells. , 2005, Molecular biology of the cell.

[9]  Roger Y Tsien,et al.  Spatio-temporal Dynamics of Protein Kinase B/Akt Signaling Revealed by a Genetically Encoded Fluorescent Reporter*♦ , 2005, Journal of Biological Chemistry.

[10]  D. Guertin,et al.  Phosphorylation and Regulation of Akt/PKB by the Rictor-mTOR Complex , 2005, Science.

[11]  D. Rawlings,et al.  Protein Kinase C βII Regulates Akt Phosphorylation on Ser-473 in a Cell Type- and Stimulus-specific Fashion*♦ , 2004, Journal of Biological Chemistry.

[12]  A. Bresnick,et al.  Cell Type-specific Regulation of RhoA Activity during Cytokinesis* , 2004, Journal of Biological Chemistry.

[13]  A. Prescott,et al.  Structural insights into the regulation of PDK1 by phosphoinositides and inositol phosphates , 2004, The EMBO journal.

[14]  B. Hemmings,et al.  Identification of a PKB/Akt Hydrophobic Motif Ser-473 Kinase as DNA-dependent Protein Kinase*♦ , 2004, Journal of Biological Chemistry.

[15]  M. Matsuda,et al.  RalA activation at nascent lamellipodia of epidermal growth factor-stimulated Cos7 cells and migrating Madin-Darby canine kidney cells. , 2004, Molecular biology of the cell.

[16]  B. Hemmings,et al.  Advances in protein kinase B signalling: AKTion on multiple fronts. , 2004, Trends in biochemical sciences.

[17]  T. Uemura,et al.  Phosphoinositide 3-Kinase-mediated Activation of Cofilin Phosphatase Slingshot and Its Role for Insulin-induced Membrane Protrusion* , 2004, Journal of Biological Chemistry.

[18]  A. Bershadsky,et al.  Lamellipodium extension and cadherin adhesion: two cell responses to cadherin activation relying on distinct signalling pathways , 2004, Journal of Cell Science.

[19]  Pamela A. Silver,et al.  A chemical genetic screen identifies inhibitors of regulated nuclear export of a Forkhead transcription factor in PTEN-deficient tumor cells. , 2003, Cancer cell.

[20]  Y. Umezawa,et al.  Production of PtdInsP3 at endomembranes is triggered by receptor endocytosis , 2003, Nature Cell Biology.

[21]  M. Sulis,et al.  PTEN: from pathology to biology. , 2003, Trends in cell biology.

[22]  Y. Umezawa,et al.  Fluorescent Indicators for Akt/Protein Kinase B and Dynamics of Akt Activity Visualized in Living Cells* , 2003, Journal of Biological Chemistry.

[23]  L. Feig.  Ral-GTPases: approaching their 15 minutes of fame. , 2003, Trends in cell biology.

[24]  M. Matsuda,et al.  Activity of Rho-family GTPases during cell division as visualized with FRET-based probes , 2003, The Journal of cell biology.

[25]  Borivoj Vojnovic,et al.  Monitoring conformational changes of proteins in cells by fluorescence lifetime imaging microscopy. , 2003, The Biochemical journal.

[26]  B. Schaffhausen,et al.  PDK1 mediates growth factor‐induced Ral‐GEF activation by a kinase‐independent mechanism , 2002, The EMBO journal.

[27]  G. Prestwich,et al.  Phosphatidylinositol (3,4,5)P3 Is Essential but Not Sufficient for Protein Kinase B (PKB) Activation; Phosphatidylinositol (3,4)P2 Is Required for PKB Phosphorylation at Ser-473 , 2002, The Journal of Biological Chemistry.

[28]  A. Saltiel,et al.  Insulin signaling pathways in time and space. , 2002, Trends in cell biology.

[29]  N. Masuyama,et al.  Akt mediates Rac/Cdc42-regulated cell motility in growth factor-stimulated cells and in invasive PTEN knockout cells , 2001, Current Biology.

[30]  M. Falasca,et al.  Specificity in pleckstrin homology (PH) domain membrane targeting: a role for a phosphoinositide–protein co‐operative mechanism , 2001, FEBS letters.

[31]  A. Miyawaki,et al.  Spatio-temporal images of growth-factor-induced activation of Ras and Rap1 , 2001, Nature.

[32]  A Miyawaki,et al.  Red fluorescent protein from Discosoma as a fusion tag and a partner for fluorescence resonance energy transfer. , 2001, Biochemistry.

[33]  D. Roymans,et al.  Phosphatidylinositol 3-kinases in tumor progression. , 2001, European journal of biochemistry.

[34]  A. Sorkin,et al.  Interaction of EGF receptor and Grb2 in living cells visualized by fluorescence resonance energy transfer (FRET) microscopy , 2000, Current Biology.

[35]  S. Dowler,et al.  Identification of pleckstrin-homology-domain-containing proteins with novel phosphoinositide-binding specificities. , 2000, The Biochemical journal.

[36]  D. Stokoe,et al.  5′ Phospholipid Phosphatase SHIP-2 Causes Protein Kinase B Inactivation and Cell Cycle Arrest in Glioblastoma Cells , 2000, Molecular and Cellular Biology.

[37]  M. Lemmon,et al.  Signal-dependent membrane targeting by pleckstrin homology (PH) domains. , 2000, The Biochemical journal.

[38]  Li Guang,et al.  Involvement of Ras and Ral in Chemotactic Migration of Skeletal Myoblasts , 2000, Molecular and Cellular Biology.

[39]  R. Mamillapalli,et al.  Genetic deletion of the Pten tumor suppressor gene promotes cell motility by activation of Rac1 and Cdc42 GTPases , 2000, Current Biology.

[40]  T. Sasaki,et al.  SHIP is a negative regulator of growth factor receptor-mediated PKB/Akt activation and myeloid cell survival. , 1999, Genes & development.

[41]  P. Cohen,et al.  Role of phosphatidylinositol 3,4,5-trisphosphate in regulating the activity and localization of 3-phosphoinositide-dependent protein kinase-1. , 1999, The Biochemical journal.

[42]  T. Kurosaki,et al.  The Inositol Phosphatase SHIP Inhibits Akt/PKB Activation in B Cells* , 1998, The Journal of Biological Chemistry.

[43]  P. Vogt,et al.  The akt kinase: molecular determinants of oncogenicity. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[44]  J. Bos.  All in the family? New insights and questions regarding interconnectivity of Ras, Rap1 and Ral , 1998, The EMBO journal.

[45]  G. Martin,et al.  Ras-independent activation of Ral by a Ca2+-dependent pathway , 1998, Current Biology.

[46]  Phillip T. Hawkins,et al.  Translocation of PDK-1 to the plasma membrane is important in allowing PDK-1 to activate protein kinase B , 1998, Current Biology.

[47]  B. Burgering,et al.  Essential Role for Protein Kinase B (PKB) in Insulin-induced Glycogen Synthase Kinase 3 Inactivation , 1998, The Journal of Biological Chemistry.

[48]  R. Wolthuis,et al.  Activation of the Small GTPase Ral in Platelets , 1998, Molecular and Cellular Biology.

[49]  J. Downward.  Mechanisms and consequences of activation of protein kinase B/Akt. , 1998, Current opinion in cell biology.

[50]  F. McCormick,et al.  Dual role of phosphatidylinositol-3,4,5-trisphosphate in the activation of protein kinase B. , 1997, Science.

[51]  M. Andjelkovic,et al.  High Affinity Binding of Inositol Phosphates and Phosphoinositides to the Pleckstrin Homology Domain of RAC/Protein Kinase B and Their Influence on Kinase Activity* , 1997, The Journal of Biological Chemistry.

[52]  David R. Kaplan,et al.  Direct Regulation of the Akt Proto-Oncogene Product by Phosphatidylinositol-3,4-bisphosphate , 1997, Science.

[53]  D. Pot,et al.  A specific product of phosphatidylinositol 3-kinase directly activates the protein kinase Akt through its pleckstrin homology domain , 1997, Molecular and cellular biology.

[54]  P. Cohen,et al.  Mechanism of activation of protein kinase B by insulin and IGF‐1. , 1996, The EMBO journal.

[55]  S. Cantor,et al.  Evidence for a Ras/Ral signaling cascade. , 1996, Trends in biochemical sciences.

[56]  D. Alessi,et al.  Specific binding of the Akt-1 protein kinase to phosphatidylinositol 3,4,5-trisphosphate without subsequent activation. , 1996, The Biochemical journal.

[57]  R. Tsien,et al.  Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer , 1996, Current Biology.

[58]  T. Jovin,et al.  FRET imaging , 2003, Nature Biotechnology.

[59]  Roger Y. Tsien,et al.  Creating new fluorescent probes for cell biology , 2003, Nature Reviews Molecular Cell Biology.

[60]  K. Mikoshiba,et al.  A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications , 2002, Nature Biotechnology.

[61]  Phosphatidylinositol (3,4,5)P 3 Is Essential but Not Sufficient for Protein Kinase B (PKB) Activation; Phosphatidylinositol (3,4)P 2 Is Required for PKB Phosphorylation at Ser-473 STUDIES USING CELLS FROM SH2-CONTAINING INOSITOL-5-PHOSPHATASE KNOCKOUT MICE* , 2022 .