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A Novel Pleckstrin Homology Domain-containing Protein Enhances Insulin-stimulated Akt Phosphorylation and GLUT4 Translocation in Adipocytes*

Open AccessPublished:June 28, 2010DOI:https://doi.org/10.1074/jbc.M110.146886
      Protein kinase B/Akt protein kinases control an array of diverse functions, including cell growth, survival, proliferation, and metabolism. We report here the identification of pleckstrin homology-like domain family B member 1 (PHLDB1) as an insulin-responsive protein that enhances Akt activation. PHLDB1 contains a pleckstrin homology domain, which we show binds phosphatidylinositol PI(3,4)P2, PI(3,5)P2, and PI(3,4,5)P3, as well as a Forkhead-associated domain and coiled coil regions. PHLDB1 expression is increased during adipocyte differentiation, and it is abundant in many mouse tissues. Both endogenous and HA- or GFP-tagged PHLDB1 displayed a cytoplasmic disposition in unstimulated cultured adipocytes but translocated to the plasma membrane in response to insulin. Depletion of PHLDB1 by siRNA inhibited insulin stimulation of Akt phosphorylation but not tyrosine phosphorylation of IRS-1. RNAi-based silencing of PHLDB1 in cultured adipocytes also attenuated insulin-stimulated deoxyglucose transport and Myc-GLUT4-EGFP translocation to the plasma membrane, whereas knockdown of the PHLDB1 isoform PHLDB2 failed to attenuate insulin-stimulated deoxyglucose transport. Furthermore, adenovirus-mediated expression of PHLDB1 in adipocytes enhanced insulin-stimulated Akt and p70 S6 kinase phosphorylation, as well as GLUT4 translocation. These results indicate that PHLDB1 is a novel modulator of Akt protein kinase activation by insulin.

      Introduction

      Akt protein kinase (also known as protein kinase B) has three isoforms that function as key cellular regulators downstream of various growth factors and hormonal signals (
      • Gonzalez E.
      • McGraw T.E.
      ). A large panel of Akt substrate proteins have been identified that regulate proliferation, growth, survival, and metabolism in many cell types (
      • Manning B.D.
      • Cantley L.C.
      ). Akt is a member of the large AGC protein kinase family, which includes protein kinase C (PKC), 3-phosphoinositide-dependent kinase 1 (PDK1), Rho-activated kinase, and p70 ribosomal S6 kinase (S6K).
      The abbreviations used are: S6K
      S6 kinase
      TIRF
      total internal reflection fluorescence
      PI
      phosphatidylinositol.
      Akt is composed of three functionally distinct regions as follows: an N-terminal pleckstrin homology (PH) domain, a central catalytic kinase domain, and a C-terminal hydrophobic motif (
      • Pearce L.R.
      • Komander D.
      • Alessi D.R.
      ). Akt activation is thought to proceed through its recruitment to the plasma membrane via interaction of its PH domain with the phosphoinositides, specifically PI(3,4,5)P3 and PI(3,4)P2 produced by p85/p110 phosphoinositide 3-kinases (PI3K) (
      • Thomas C.C.
      • Deak M.
      • Alessi D.R.
      • van Aalten D.M.
      ,
      • Milburn C.C.
      • Deak M.
      • Kelly S.M.
      • Price N.C.
      • Alessi D.R.
      • Van Aalten D.M.
      ). The lipid-bound Akt is phosphorylated by two protein kinases, PDK1, which is also recruited through its PH domain binding to PI(3,4,5)P3 and phosphorylates Thr-308 in the activation T-loop of Akt (
      • Alessi D.R.
      • James S.R.
      • Downes C.P.
      • Holmes A.B.
      • Gaffney P.R.
      • Reese C.B.
      • Cohen P.
      ), and mammalian target of rapamycin complex 2 (mTORC2) (
      • Sarbassov D.D.
      • Guertin D.A.
      • Ali S.M.
      • Sabatini D.M.
      ), which phosphorylates Akt Ser-473 in the hydrophobic motif. Both phosphorylations are necessary to fully activate the Akt protein kinase (
      • Alessi D.R.
      • Andjelkovic M.
      • Caudwell B.
      • Cron P.
      • Morrice N.
      • Cohen P.
      • Hemmings B.A.
      ). The phosphorylated hydrophobic motif of Akt provides a docking site for PDK1 (
      • Balendran A.
      • Biondi R.M.
      • Cheung P.C.
      • Casamayor A.
      • Deak M.
      • Alessi D.R.
      ,
      • Frödin M.
      • Antal T.L.
      • Dümmler B.A.
      • Jensen C.J.
      • Deak M.
      • Gammeltoft S.
      • Biondi R.M.
      ,
      • Biondi R.M.
      • Kieloch A.
      • Currie R.A.
      • Deak M.
      • Alessi D.R.
      ).
      Recently a more detailed “PH-in/PH-out” model for Akt activation has been proposed (
      • Calleja V.
      • Alcor D.
      • Laguerre M.
      • Park J.
      • Vojnovic B.
      • Hemmings B.A.
      • Downward J.
      • Parker P.J.
      • Larijani B.
      ). Forster resonance energy transfer (FRET)/two-photon fluorescence lifetime imaging microscopy suggested that the preactivation complex of Akt and PDK1 is maintained in an inactive state through an Akt intramolecular interaction between its PH domain and kinase domains (PH-in conformer). This domain-domain interaction apparently prevents the Akt activation loop from being phosphorylated by PDK1. Upon stimulation, Akt PH-domain interaction with phosphoinositides and the concomitant change in Akt conformation purportedly cause the separation of the PH domain and kinase domain (PH-out conformer), allowing the associated PDK1 to phosphorylate Akt Thr-308 (
      • Calleja V.
      • Laguerre M.
      • Parker P.J.
      • Larijani B.
      ). This model is also consistent with data showing phosphorylation of Akt by PDK1 is associated with membrane recruitment and a conformational change in Akt (
      • Calleja V.
      • Laguerre M.
      • Larijani B.
      ). Akt also contains a turn motif phosphorylation site in the kinase domain (Thr-450 in Akt1, Thr-451 in Akt2, and Thr-447 in Akt3). The mTORC2-dependent phosphorylation of the Akt turn motif is required for kinase stability and may also protect the hydrophobic motif site from dephosphorylation (
      • Hauge C.
      • Antal T.L.
      • Hirschberg D.
      • Doehn U.
      • Thorup K.
      • Idrissova L.
      • Hansen K.
      • Jensen O.N.
      • Jørgensen T.J.
      • Biondi R.M.
      • Frödin M.
      ,
      • Ikenoue T.
      • Inoki K.
      • Yang Q.
      • Zhou X.
      • Guan K.L.
      ,
      • Facchinetti V.
      • Ouyang W.
      • Wei H.
      • Soto N.
      • Lazorchak A.
      • Gould C.
      • Lowry C.
      • Newton A.C.
      • Mao Y.
      • Miao R.Q.
      • Sessa W.C.
      • Qin J.
      • Zhang P.
      • Su B.
      • Jacinto E.
      ).
      One of the established functions of Akt is to mediate insulin signaling to cause GLUT4 glucose transporter translocation from intracellular membranes to the cell surface membrane, thereby stimulating glucose uptake into muscle and adipose tissues (
      • Kohn A.D.
      • Summers S.A.
      • Birnbaum M.J.
      • Roth R.A.
      ,
      • Cho H.
      • Mu J.
      • Kim J.K.
      • Thorvaldsen J.L.
      • Chu Q.
      • Crenshaw 3rd, E.B.
      • Kaestner K.H.
      • Bartolomei M.S.
      • Shulman G.I.
      • Birnbaum M.J.
      ,
      • Jiang Z.Y.
      • Zhou Q.L.
      • Coleman K.A.
      • Chouinard M.
      • Boese Q.
      • Czech M.P.
      ). The aim of this study was to search for new proteins that may function to enhance insulin signaling to Akt and GLUT4 translocation. Here, we report the identification of a novel, uncharacterized protein denoted pleckstrin homology-like domain, family B, member 1 (PHLDB1) that displays such a function. PHLDB1 (also known as LL5α) is a protein that was first identified in a bioinformatics screen (
      • Katoh M.
      • Katoh M.
      ) and has an isoform (PHLDB2/LL5β) with 70% identity at the protein level containing a Forkhead-associated (FHA) domain and a C-terminal PH domain. LL5β has been suggested to interact with filament C (
      • Paranavitane V.
      • Coadwell W.J.
      • Eguinoa A.
      • Hawkins P.T.
      • Stephens L.
      ,
      • Paranavitane V.
      • Stephens L.R.
      • Hawkins P.T.
      ), a confirmed direct target of Akt (
      • Murray J.T.
      • Campbell D.G.
      • Peggie M.
      • Mora A.
      • Alfonso M.
      • Cohen P.
      ). LL5β is also implicated as a binding partner for CLASPs that are mammalian microtubule-stabilizing proteins involved in the interaction between distal microtubule ends and the cell cortex (
      • Lansbergen G.
      • Grigoriev I.
      • Mimori-Kiyosue Y.
      • Ohtsuka T.
      • Higa S.
      • Kitajima I.
      • Demmers J.
      • Galjart N.
      • Houtsmuller A.B.
      • Grosveld F.
      • Akhmanova A.
      ). In a screen for synaptically enriched transcripts at the neuromuscular junction, LL5β was identified as a regulator of postsynaptic differentiation and is required for assembly of the postsynaptic apparatus (
      • Kishi M.
      • Kummer T.T.
      • Eglen S.J.
      • Sanes J.R.
      ). In this study, we found that PHLDB1 is highly expressed in cultured adipocytes and adipose tissues. We also demonstrated that PHLDB1 binds PI(3,4,5)P3 through its PH domain, functions in adipocytes as a positive regulator of Akt activation, and is required for optimal insulin-induced glucose transport and GLUT4 translocation.

      Acknowledgments

      We thank Karl D. Bellve, Clive Standley, and Lawrence M. Lifshitz from the Biomedical Imaging Group at University of Massachusetts Medical School for the support with the TIRF microscopy and imaging analysis. We thank Paul S. Furcinitti from Digital Imaging Core Facility for the support with the immunofluorescence microscopy.

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