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Thematic Review Series Thematic Review Series: Seeing 2020: Lipids and Lipid-Soluble Molecules in the Eye| Volume 62, 100041, January 01, 2021

Signaling roles of phosphoinositides in the retina

  • Raju V.S. Rajala
    Correspondence
    For correspondence: Raju V. S. Rajala
    Affiliations
    Departments of Ophthalmology, Physiology, and Cell Biology, and Dean McGee Eye Institute, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104
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Open AccessPublished:February 05, 2021DOI:https://doi.org/10.1194/jlr.TR120000806

      Abstract

      The field of phosphoinositide signaling has expanded significantly in recent years. Phosphoinositides (also known as phosphatidylinositol phosphates or PIPs) are universal signaling molecules that directly interact with membrane proteins or with cytosolic proteins containing domains that directly bind phosphoinositides and are recruited to cell membranes. Through the activities of phosphoinositide kinases and phosphoinositide phosphatases, seven distinct phosphoinositide lipid molecules are formed from the parent molecule, phosphatidylinositol. PIP signals regulate a wide range of cellular functions, including cytoskeletal assembly, membrane budding and fusion, ciliogenesis, vesicular transport, and signal transduction. Given the many excellent reviews on phosphoinositide kinases, phosphoinositide phosphatases, and PIPs in general, in this review, we discuss recent studies and advances in PIP lipid signaling in the retina. We specifically focus on PIP lipids from vertebrate (e.g., bovine, rat, mouse, toad, and zebrafish) and invertebrate (e.g., Drosophila, horseshoe crab, and squid) retinas. We also discuss the importance of PIPs revealed from animal models and human diseases, and methods to study PIP levels both in vitro and in vivo. We propose that future studies should investigate the function and mechanism of activation of PIP-modifying enzymes/phosphatases and further unravel PIP regulation and function in the different cell types of the retina.

      Supplementary key words

      Abbreviations:

      CNG (cyclic nucleotide-gated), CNGA1 (cyclic nucleotidegated channel subunit α1), CNTF (ciliary neurotrophic factor), EPO (erythropoietin), FYCO1 (FYVE and coiled-coil domain autophagy adaptor 1), GPCR (G protein-coupled receptor), Grb14 (growth factor receptor-bound protein 14), GSK3β (glycogen synthase kinase 3β), IGF (insulin-like growth factor), INPP5E (inositol polyphosphate 5-phosphatase), IP3 (inositol triphosphate), IR (insulin receptor), IRS (insulin receptor substrate), mTOR (mammalian target of rapamycin), OCRL (oculocerebrorenal syndrome of Lowe), PDGF (platelet-derived growth factor), PH (pleckstrin homology), PI (phosphatidylinositol), PIKfyve (FYVE-type zinc finger containing phosphoinositide kinase), PI3K (phosphoinositide 3-kinase), PI4K (phosphatidylinositol 4-kinase), PIP (phosphatidylinositol phosphate), PI(3)P (phosphatidylinositol 3-phosphate), PI(4)P (phosphatidylinositol 4-phosphate), PI(5)P (phosphatidylinositol 5-phosphate), PI(3,4) P2 (phosphatidylinositol 3,4-bisphosphate), PI(3,5)P2 (phosphatidylinositol 3,5-bisphosphate), PI(4,5)P2 (phosphatidylinositol 4,5-bisphosphate), PI(3,4,5)P2 (phosphatidylinositol 3,4,5-trisphosphate), PIPK (phosphatidylinositol phosphate kinase), PITP (phosphatidylinositol transfer protein), PKC (protein kinase C), PLC (phospholipase C), PTEN (phosphatase and tensin homolog), PTP1B (protein tyrosine phosphatase 1B), RA (Ras-associating), ROS (rod outer segment), RPE (retinal pigment epithelium), SH2 (Src homology 2), SHIP (Src homology 2 domain-containing inositol polyphosphate 5-phosphatase), TRP (transient receptor potential), Vps (vacuolar protein sorting), VDAC (voltage-dependent anion channel)
      Phosphatidylinositol (PI) is a minor constituent (∼0.5–1%) of the total phospholipid pool in the cell membrane (
      • Balla T.
      Phosphoinositides: tiny lipids with giant impact on cell regulation.
      ). Phosphatidylinositol consists of a head group of D-myo-inositol and a backbone of the trihydroxy alcohol, glycerol, in which the C1 and C2 positions of glycerol are occupied with two fatty acids (Fig. 1). The free-OH groups (3, 4, and 5) in the inositol head group undergo phosphorylation by specific phosphoinositide kinases. These phosphorylated phosphatidylinositol phosphates are collectively called phosphoinositides (PIPs).
      Figure thumbnail gr1
      Fig. 1Structure of phosphatidylinositol (PI). D-myo-inositol presented as a Haworth projection (A). Phosphatidylinositol contains D-myo-inositol attached to a glycerol backbone and two fatty acids attached to the C1 and C2 positions of the glycerol (B).
      The intracellular pools of PIPs are dynamically converted from one form to the other through the action of specific phosphoinositide kinases, and phosphoinositide phosphatases can generate seven distinct PIPs (
      • Balla T.
      Phosphoinositides: tiny lipids with giant impact on cell regulation.
      ,
      • Sasaki T.
      • Takasuga S.
      • Sasaki J.
      • Kofuji S.
      • Eguchi S.
      • Yamazaki M.
      • Suzuki A.
      Mammalian phosphoinositide kinases and phosphatases.
      ,
      • Rusten T.E.
      • Stenmark H.
      Analyzing phosphoinositides and their interacting proteins.
      ) (Fig. 2). These molecules include PI(3)P, PI(4)P, PI(5)P, PI(3,4)P2, PI(3,5)P2, PI(4,5)P2, and PI(3,4,5)P3. Of these PIPs, the D3-phosphoinositides include PI(3)P, PI(3,4)P2, and PI(3,4,5)P3; the D4-phosphoinositides include PI(4)P and PI(4,5)P2; and the D5-phosphoinositides include PI(5)P and PI(3,5)P2. The D3-PIPs are formed by the action of the phosphoinositide 3-kinases (PI3Ks), which use PI, PI(4)P, and PI(4,5)P2 to generate PI(3)P, PI(3,4)P2, and PI(3,4,5)P3. The D4-PIPs are formed by the action of the PI4 kinases (PI4Ks), which use PI and PI(5)P to generate PI(4)P and PI(4,5)P2. The D5-PIPs are formed by the action of the enzyme PI5K, which uses PI and PI(3)P to generate PI(5)P and PI(3,5)P2 (
      • Balla T.
      Phosphoinositides: tiny lipids with giant impact on cell regulation.
      ,
      • Rusten T.E.
      • Stenmark H.
      Analyzing phosphoinositides and their interacting proteins.
      ). These seven distinct PIPs are present in all mammalian cells (
      • Rameh L.E.
      • Cantley L.C.
      The role of phosphoinositide 3-kinase lipid products in cell function.
      ), and their formation is also present in the retina/photoreceptor cells (
      • Ghalayini A.J.
      • Anderson R.E.
      Light adaptation of bovine retinas in situ stimulates phosphatidylinositol synthesis in rod outer segments in vitro.
      ,
      • Huang Z.
      • Ghalayini A.
      • Guo X.X.
      • Alvarez K.M.
      • Anderson R.E.
      Light-mediated activation of diacylglycerol kinase in rat and bovine rod outer segments.
      ,
      • Guo X.
      • Ghalayini A.J.
      • Chen H.
      • Anderson R.E.
      Phosphatidylinositol 3-kinase in bovine photoreceptor rod outer segments.
      ,
      • Guo X.X.
      • Huang Z.
      • Bell M.W.
      • Chen H.
      • Anderson R.E.
      Tyrosine phosphorylation is involved in phosphatidylinositol 3-kinase activation in bovine rod outer segments.
      ). The D3-, D4-, and D5-phosphoinositides regulate cytoskeletal organization, membrane fusion and budding, ciliogenesis, vesicular transport, and signal transduction (
      • Martin T.F.
      Phosphoinositide lipids as signaling molecules: common themes for signal transduction, cytoskeletal regulation, and membrane trafficking.
      ,
      • Fruman D.A.
      • Meyers R.E.
      • Cantley L.C.
      Phosphoinositide kinases.
      ). PI(3)P is involved in the vesicle export from the Golgi. PI(4,5)P2 is involved in exocytosis, cytoskeletal regulation, and regulation of phospholipase D/A2. PI(3,5)P2 regulates Golgi, lysosomal/endosome trafficking, and osmoprotection. PI(3,4,5)P3 is involved in cell survival, solute transport, and regulation of ARF/Rac protein (
      • Balla T.
      Phosphoinositides: tiny lipids with giant impact on cell regulation.
      ,
      • Martin T.F.
      Phosphoinositide lipids as signaling molecules: common themes for signal transduction, cytoskeletal regulation, and membrane trafficking.
      ).
      Figure thumbnail gr2
      Fig. 2Generation of seven distinct phosphoinositides. The inositol head group contains several free hydroxyls that undergo phosphorylation by phosphoinositide kinases and dephosphorylation by phosphoinositide phosphatases. Phosphorylation of free hydroxyls at the 3, 4, 5 positions facilitates the generation of seven PPIs. PI(4,5)P2 undergoes hydrolysis by phospholipase C (PLC) generates two-second messenger signaling molecules, diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). IP3 binds to IP3-sensitive Ca2+ channels on the endoplasmic reticulum and mobilizes the release of Ca2+. Both, DAG and Ca2+ activate protein kinase C (PKC). Activated PKC phosphorylates its downstream target proteins. MTMl, myotubularin l (3’phosphatase); MTMRs, myotubularin-related phospholipid phosphatase; PI(3)P, phosphatidylinositol 3-phosphate; PI(4)P, phosphatidylinositol 4-phosphate; PI(5)P, phosphatidylinositol 5-phosphate; PI(3,4)P2, phosphatidylinositol 3,4-bisphosphate; PI(3,5)P2, phosphatidylinositol 3,5-bisphosphate; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PI(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate. PIP4K, phosphatidylinositol 5-phosphate 4-kinase; PI3K, phosphoinositide 3-kinase; PIP5K, phosphatidylinositol 4-phosphate 5-kinase; PTEN, phosphatase and tensin homolog; SHIP, Src homology 2 (SH2) domain-containing inositol polyphosphate 5-phosphatase; PIKfyve, FYVE-type zinc finger containing phosphoinositide kinase; OCRL, oculocerebrorenal syndrome of Lowe; INPP5E, inositol polyphosphate 5-phosphatase; PI4K, phosphatidylinositol 4-kinase.
      There are many excellent reviews on phosphoinositide kinases, phosphoinositide phosphatases, and PIPs in general. This review describes the recent studies and advances in PIP lipids and their signaling in the retina. The PIP lipids from vertebrate (e.g., bovine, rat, mouse, toad, zebrafish) and invertebrate [e.g., Drosophila, limulus (horseshoe crab), squid] retina are discussed. The importance of PIPs revealed from animal models and human diseases and methods to study PIP levels in vitro and in vivo are also described.

      Phosphoinositides in the retina

      In 1981, Anderson and Hollyfield (
      • Anderson R.E.
      • Hollyfield J.G.
      Light stimulates the incorporation of inositol into phosphatidylinositol in the retina.
      ) reported that light stimulates the incorporation of inositol in the vertebrate retina. Light has also been shown to stimulate the generation of PIs in horizontal cells of the retina (
      • Anderson R.E.
      • Maude M.B.
      • Kelleher P.A.
      • Rayborn M.E.
      • Hollyfield J.G.
      Phosphoinositide metabolism in the retina: localization to horizontal cells and regulation by light and divalent cations.
      ,
      • Anderson R.E.
      • Hollyfield J.G.
      Inositol incorporation into phosphoinositides in retinal horizontal cells of Xenopus laevis: enhancement by acetylcholine, inhibition by glycine.
      ). Subsequent studies showed that light adaptation of bovine retinas in situ stimulates PI synthesis in retinal rod outer segment (ROS) membranes in vitro (
      • Ghalayini A.J.
      • Anderson R.E.
      Light adaptation of bovine retinas in situ stimulates phosphatidylinositol synthesis in rod outer segments in vitro.
      ). The work described in 1983 and 1984 by Anderson and colleagues revealed a functional significance in photoreceptor horizontal cell synapses (
      • Anderson R.E.
      • Maude M.B.
      • Kelleher P.A.
      • Rayborn M.E.
      • Hollyfield J.G.
      Phosphoinositide metabolism in the retina: localization to horizontal cells and regulation by light and divalent cations.
      ,
      • Anderson R.E.
      • Hollyfield J.G.
      Inositol incorporation into phosphoinositides in retinal horizontal cells of Xenopus laevis: enhancement by acetylcholine, inhibition by glycine.
      ). Light stimulates the PI metabolism in these cells, and these PIPs are important for synaptic ribbon formation, glutamate release, and signaling (
      • Anderson R.E.
      • Maude M.B.
      • Kelleher P.A.
      • Rayborn M.E.
      • Hollyfield J.G.
      Phosphoinositide metabolism in the retina: localization to horizontal cells and regulation by light and divalent cations.
      ,
      • Anderson R.E.
      • Hollyfield J.G.
      Inositol incorporation into phosphoinositides in retinal horizontal cells of Xenopus laevis: enhancement by acetylcholine, inhibition by glycine.
      ).
      Phosphatidylinositol is a comparatively large component of the membranes of most cells in metazoans ranging from 4 to 20 mol% of total phospholipid (
      • Balla T.
      Phosphoinositides: tiny lipids with giant impact on cell regulation.
      ,
      • Wensel T.G.
      Phosphoinositides in retinal function and disease.
      ), and analysis of phosphatidylinositol in six different mammalian species shows 4.4–6.4% mol% of total retina phospholipid (
      • Wensel T.G.
      Phosphoinositides in retinal function and disease.
      ,
      • Broekhuyse R.M.
      Phospholipids in tissues of the eye. I. Isolation, characterization and quantitative analysis by two-dimensional thin-layer chromatography of diacyl and vinyl-ether phospholipids.
      ,
      • Anderson R.E.
      • Feldman L.S.
      • Feldman G.L.
      Lipids of ocular tissues. II. The phospholipids of mature bovine and rabbit whole retina.
      ,
      • Anderson R.E.
      Lipids of ocular tissues. IV. A comparison of the phospholipids from the retina of six mammalian species.
      ). Studies have shown that retinal ROS membranes contain lower levels of PI than do retinal pigment epithelium (RPE) cells (
      • Anderson R.E.
      • Lissandrello P.M.
      • Maude M.B.
      • Matthes M.T.
      Lipids of bovine retinal pigment epithelium.
      ) and ER membranes isolated from bovine retinas (
      • Anderson R.E.
      • Maude M.B.
      • Zimmerman W.
      Lipids of ocular tissues–X. Lipid composition of subcellular fractions of bovine retina.
      ). Recently, Wensel’s laboratory reported that ROSs and fragments of inner segments contain PI(3)P at 0.0035 mol% and PI(4)P and PI(4,5)P2 on the order of 0.04 mol%, 10-fold higher than the levels of PI(3)P of total phospholipid (
      • Wensel T.G.
      Phosphoinositides in retinal function and disease.
      ).
      Phototransduction is modulated by phosphoinositides within photoreceptors (
      • Ghalayini A.J.
      • Anderson R.E.
      Light adaptation of bovine retinas in situ stimulates phosphatidylinositol synthesis in rod outer segments in vitro.
      ,
      • Schmidt S.Y.
      Light- and cytidine-dependent phosphatidylinositol synthesis in photoreceptor cells of the rat.
      ), which increase phospholipase C (PLC) activity in the outer segment membranes, increase the uptake of radiolabeled inositol and phosphoinositide turnover in photoreceptor cells (
      • Schmidt S.Y.
      Light- and cytidine-dependent phosphatidylinositol synthesis in photoreceptor cells of the rat.
      ,
      • Schmidt S.Y.
      Light enhances the turnover of phosphatidylinositol in rat retinas.
      ), and release inositol 1,4,5-trisphosphate (IP3) from the retina (
      • Jung H.H.
      • Reme C.E.
      • Pfeilschifter J.
      Light evoked inositol trisphosphate release in the rat retina in vitro.
      ). Furthermore, IP3 receptors (
      • Day N.S.
      • Koutz C.A.
      • Anderson R.E.
      Inositol-1,4,5-trisphosphate receptors in the vertebrate retina.
      ) and PLC (
      • Ghalayini A.J.
      • Tarver A.P.
      • Mackin W.M.
      • Koutz C.A.
      • Anderson R.E.
      Identification and immunolocalization of phospholipase C in bovine rod outer segments.
      ,
      • Peng Y.W.
      • Rhee S.G.
      • Yu W.P.
      • Ho Y.K.
      • Schoen T.
      • Chader G.J.
      • Yau K.W.
      Identification of components of a phosphoinositide signaling pathway in retinal rod outer segments.
      ) are expressed in the outer segments. In invertebrates, phototransduction is mediated through PLC activation, while in the vertebrate retina, the phototransduction cascade is mediated through rhodopsin activation and subsequent hydrolysis of cGMP (
      • Kaupp U.B.
      • Seifert R.
      Cyclic nucleotide-gated ion channels.
      ,
      • Yau K.W.
      • Hardie R.C.
      Phototransduction motifs and variations.
      ). Interestingly, in the intrinsically photosensitive retinal ganglion cells, PLC-mediated hydrolysis is activated by the photopigment, melanopsin, which couples Gq to open the transient receptor potential (TRP) channels (
      • Schmidt T.M.
      • Chen S.K.
      • Hattar S.
      Intrinsically photosensitive retinal ganglion cells: many subtypes, diverse functions.
      ), suggesting evolutionarily conserved pathways that use PI(4,5)P2 as a substrate for the modulation of phototransduction.
      One of the potential functions of PLC is involvement in the translocation of arrestin from inner to outer segments of photoreceptor cells (
      • Orisme W.
      • Li J.
      • Goldmann T.
      • Bolch S.
      • Wolfrum U.
      • Smith W.C.
      Light-dependent translocation of arrestin in rod photoreceptors is signaled through a phospholipase C cascade and requires ATP.
      ). Furthermore, activators of the downstream effector of PLC, protein kinase C (PKC), and PLC facilitate arrestin translocation to outer segment membranes, independent of light (
      • Orisme W.
      • Li J.
      • Goldmann T.
      • Bolch S.
      • Wolfrum U.
      • Smith W.C.
      Light-dependent translocation of arrestin in rod photoreceptors is signaled through a phospholipase C cascade and requires ATP.
      ). Physiological studies also show that phototransduction is modulated by PIPs (
      • Wensel T.G.
      Phosphoinositides in retinal function and disease.
      ,
      • He F.
      • Mao M.
      • Wensel T.G.
      Enhancement of phototransduction g protein-effector interactions by phosphoinositides.
      ). The cone cyclic nucleotide-gated (CNG) (
      • Bright S.R.
      • Rich E.D.
      • Varnum M.D.
      Regulation of human cone cyclic nucleotide-gated channels by endogenous phospholipids and exogenously applied phosphatidylinositol 3,4,5-trisphosphate.
      ) and olfactory (
      • Brady J.D.
      • Rich E.D.
      • Martens J.R.
      • Karpen J.W.
      • Varnum M.D.
      • Brown R.L.
      Interplay between PIP3 and calmodulin regulation of olfactory cyclic nucleotide-gated channels.
      ) channels are known to be inhibited by PI3K-generated PI(3,4,5)P3.
      It has been suggested that phosphoinositides play an important role in photoreceptor cell processes, such as disk morphogenesis, endocytosis, exocytosis, membrane budding, endosomal sorting, and post-Golgi vesicle trafficking. In rhodopsin trafficking, the involvement of PI(3)P and PI(4,5)P2 has been demonstrated (
      • Deretic D.
      • Traverso V.
      • Parkins N.
      • Jackson F.
      • Rodriguez de Turco E.B.
      • Ransom N.
      Phosphoinositides, ezrin/moesin, and rac1 regulate fusion of rhodopsin transport carriers in retinal photoreceptors.
      ,
      • Spencer W.J.
      • Lewis T.R.
      • Phan S.
      • Cady M.A.
      • Serebrovskaya E.O.
      • Schneider N.F.
      • Kim K.Y.
      • Cameron L.A.
      • Skiba N.P.
      • Ellisman M.H.
      • et al.
      Photoreceptor disc membranes are formed through an Arp2/3-dependent lamellipodium-like mechanism.
      ). The actin-nucleating proteins Arp2 and Arp3 are demonstrated to be involved in basal disc extensions (
      • Spencer W.J.
      • Lewis T.R.
      • Phan S.
      • Cady M.A.
      • Serebrovskaya E.O.
      • Schneider N.F.
      • Kim K.Y.
      • Cameron L.A.
      • Skiba N.P.
      • Ellisman M.H.
      • et al.
      Photoreceptor disc membranes are formed through an Arp2/3-dependent lamellipodium-like mechanism.
      ), and local pools of PI(4,5)P2 may be important for their function (
      • Bucki R.
      • Wang Y.H.
      • Yang C.
      • Kandy S.K.
      • Fatunmbi O.
      • Bradley R.
      • Pogoda K.
      • Svitkina T.
      • Radhakrishnan R.
      • Janmey P.A.
      Lateral distribution of phosphatidylinositol 4,5-bisphosphate in membranes regulates formin- and ARP2/3-mediated actin nucleation.
      ,
      • Daste F.
      • Walrant A.
      • Holst M.R.
      • Gadsby J.R.
      • Mason J.
      • Lee J.E.
      • Brook D.
      • Mettlen M.
      • Larsson E.
      • Lee S.F.
      • et al.
      Control of actin polymerization via the coincidence of phosphoinositides and high membrane curvature.
      ).

      Metabolism of phosphoinositides in the retina

      There is an active PI metabolism in the vertebrate retina and ROSs (
      • Huang Z.
      • Ghalayini A.
      • Guo X.X.
      • Alvarez K.M.
      • Anderson R.E.
      Light-mediated activation of diacylglycerol kinase in rat and bovine rod outer segments.
      ,
      • Guo X.
      • Ghalayini A.J.
      • Chen H.
      • Anderson R.E.
      Phosphatidylinositol 3-kinase in bovine photoreceptor rod outer segments.
      ,
      • Guo X.X.
      • Huang Z.
      • Bell M.W.
      • Chen H.
      • Anderson R.E.
      Tyrosine phosphorylation is involved in phosphatidylinositol 3-kinase activation in bovine rod outer segments.
      ,
      • Anderson R.E.
      • Maude M.B.
      • Kelleher P.A.
      • Rayborn M.E.
      • Hollyfield J.G.
      Phosphoinositide metabolism in the retina: localization to horizontal cells and regulation by light and divalent cations.
      ,
      • Anderson R.E.
      • Hollyfield J.G.
      Inositol incorporation into phosphoinositides in retinal horizontal cells of Xenopus laevis: enhancement by acetylcholine, inhibition by glycine.
      ,
      • Schmidt S.Y.
      Light- and cytidine-dependent phosphatidylinositol synthesis in photoreceptor cells of the rat.
      ,
      • Schmidt S.Y.
      Light enhances the turnover of phosphatidylinositol in rat retinas.
      ,
      • Jung H.H.
      • Reme C.E.
      • Pfeilschifter J.
      Light evoked inositol trisphosphate release in the rat retina in vitro.
      ,
      • Peng Y.W.
      • Rhee S.G.
      • Yu W.P.
      • Ho Y.K.
      • Schoen T.
      • Chader G.J.
      • Yau K.W.
      Identification of components of a phosphoinositide signaling pathway in retinal rod outer segments.
      ,
      • Giusto N.M.
      • Pasquare S.J.
      • Salvador G.A.
      • Ilincheta de Boschero M.G.
      Lipid second messengers and related enzymes in vertebrate rod outer segments.
      ,
      • Anderson R.E.
      • Maude M.B.
      • Kelleher P.A.
      • Maida T.M.
      • Basinger S.F.
      Metabolism of phosphatidylcholine in the frog retina.
      ,
      • Anderson R.E.
      • Kelleher P.A.
      • Maude M.B.
      Metabolism of phosphatidylethanolamine in the frog retina.
      ,
      • Anderson R.E.
      • Maude M.B.
      • Pu G.A.
      • Hollyfield J.G.
      Effect of light on the metabolism of lipids in the rat retina.
      ,
      • Schmidt S.Y.
      Phosphatidylinositol synthesis and phosphorylation are enhanced by light in rat retinas.
      ,
      • Choe H.G.
      • Ghalayini A.J.
      • Anderson R.E.
      Phosphoinositide metabolism in frog rod outer segments.
      ,
      • Ghalayini A.J.
      • Guo X.X.
      • Koutz C.A.
      • Anderson R.E.
      Light stimulates tyrosine phosphorylation of rat rod outer segments In vivo.
      ,
      • Brown J.E.
      • Blazynski C.
      • Cohen A.I.
      Light induces a rapid and transient increase in inositol-trisphosphate in toad rod outer segments.
      ,
      • Das N.D.
      • Yoshioka T.
      • Samuelson D.
      • Shichi H.
      Immunocytochemical localization of phosphatidylinositol-4,5-bisphosphate in dark- and light-adapted rat retinas.
      ,
      • Ferreira P.A.
      • Shortridge R.D.
      • Pak W.L.
      Distinctive subtypes of bovine phospholipase C that have preferential expression in the retina and high homology to the norpA gene product of Drosophila.
      ,
      • Gehm B.D.
      • Mc Connell D.G.
      Phosphoinositide synthesis in bovine rod outer segments.
      ,
      • Gehm B.D.
      • Pinke R.M.
      • Laquerre S.
      • Chafouleas J.G.
      • Schultz D.A.
      • Pepperl D.J.
      • McConnell D.G.
      Activation of bovine rod outer segment phosphatidylinositol-4,5-bisphosphate phospholipase C by calmodulin antagonists does not depend on calmodulin.
      ,
      • Hayashi F.
      • Amakawa T.
      Light-mediated breakdown of phosphatidylinositol-4,5-bisphosphate in isolated rod outer segments of frog photoreceptor.
      ,
      • Jelsema C.L.
      Regulation of phospholipase A2 and phospholipase C in rod outer segments of bovine retina involves a common GTP-binding protein but different mechanisms of action.
      ,
      • Millar F.A.
      • Fisher S.C.
      • Muir C.A.
      • Edwards E.
      • Hawthorne J.N.
      Polyphosphoinositide hydrolysis in response to light stimulation of rat and chick retina and retinal rod outer segments.
      ). Light has been shown to stimulate several enzymes that are involved in PI metabolism, such as class I PI3K (
      • Guo X.
      • Ghalayini A.J.
      • Chen H.
      • Anderson R.E.
      Phosphatidylinositol 3-kinase in bovine photoreceptor rod outer segments.
      ,
      • Rajala A.
      • Anderson R.E.
      • Ma J.X.
      • Lem J.
      • Al Ubaidi M.R.
      • Rajala R.V.
      G-protein-coupled receptor rhodopsin regulates the phosphorylation of retinal insulin receptor.
      ), class III PI3K (
      • He F.
      • Agosto M.A.
      • Anastassov I.A.
      • Tse D.Y.
      • Wu S.M.
      • Wensel T.G.
      Phosphatidylinositol-3-phosphate is light-regulated and essential for survival in retinal rods.
      ,
      • Rajala R.V.
      • Rajala A.
      • Morris A.J.
      • Anderson R.E.
      Phosphoinositides: minor lipids make a major impact on photoreceptor cell functions.
      ), PI synthetase (
      • Ghalayini A.J.
      • Anderson R.E.
      Light adaptation of bovine retinas in situ stimulates phosphatidylinositol synthesis in rod outer segments in vitro.
      ), DAG kinase (
      • Huang Z.
      • Ghalayini A.
      • Guo X.X.
      • Alvarez K.M.
      • Anderson R.E.
      Light-mediated activation of diacylglycerol kinase in rat and bovine rod outer segments.
      ), phosphatidylethanolamine N-methyltransferase (
      • Roque M.E.
      • Salvador G.A.
      • Giusto N.M.
      Light activation of phosphatidylethanolamine N-methyltransferase in rod outer segments and its modulation by association states of transducin.
      ), phospholipase A2 (
      • Castagnet P.I.
      • Giusto N.M.
      Properties of phospholipase A2 activity from bovine retinal rod outer segments.
      ), phospholipase D (
      • Salvador G.A.
      • Giusto N.M.
      Phospholipase D from photoreceptor rod outer segments is a downstream effector of RhoA: evidence of a light-dependent mechanism.
      ), PLC (
      • Giusto N.M.
      • Pasquare S.J.
      • Salvador G.A.
      • Castagnet P.I.
      • Roque M.E.
      • Ilincheta de Boschero M.G.
      Lipid metabolism in vertebrate retinal rod outer segments.
      ,
      • Ghalayini A.
      • Anderson R.E.
      Phosphatidylinositol 4,5-bisphosphate: light-mediated breakdown in the vertebrate retina.
      ,
      • Ghalayini A.J.
      • Anderson R.E.
      Activation of bovine rod outer segment phospholipase C by arrestin.
      ,
      • Ghalayini A.J.
      • Weber N.R.
      • Rundle D.R.
      • Koutz C.A.
      • Lambert D.
      • Guo X.X.
      • Anderson R.E.
      Phospholipase Cgamma1 in bovine rod outer segments: immunolocalization and light-dependent binding to membranes.
      ), PKC (
      • Natalini P.M.
      • Mateos M.V.
      • Ilincheta de Boschero M.G.
      • Giusto N.M.
      A novel light-dependent activation of DAGK and PKC in bovine photoreceptor nuclei.
      ), lipid phosphate phosphatase (
      • Pasquaré S.J.
      • Salvador G.A.
      • Roque M.E.
      • Giusto N.M.
      Effect of light on phosphatidate phosphohydrolase activity of retina rod outer segments: the role of transducin.
      ), and DAG lipase (
      • Giusto N.M.
      • Pasquare S.J.
      • Salvador G.A.
      • Ilincheta de Boschero M.G.
      Lipid second messengers and related enzymes in vertebrate rod outer segments.
      ). Light also modulates the second messengers generated in the retina from DAG, PC, and PA (
      • Giusto N.M.
      • Pasquare S.J.
      • Salvador G.A.
      • Ilincheta de Boschero M.G.
      Lipid second messengers and related enzymes in vertebrate rod outer segments.
      ). Further, light activates proteins regulated by insulin signaling (
      • Balasubramanian N.
      • Slepak V.Z.
      Light-mediated activation of Rac-1 in photoreceptor outer segments.
      ,
      • Natalini P.M.
      • Mateos M.V.
      • Ilincheta de Boschero M.G.
      • Giusto N.M.
      Insulin-related signaling pathways elicited by light in photoreceptor nuclei from bovine retina.
      ,
      • Li G.
      • Rajala A.
      • Wiechmann A.F.
      • Anderson R.E.
      • Rajala R.V.
      Activation and membrane binding of retinal protein kinase Balpha/Akt1 is regulated through light-dependent generation of phosphoinositides.
      ,
      • Rajala A.
      • Daly R.J.
      • Tanito M.
      • Allen D.T.
      • Holt L.J.
      • Lobanova E.S.
      • Arshavsky V.Y.
      • Rajala R.V.
      Growth factor receptor-bound protein 14 undergoes light-dependent intracellular translocation in rod photoreceptors: functional role in retinal insulin receptor activation.
      ).

      Phosphoinositide kinases and phosphatases

      Forty-seven genes encode 19 phosphoinositide kinases and phosphatidylinositol phosphate (PIP) kinases (PIPKs) and 28 PIP phosphatases in mammals (
      • Sasaki T.
      • Takasuga S.
      • Sasaki J.
      • Kofuji S.
      • Eguchi S.
      • Yamazaki M.
      • Suzuki A.
      Mammalian phosphoinositide kinases and phosphatases.
      ). The action of the lipid kinases, PI3K, PI4K, and PI5K, and PI3-, PI4- and PI5-specific lipid phosphatases can generate seven distinct phosphoinositide lipids in mammalian cells (
      • Balla T.
      Phosphoinositides: tiny lipids with giant impact on cell regulation.
      ,
      • Sasaki T.
      • Takasuga S.
      • Sasaki J.
      • Kofuji S.
      • Eguchi S.
      • Yamazaki M.
      • Suzuki A.
      Mammalian phosphoinositide kinases and phosphatases.
      ,
      • Rusten T.E.
      • Stenmark H.
      Analyzing phosphoinositides and their interacting proteins.
      ). The phosphoinositide kinase isoforms are divided into three major families: the PI 3-kinases (PI3Ks), PI 4-kinases (PI4Ks), and PIPKs (
      • Brown J.R.
      • Auger K.R.
      Phylogenomics of phosphoinositide lipid kinases: perspectives on the evolution of second messenger signaling and drug discovery.
      ).

      Phosphoinositide 3-kinases

      The phosphoinositide 3-kinases have been broadly divided into four classes: class I, class II, class III, and class IV. Members of class I PI3K enzymes are heterodimers and consist of a p110 kDa catalytic subunit and a p85 regulatory subunit that contains two Src-homology regions capable of binding to phosphotyrosine sequences on the growth factor receptors (
      • Fruman D.A.
      • Meyers R.E.
      • Cantley L.C.
      Phosphoinositide kinases.
      ). The growth factors, such as platelet-derived growth factor (PDGF), insulin, insulin-like growth factor (IGF)-1, and nerve growth factors, bind to its cognate receptor tyrosine kinase(s) and activate class I PI3Ks (
      • Cantley L.C.
      The phosphoinositide 3-kinase pathway.
      ) (Fig. 3).
      Figure thumbnail gr3
      Fig. 3Activation of phosphoinositide 3-kinases. PI3Ks are activated through tyrosine kinase receptor activation, interaction with RAS, through GPCR activation, or are integrin-mediated. The PIPs generated at the membrane attract phospholipid-binding proteins in the cytosol, activate the downstream signaling (A–C), and regulate various aspects of cellular functions. In the case of the IR, IRS adaptor proteins bind to the IR by facilitating a platform for the binding of multiple PI3K molecules and amplify the signal.
      In mammals, class I PI3K-p110 catalytic subunits are encoded by four genes: Pik3ca, Pik3cb, Pik3cg, and Pik3cd, which are referred to as PI3Kα, -β, -γ, and -δ (
      • Zhao L.
      • Vogt P.K.
      Class I PI3K in oncogenic cellular transformation.
      ). The expression profile of these genes in tissues is not uniform. Ubiquitous expression of Pik3ca and Pik3cb genes has been demonstrated, whereas leukocytes specifically express Pik3cg and Pik3cd genes (
      • Zhao L.
      • Vogt P.K.
      Class I PI3K in oncogenic cellular transformation.
      ). Pik3cg is also expressed in cardiac tissues (
      • Hirsch E.
      • Lembo G.
      • Montrucchio G.
      • Rommel C.
      • Costa C.
      • Barberis L.
      Signaling through PI3Kgamma: a common platform for leukocyte, platelet and cardiovascular stress sensing.
      ). All of the PI3Kα, -β, -γ, and -δ-p110 subunits have the phosphatidylinositol kinase domain, a catalytic domain, a C2 lipid-binding domain, and a GTPase Ras domain; these proteins all have a significant homology at the N-terminal end of the molecule (
      • Hirsch E.
      • Costa C.
      • Ciraolo E.
      Phosphoinositide 3-kinases as a common platform for multi-hormone signaling.
      ). In mammalian cells, the catalytic subunits of p110α, p110β, and p110δ are associated with any of five different regulatory subunits, p85α, p85β, p55γ, p55α, and p50α, referred to as “p85 subunits,” for phosphorylation of the lipid substrates (
      • Geering B.
      • Cutillas P.R.
      • Nock G.
      • Gharbi S.I.
      • Vanhaesebroeck B.
      Class IA phosphoinositide 3-kinases are obligate p85-p110 heterodimers.
      ). The p85 regulatory subunits are encoded by three genes, Pik3r1, Pik3r2, and Pik3r3 (
      • Geering B.
      • Cutillas P.R.
      • Nock G.
      • Gharbi S.I.
      • Vanhaesebroeck B.
      Class IA phosphoinositide 3-kinases are obligate p85-p110 heterodimers.
      ). Pik3r1 encodes the p85α, p55α, and p50α subunits. Both regulatory subunits, p85α and p85β, are universally expressed in all tissues (
      • Hirsch E.
      • Costa C.
      • Ciraolo E.
      Phosphoinositide 3-kinases as a common platform for multi-hormone signaling.
      ), including retina (
      • Ivanovic I.
      • Allen D.T.
      • Dighe R.
      • Le Y.Z.
      • Anderson R.E.
      • Rajala R.V.
      Phosphoinositide 3-kinase signaling in retinal rod photoreceptors.
      ), whereas p55γ is specifically expressed in brain tissues (
      • Pons S.
      • Asano T.
      • Glasheen E.
      • Miralpeix M.
      • Zhang Y.
      • Fisher T.L.
      • Myers Jr., M.G.
      • Sun X.J.
      • White M.F.
      The structure and function of p55PIK reveal a new regulatory subunit for phosphatidylinositol 3-kinase.
      ). The expression of p50α and p55α has been demonstrated in fat, muscle, liver, and brain tissues (
      • Antonetti D.A.
      • Algenstaedt P.
      • Kahn C.R.
      Insulin receptor substrate 1 binds two novel splice variants of the regulatory subunit of phosphatidylinositol 3-kinase in muscle and brain.
      ,
      • Inukai K.
      • Anai M.
      • Van Breda E.
      • Hosaka T.
      • Katagiri H.
      • Funaki M.
      • Fukushima Y.
      • Ogihara T.
      • Yazaki Y.
      • Kikuchi M.
      • et al.
      A novel 55-kDa regulatory subunit for phosphatidylinositol 3-kinase structurally similar to p55PIK Is generated by alternative splicing of the p85alpha gene.
      ). The regulatory p85 subunits interact with a unique domain present at the N-terminal end of the p110 catalytic subunits (
      • Hiles I.D.
      • Otsu M.
      • Volinia S.
      • Fry M.J.
      • Gout I.
      • Dhand R.
      • Panayotou G.
      • Ruiz-Larrea F.
      • Thompson A.
      • Totty N.F.
      Phosphatidylinositol 3-kinase: structure and expression of the 110 kd catalytic subunit.
      ). In addition to the Src homology 2 (SH2) domain, the p85 regulatory subunit also contains an SH3 domain, which is capable of binding to proline-rich sequences and also contains a region with high conservation to the breakpoint cluster region (
      • Hiles I.D.
      • Otsu M.
      • Volinia S.
      • Fry M.J.
      • Gout I.
      • Dhand R.
      • Panayotou G.
      • Ruiz-Larrea F.
      • Thompson A.
      • Totty N.F.
      Phosphatidylinositol 3-kinase: structure and expression of the 110 kd catalytic subunit.
      ). Extensive studies on class I PI3K have been carried out in the retina, especially in photoreceptor cells. Functionally, class I PI3K is essential for cone photoreceptor structure and function but not for rod photoreceptor cell survival (
      • Ivanovic I.
      • Allen D.T.
      • Dighe R.
      • Le Y.Z.
      • Anderson R.E.
      • Rajala R.V.
      Phosphoinositide 3-kinase signaling in retinal rod photoreceptors.
      ,
      • Ivanovic I.
      • Anderson R.E.
      • Le Y.Z.
      • Fliesler S.J.
      • Sherry D.M.
      • Rajala R.V.
      Deletion of the p85alpha regulatory subunit of phosphoinositide 3-kinase in cone photoreceptor cells results in cone photoreceptor degeneration.
      ,
      • Rajala R.V.
      Phosphoinositide 3-kinase signaling in the vertebrate retina.
      ).
      Research also indicates that class IA PI3Kβ activation is regulated both by the G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (
      • Murga C.
      • Fukuhara S.
      • Gutkind J.S.
      A novel role for phosphatidylinositol 3-kinase beta in signaling from G protein-coupled receptors to Akt.
      ). The class I PI3Kγ lacks the p85-binding motif; hence, it interacts with p101 (
      • Stephens L.R.
      • Eguinoa A.
      • Erdjument-Bromage H.
      • Lui M.
      • Cooke F.
      • Coadwell J.
      • Smrcka A.S.
      • Thelen M.
      • Cadwallader K.
      • Tempst P.
      • et al.
      The G beta gamma sensitivity of a PI3K is dependent upon a tightly associated adaptor, p101.
      ) and p84/87 adaptor proteins for regulation (
      • Suire S.
      • Hawkins P.
      • Stephens L.
      Activation of phosphoinositide 3-kinase gamma by Ras.
      ,
      • Voigt P.
      • Brock C.
      • Nurnberg B.
      • Schaefer. M.
      Assigning functional domains within the p101 regulatory subunit of phosphoinositide 3-kinase gamma.
      ). The class IB PI3Kγ is activated through GPCR signaling (Fig. 3). Upon GPCR activation by the ligand(s), heterotrimeric G protein composed of α-, β-, and γ-subunits binds to the GPCR (
      • Weis W.I.
      • Kobilka B.K.
      The molecular basis of G protein-coupled receptor activation.
      ). The α-subunit of G protein in its bound GDP readily exchanges with the cytosolic GTP, and upon GPCR activation dissociates from the rest of the βγ-subunits and activates the downstream effector molecules (
      • Smrcka A.V.
      G protein betagamma subunits: central mediators of G protein-coupled receptor signaling.
      ). The free Gβγ-subunits of heterotrimeric G proteins, mostly the Gi subtype, activate PI3K signaling (
      • Hirsch E.
      • Lembo G.
      • Montrucchio G.
      • Rommel C.
      • Costa C.
      • Barberis L.
      Signaling through PI3Kgamma: a common platform for leukocyte, platelet and cardiovascular stress sensing.
      ,
      • Murga C.
      • Laguinge L.
      • Wetzker R.
      • Cuadrado A.
      • Gutkind J.S.
      Activation of Akt/protein kinase B by G protein-coupled receptors. A role for alpha and beta gamma subunits of heterotrimeric G proteins acting through phosphatidylinositol-3-OH kinasegamma.
      ) (Fig. 3). The PI3K family of lipid kinases has also been shown to be stimulated by chemokines in regulating the chemotaxis of inflammatory T lymphocytes, eosinophils, neutrophils, and macrophages, and PI3K signaling regulates the chemokine-activated cell migration (
      • Curnock A.P.
      • Logan M.K.
      • Ward S.G.
      Chemokine signalling: pivoting around multiple phosphoinositide 3-kinases.
      ).
      The structure and function of class II PI3Ks are distinct from those of class I PI3Ks. The class II PI3Ks have a C-terminal C2 domain, which lacks a critical aspartate residue that coordinates the binding of Ca2+. The class II PI3K binds to lipid in a Ca2+-independent manner (
      • Margaria J.P.
      • Ratto E.
      • Gozzelino L.
      • Li H.
      • Hirsch E.
      Class II PI3Ks at the intersection between signal transduction and membrane trafficking.
      ,
      • Gulluni F.
      • De Santis M.C.
      • Margaria J.P.
      • Martini M.
      • Hirsch E.
      Class II PI3K functions in cell biology and disease.
      ). The class II PI3K consists of three catalytic isoforms, which include C2α, C2β, and C2γ, but is distinct from class I and III, as they do not have regulator subunits (
      • Margaria J.P.
      • Ratto E.
      • Gozzelino L.
      • Li H.
      • Hirsch E.
      Class II PI3Ks at the intersection between signal transduction and membrane trafficking.
      ,
      • Gulluni F.
      • De Santis M.C.
      • Margaria J.P.
      • Martini M.
      • Hirsch E.
      Class II PI3K functions in cell biology and disease.
      ). The class II enzyme catalyzes the conversion of PI to PI(3)P and PI(4)P to PI(3,4)P2 (
      • Margaria J.P.
      • Ratto E.
      • Gozzelino L.
      • Li H.
      • Hirsch E.
      Class II PI3Ks at the intersection between signal transduction and membrane trafficking.
      • Gulluni F.
      • De Santis M.C.
      • Margaria J.P.
      • Martini M.
      • Hirsch E.
      Class II PI3K functions in cell biology and disease.
      ). PI(3,4)P2 has been shown to play a role in the invagination step of clathrin-mediated endocytosis (
      • Posor Y.
      • Eichhorn-Grunig M.
      • Haucke V.
      Phosphoinositides in endocytosis.
      ). The class II PI3K C2α and C2β isoforms are ubiquitously expressed in all tissues, but the expression of the C2γ isoform is restricted to hepatocytes (
      • Posor Y.
      • Eichhorn-Grunig M.
      • Haucke V.
      Phosphoinositides in endocytosis.
      ). To date, there are no available studies of class II PI3K in the retina.
      The structure and function of class III PI3K are distinct from those of class I and class II PI3K. Class III PI3K, also known as vacuolar protein sorting (Vps)34, was first identified in budding yeast, Saccharomyces cerevisiae, and screens for proteins involved in the regulation of vesicle-mediated Vps (
      • Backer J.M.
      The regulation and function of class III PI3Ks: novel roles for Vps34.
      ). Several PI(3)P binding proteins have been identified, and all are involved in protein trafficking (
      • Marat A.L.
      • Haucke V.
      Phosphatidylinositol 3-phosphates–at the interface between cell signalling and membrane traffic.
      ). The crystal structure of Vps34 has been solved (
      • Miller S.
      • Tavshanjian B.
      • Oleksy A.
      • Perisic O.
      • Houseman B.T.
      • Shokat K.M.
      • Williams R.L.
      Shaping development of autophagy inhibitors with the structure of the lipid kinase Vps34.
      ). Class III enzymes, which phosphorylate only PI, are heterodimers of a catalytic subunit associated with the serine/threonine-protein kinase adaptor subunit that is required for membrane recruitment (
      • Martin T.F.
      Phosphoinositide lipids as signaling molecules: common themes for signal transduction, cytoskeletal regulation, and membrane trafficking.
      ,
      • Fruman D.A.
      • Meyers R.E.
      • Cantley L.C.
      Phosphoinositide kinases.
      ). The class III PI3K, Vps34p, is responsible for producing the majority of the cellular PI(3)P and is involved in protein trafficking through the lysosome (
      • Marat A.L.
      • Haucke V.
      Phosphatidylinositol 3-phosphates–at the interface between cell signalling and membrane traffic.
      ). Class III PI3K Vps34 is closer to class I PI3K in terms of heterodimers of catalytic (Vps34) and regulatory (Vps15, a 150 kDa protein) subunits (
      • Stjepanovic G.
      • Baskaran S.
      • Lin M.G.
      • Hurley J.H.
      Vps34 kinase domain dynamics regulate the autophagic PI 3-kinase complex.
      ). Class III enzyme-generated PI(3)P is mainly involved in the trafficking of vesicles and proteins (
      • Mayinger P.
      Phosphoinositides and vesicular membrane traffic.
      ). Some studies show that PI(3)P regulates immune cell function and phagocytosis (
      • Gillooly D.J.
      • Simonsen A.
      • Stenmark H.
      Phosphoinositides and phagocytosis.
      ,
      • Kale S.D.
      • Gu B.
      • Capelluto D.G.
      • Dou D.
      • Feldman E.
      • Rumore A.
      • Arredondo F.D.
      • Hanlon R.
      • Fudal I.
      • Rouxel T.
      • et al.
      External lipid PI3P mediates entry of eukaryotic pathogen effectors into plant and animal host cells.
      ). Class III PI3K plays an important role in rod, bipolar, and RPE cell functions in the retina (
      • Wensel T.G.
      Phosphoinositides in retinal function and disease.
      ,
      • He F.
      • Agosto M.A.
      • Anastassov I.A.
      • Tse D.Y.
      • Wu S.M.
      • Wensel T.G.
      Phosphatidylinositol-3-phosphate is light-regulated and essential for survival in retinal rods.
      ,
      • He F.
      • Nichols R.M.
      • Kailasam L.
      • Wensel T.G.
      • Agosto M.A.
      Critical role for phosphatidylinositol-3 kinase Vps34/PIK3C3 in ON-bipolar cells.
      ,
      • He F.
      • Agosto M.A.
      • Nichols R.M.
      • Wensel T.G.
      Multiple phosphatidylinositol(3)phosphate roles in retinal pigment epithelium membrane recycling.
      ).
      Class IV is a collection of enzymes, which include ataxia-telangiectasia mutated, ataxia telangiectasia and Rad3-related, DNA-dependent protein kinase, and mammalian target of rapamycin (mTOR). These enzymes are occasionally referred to as the class IV PI3Ks. Unlike class I, II, and III PI3Ks, which are lipid kinases, class IV PI3Ks are protein serine/threonine kinases (
      • Takai H.
      • Wang R.C.
      • Takai K.K.
      • Yang H.
      • de Lange T.
      Tel2 regulates the stability of PI3K-related protein kinases.
      ). However, in vitro, class I PI3K has been shown to have protein kinase activity (
      • Thomas D.
      • Powell J.A.
      • Green B.D.
      • Barry E.F.
      • Ma Y.
      • Woodcock J.
      • Fitter S.
      • Zannettino A.C.
      • Pitson S.M.
      • Hughes T.P.
      • et al.
      Protein kinase activity of phosphoinositide 3-kinase regulates cytokine-dependent cell survival.
      ).

      Insulin receptor-regulated class i PI3K in photoreceptor cells

      In 1997, class I phosphoinositide 3-kinase (PI3K) was reported to be responsible for the generation of D3-PIPs in retinal ROSs (
      • Guo X.
      • Ghalayini A.J.
      • Chen H.
      • Anderson R.E.
      Phosphatidylinositol 3-kinase in bovine photoreceptor rod outer segments.
      ). Furthermore, tyrosine phosphorylation in vitro was shown to stimulate the PI3K activity in isolated bovine retinal outer segment membranes (
      • Guo X.X.
      • Huang Z.
      • Bell M.W.
      • Chen H.
      • Anderson R.E.
      Tyrosine phosphorylation is involved in phosphatidylinositol 3-kinase activation in bovine rod outer segments.
      ). The class I PI3K is composed of two subunits, a p110 catalytic subunit and a p85 regulatory subunit (
      • Fruman D.A.
      • Meyers R.E.
      • Cantley L.C.
      Phosphoinositide kinases.
      ). With the application of p85 regulatory subunits that contain both N-terminal and C-terminal SH2 domains, Rajala and Anderson (
      • Rajala R.V.
      • Anderson R.E.
      Interaction of the insulin receptor beta-subunit with phosphatidylinositol 3-kinase in bovine ROS.
      ) identified that the insulin receptor (IR) in the retina/ROS is the receptor that regulates P13K activity. The authors also observed that in vitro tyrosine phosphorylation enhanced the phosphorylation of the IR, which results in the recruitment of the p85-N-SH2 domain, binds to the tyrosine-phosphorylated IR, and activates PI3K (
      • Rajala R.V.
      • Anderson R.E.
      Interaction of the insulin receptor beta-subunit with phosphatidylinositol 3-kinase in bovine ROS.
      ).
      In rod photoreceptors, PI3K activation is mediated through light-dependent tyrosine phosphorylation of the IR (
      • Rajala A.
      • Anderson R.E.
      • Ma J.X.
      • Lem J.
      • Al Ubaidi M.R.
      • Rajala R.V.
      G-protein-coupled receptor rhodopsin regulates the phosphorylation of retinal insulin receptor.
      ,
      • Rajala R.V.
      • McClellan M.E.
      • Ash J.D.
      • Anderson R.E.
      In vivo regulation of phosphoinositide 3-kinase in retina through light-induced tyrosine phosphorylation of the insulin receptor beta-subunit.
      ) (Fig. 4). The IR activation has been shown to be independent of G protein transducin activation and dependent on the photobleaching of rhodopsin (
      • Rajala A.
      • Anderson R.E.
      • Ma J.X.
      • Lem J.
      • Al Ubaidi M.R.
      • Rajala R.V.
      G-protein-coupled receptor rhodopsin regulates the phosphorylation of retinal insulin receptor.
      ). This IR/PI3K activation is a noncanonical rhodopsin activation (
      • Rajala A.
      • Rajala R.V.S.
      A non-canonical rhodopsin-mediated insulin receptor signaling pathway in retinal photoreceptor neurons.
      ) (Fig. 4). In the retina, the IR is constitutively phosphorylated (
      • Reiter C.E.
      • Sandirasegarane L.
      • Wolpert E.B.
      • Klinger M.
      • Simpson I.A.
      • Barber A.J.
      • Antonetti D.A.
      • Kester M.
      • Gardner T.W.
      Characterization of insulin signaling in rat retina in vivo and ex vivo.
      ,
      • Rajala R.V.
      • Wiskur B.
      • Tanito M.
      • Callegan M.
      • Rajala A.
      Diabetes reduces autophosphorylation of retinal insulin receptor and increases protein-tyrosine phosphatase-1B activity.
      ). However, the phosphorylation state of the IR is regulated through dark and light adaptation (
      • Rajala R.V.
      • Tanito M.
      • Neel B.G.
      • Rajala A.
      Enhanced retinal insulin receptor-activated neuroprotective survival signal in mice lacking the protein-tyrosine phosphatase-1B gene.
      ). In dark-adapted conditions, the IR is in the inactive state because of increased protein tyrosine phosphatase 1B (PTP1B) activity, which dephosphorylates the IR and results in the reduced association of PI3K with the IR (
      • Rajala R.V.
      • Tanito M.
      • Neel B.G.
      • Rajala A.
      Enhanced retinal insulin receptor-activated neuroprotective survival signal in mice lacking the protein-tyrosine phosphatase-1B gene.
      ). Upon rhodopsin activation, adaptor protein growth factor receptor-bound protein 14 (Grb14) localized to rod inner segments in the dark translocates to the outer segments in light and undergoes tyrosine phosphorylation by a nonreceptor tyrosine kinase, Src, in a rhodopsin-dependent manner (
      • Rajala A.
      • Daly R.J.
      • Tanito M.
      • Allen D.T.
      • Holt L.J.
      • Lobanova E.S.
      • Arshavsky V.Y.
      • Rajala R.V.
      Growth factor receptor-bound protein 14 undergoes light-dependent intracellular translocation in rod photoreceptors: functional role in retinal insulin receptor activation.
      ,
      • Basavarajappa D.K.
      • Gupta V.K.
      • Dighe R.
      • Rajala A.
      • Rajala R.V.
      Phosphorylated Grb14 is an endogenous inhibitor of retinal protein tyrosine phosphatase 1B, and light-dependent activation of Src phosphorylates Grb14.
      ) (Fig. 4). The Src phosphorylation was abolished in animals deficient in the photobleaching of rhodopsin (
      • Basavarajappa D.K.
      • Gupta V.K.
      • Dighe R.
      • Rajala A.
      • Rajala R.V.
      Phosphorylated Grb14 is an endogenous inhibitor of retinal protein tyrosine phosphatase 1B, and light-dependent activation of Src phosphorylates Grb14.
      ). The tyrosine-phosphorylated-Grb14 binds to PTP1B and inactivates its activity (Fig. 4). Thus, the IR becomes active, which then activates PI3K and promotes photoreceptor survival (
      • Rajala R.V.
      • Tanito M.
      • Neel B.G.
      • Rajala A.
      Enhanced retinal insulin receptor-activated neuroprotective survival signal in mice lacking the protein-tyrosine phosphatase-1B gene.
      ,
      • Basavarajappa D.K.
      • Gupta V.K.
      • Dighe R.
      • Rajala A.
      • Rajala R.V.
      Phosphorylated Grb14 is an endogenous inhibitor of retinal protein tyrosine phosphatase 1B, and light-dependent activation of Src phosphorylates Grb14.
      ,
      • Rajala R.V.
      • Anderson R.E.
      Rhodopsin-regulated insulin receptor signaling pathway in rod photoreceptor neurons.
      ) (Fig. 4). Conditional deletion of the IR in rod photoreceptors resulted in stress-induced photoreceptor degeneration (
      • Rajala A.
      • Tanito M.
      • Le Y.Z.
      • Kahn C.R.
      • Rajala R.V.
      Loss of neuroprotective survival signal in mice lacking insulin receptor gene in rod photoreceptor cells.
      ). The serine/threonine kinase Akt2, the downstream effector of IR, and PI3K deletion result in stress-induced photoreceptor degeneration (
      • Li G.
      • Anderson R.E.
      • Tomita H.
      • Adler R.
      • Liu X.
      • Zack D.J.
      • Rajala R.V.
      Nonredundant role of Akt2 for neuroprotection of rod photoreceptor cells from light-induced cell death.
      ). The proteins that regulate IR singing pathway in rods are also expressed in cones (
      • Rajala A.
      • Wang Y.
      • Rajala R.V.
      Activation of oncogenic tyrosine kinase signaling promotes insulin receptor-mediated cone photoreceptor survival.
      ,
      • Rajala A.
      • Dighe R.
      • Agbaga M.P.
      • Anderson R.E.
      • Rajala R.V.
      Insulin receptor signaling in cones.
      ). Interestingly, deletion of the IR in cones resulted in cone degeneration without added stress (
      • Rajala A.
      • Wang Y.
      • Rajala R.V.
      Activation of oncogenic tyrosine kinase signaling promotes insulin receptor-mediated cone photoreceptor survival.
      ).
      Figure thumbnail gr4
      Fig. 4Noncanonical IR signaling-mediated activation of PI3K in the retina/photoreceptor cells. The IR in the retina is constitutively activated. However, the activated (phosphorylation) state of the IR is light-dependent. Under a dark-adapted state, the IR undergoes dephosphorylation by PTP1B and keeps the IR in an inactive state. Upon illumination, rhodopsin activation facilitates the translocation of Grb14 from inner segments to outer segments, where it undergoes a light- and rhodopsin-dependent phosphorylation by a nonreceptor tyrosine kinase, Src. Phosphorylated Grb14 binds to PTP1B and inhibits its activity, thus preserving the phosphorylation of the IR, which can bind to PI3K and generate PIPs. RIS, rod inner segments; Pi, inorganic phosphate; P, phosphorylation.

      Interaction between phosphoinositides and phospholipid-binding proteins

      Several pleckstrin homology (PH) domain-containing proteins are expressed in the retina. PIPs regulate these proteins directly or indirectly. These proteins include PH domain retinal protein 1 (PHR1) (
      • Xu S.
      • Ladak R.
      • Swanson D.A.
      • Soltyk A.
      • Sun H.
      • Ploder L.
      • Vidgen D.
      • Duncan A.M.
      • Garami E.
      • Valle D.
      • et al.
      PHR1 encodes an abundant, pleckstrin homology domain-containing integral membrane protein in the photoreceptor outer segments.
      ,
      • Xu S.
      • Wang Y.
      • Zhao H.
      • Zhang L.
      • Xiong W.
      • Yau K.W.
      • Hiel H.
      • Glowatzki E.
      • Ryugo D.K.
      • Valle D.
      PHR1, a PH domain-containing protein expressed in primary sensory neurons.
      ), Evectin-1 (
      • Krappa R.
      • Nguyen A.
      • Burrola P.
      • Deretic D.
      • Lemke G.
      Evectins: vesicular proteins that carry a pleckstrin homology domain and localize to post-Golgi membranes.
      ), Grb14 (
      • Rajala R.V.
      • Chan M.D.
      • Rajala A.
      Lipid-protein interactions of growth factor receptor-bound protein 14 in insulin receptor signaling.
      ), IR substrate (IRS)-1 and IRS-2 (
      • Reiter C.E.
      • Sandirasegarane L.
      • Wolpert E.B.
      • Klinger M.
      • Simpson I.A.
      • Barber A.J.
      • Antonetti D.A.
      • Kester M.
      • Gardner T.W.
      Characterization of insulin signaling in rat retina in vivo and ex vivo.
      ,
      • Rajala R.V.
      • McClellan M.E.
      • Chan M.D.
      • Tsiokas L.
      • Anderson R.E.
      Interaction of the retinal insulin receptor beta-subunit with the P85 subunit of phosphoinositide 3-kinase.
      ,
      • Yi X.
      • Schubert M.
      • Peachey N.S.
      • Suzuma K.
      • Burks D.J.
      • Kushner J.A.
      • Suzuma I.
      • Cahill C.
      • Flint C.L.
      • Dow M.A.
      • et al.
      Insulin receptor substrate 2 is essential for maturation and survival of photoreceptor cells.
      ), and three isoforms of Akt (Akt1, Akt2, and Akt3) (
      • Li G.
      • Rajala A.
      • Wiechmann A.F.
      • Anderson R.E.
      • Rajala R.V.
      Activation and membrane binding of retinal protein kinase Balpha/Akt1 is regulated through light-dependent generation of phosphoinositides.
      ,
      • Reiter C.E.
      • Sandirasegarane L.
      • Wolpert E.B.
      • Klinger M.
      • Simpson I.A.
      • Barber A.J.
      • Antonetti D.A.
      • Kester M.
      • Gardner T.W.
      Characterization of insulin signaling in rat retina in vivo and ex vivo.
      ,
      • Li G.
      • Anderson R.E.
      • Tomita H.
      • Adler R.
      • Liu X.
      • Zack D.J.
      • Rajala R.V.
      Nonredundant role of Akt2 for neuroprotection of rod photoreceptor cells from light-induced cell death.
      ). Evectin-1 is involved in the trafficking of post-Golgi membranes in photoreceptor cells (
      • Krappa R.
      • Nguyen A.
      • Burrola P.
      • Deretic D.
      • Lemke G.
      Evectins: vesicular proteins that carry a pleckstrin homology domain and localize to post-Golgi membranes.
      ). The PH domain retinal protein 1 PH domain is identical to serine/threonine kinase Akt and does not bind to inositol phosphates but interacts with transducin Gβγ subunits (
      • Xu S.
      • Ladak R.
      • Swanson D.A.
      • Soltyk A.
      • Sun H.
      • Ploder L.
      • Vidgen D.
      • Duncan A.M.
      • Garami E.
      • Valle D.
      • et al.
      PHR1 encodes an abundant, pleckstrin homology domain-containing integral membrane protein in the photoreceptor outer segments.
      ,
      • Xu S.
      • Wang Y.
      • Zhao H.
      • Zhang L.
      • Xiong W.
      • Yau K.W.
      • Hiel H.
      • Glowatzki E.
      • Ryugo D.K.
      • Valle D.
      PHR1, a PH domain-containing protein expressed in primary sensory neurons.
      ). In the retina, the Akt-PH domain binds to PIPs (
      • Li G.
      • Rajala A.
      • Wiechmann A.F.
      • Anderson R.E.
      • Rajala R.V.
      Activation and membrane binding of retinal protein kinase Balpha/Akt1 is regulated through light-dependent generation of phosphoinositides.
      ). The PH domain of Akt binds to the cytoskeletal protein myosin II (
      • Tanaka M.
      • Konishi H.
      • Touhara K.
      • Sakane F.
      • Hirata M.
      • Ono Y.
      • Kikkawa U.
      Identification of myosin II as a binding protein to the PH domain of protein kinase B.
      ). This protein is mutated in patients with Usher syndrome (
      • Weil D.
      • Blanchard S.
      • Kaplan J.
      • Guilford P.
      • Gibson F.
      • Walsh J.
      • Mburu P.
      • Varela A.
      • Levilliers J.
      • Weston M.D.
      Defective myosin VIIA gene responsible for Usher syndrome type 1B.
      ). However, the interaction between myosin and Akt has not been tested in the retina. PH domain and leucine-rich repeat protein phosphatase (PHLPP) and PH domain and leucine-rich repeat protein phosphatase-like (PHLPPL) are PH domain-containing proteins known to dephosphorylate Akt isoforms (
      • Brognard J.
      • Sierecki E.
      • Gao T.
      • Newton A.C.
      PHLPP and a second isoform, PHLPP2, differentially attenuate the amplitude of Akt signaling by regulating distinct Akt isoforms.
      ,
      • Gao T.
      • Furnari F.
      • Newton A.C.
      PHLPP: a phosphatase that directly dephosphorylates Akt, promotes apoptosis, and suppresses tumor growth.
      ) and are also expressed in the retina (
      • Rajala A.
      • Gupta V.K.
      • Anderson R.E.
      • Rajala R.V.
      Light activation of the insulin receptor regulates mitochondrial hexokinase. A possible mechanism of retinal neuroprotection.
      ,
      • Rajala R.V.
      • Kanan Y.
      • Anderson R.E.
      Photoreceptor neuroprotection: regulation of Akt activation through serine/threonine phosphatases, PHLPP and PHLPPL.
      ,
      • Kanan Y.
      • Matsumoto H.
      • Song H.
      • Sokolov M.
      • Anderson R.E.
      • Rajala R.V.
      Serine/threonine kinase Akt activation regulates the activity of retinal serine/threonine phosphatases, PHLPP and PHLPPL.
      ).

      Downstream effects of PI3K signaling in the retina

      In vivo, light stimulates the tyrosine phosphorylation of the IR, which results in the recruitment of PI3K and subsequent activation of Akt, the downstream effector of PI3K regulated through PI3K-generated PIPs (
      • Rajala R.V.
      • McClellan M.E.
      • Ash J.D.
      • Anderson R.E.
      In vivo regulation of phosphoinositide 3-kinase in retina through light-induced tyrosine phosphorylation of the insulin receptor beta-subunit.
      ). The addition of insulin as the ligand for the IR also stimulates the tyrosine phosphorylation of the IR, which results in the activation of PI3K ex vivo (
      • Li G.
      • Rajala A.
      • Wiechmann A.F.
      • Anderson R.E.
      • Rajala R.V.
      Activation and membrane binding of retinal protein kinase Balpha/Akt1 is regulated through light-dependent generation of phosphoinositides.
      ,
      • Rajala R.V.
      • Anderson R.E.
      Interaction of the insulin receptor beta-subunit with phosphatidylinositol 3-kinase in bovine ROS.
      ). In the retina, light stress induces the phosphorylation of the IR, which results in the activation of the PI3K/Akt pathway through increased tyrosine phosphorylation of the IR (
      • Rajala A.
      • Tanito M.
      • Le Y.Z.
      • Kahn C.R.
      • Rajala R.V.
      Loss of neuroprotective survival signal in mice lacking insulin receptor gene in rod photoreceptor cells.
      ). Addition of insulin growth factor-1, the ligand for IGF-1R, ex vivo to retinas stimulates the tyrosine phosphorylation of the IGF-1R, which results in the activation of the PI3K/Akt pathway (
      • Dilly A.K.
      • Rajala R.V.
      Insulin growth factor 1 receptor/PI3K/AKT survival pathway in outer segment membranes of rod photoreceptors.
      ). Interestingly, light stress, but not physiological light, stimulates the tyrosine phosphorylation of the IGF-1R that results in the activation of the PI3k/Akt downstream signaling pathway (
      • Dilly A.K.
      • Rajala R.V.
      Insulin growth factor 1 receptor/PI3K/AKT survival pathway in outer segment membranes of rod photoreceptors.
      ). In the retina, PI3K activation stimulates serine/threonine kinase Akt in vitro and in vivo (
      • Li G.
      • Rajala A.
      • Wiechmann A.F.
      • Anderson R.E.
      • Rajala R.V.
      Activation and membrane binding of retinal protein kinase Balpha/Akt1 is regulated through light-dependent generation of phosphoinositides.
      ,
      • Reiter C.E.
      • Sandirasegarane L.
      • Wolpert E.B.
      • Klinger M.
      • Simpson I.A.
      • Barber A.J.
      • Antonetti D.A.
      • Kester M.
      • Gardner T.W.
      Characterization of insulin signaling in rat retina in vivo and ex vivo.
      ,
      • Li G.
      • Anderson R.E.
      • Tomita H.
      • Adler R.
      • Liu X.
      • Zack D.J.
      • Rajala R.V.
      Nonredundant role of Akt2 for neuroprotection of rod photoreceptor cells from light-induced cell death.
      ).
      Akt kinase exists as three isoforms: Ak1, Akt2, and Akt3. All three isoforms are expressed in retina and rod photoreceptor cells (
      • Li G.
      • Rajala A.
      • Wiechmann A.F.
      • Anderson R.E.
      • Rajala R.V.
      Activation and membrane binding of retinal protein kinase Balpha/Akt1 is regulated through light-dependent generation of phosphoinositides.
      ,
      • Reiter C.E.
      • Sandirasegarane L.
      • Wolpert E.B.
      • Klinger M.
      • Simpson I.A.
      • Barber A.J.
      • Antonetti D.A.
      • Kester M.
      • Gardner T.W.
      Characterization of insulin signaling in rat retina in vivo and ex vivo.
      ). Membrane binding of Akt1 is mediated through its PH domain binding to PI3K-generated PIPs (
      • Li G.
      • Rajala A.
      • Wiechmann A.F.
      • Anderson R.E.
      • Rajala R.V.
      Activation and membrane binding of retinal protein kinase Balpha/Akt1 is regulated through light-dependent generation of phosphoinositides.
      ). In transgenic Xenopus laevis, the PH domain fused to GFP under the control of the Xenopus opsin promoter binds to PIPs in a light-dependent manner (
      • Li G.
      • Rajala A.
      • Wiechmann A.F.
      • Anderson R.E.
      • Rajala R.V.
      Activation and membrane binding of retinal protein kinase Balpha/Akt1 is regulated through light-dependent generation of phosphoinositides.
      ). In dark-adapted conditions, the actin exists as a stress fiber phenotype. Upon light-illumination, reorganization of the actin cytoskeleton was found to colocalize with PIPs in a X. laevis model (
      • Li G.
      • Rajala A.
      • Wiechmann A.F.
      • Anderson R.E.
      • Rajala R.V.
      Activation and membrane binding of retinal protein kinase Balpha/Akt1 is regulated through light-dependent generation of phosphoinositides.
      ). The expression of a mutant version of the PH domain in which the arginine 25 residue is substituted with cysteine failed to bind to PIPs (
      • Li G.
      • Rajala A.
      • Wiechmann A.F.
      • Anderson R.E.
      • Rajala R.V.
      Activation and membrane binding of retinal protein kinase Balpha/Akt1 is regulated through light-dependent generation of phosphoinositides.
      ).
      The IR/PI3K/Akt signaling proteins are also expressed in the rod inner segments. These proteins may play an important role in cellular signaling events (
      • Rajala A.
      • Rajala R.V.S.
      A non-canonical rhodopsin-mediated insulin receptor signaling pathway in retinal photoreceptor neurons.
      ). One of the functions of PI3K-activated Akt in the inner segment is to regulate hexokinase 2 as it interacts with mitochondria in the photoreceptors (
      • Rajala A.
      • Gupta V.K.
      • Anderson R.E.
      • Rajala R.V.
      Light activation of the insulin receptor regulates mitochondrial hexokinase. A possible mechanism of retinal neuroprotection.
      ). Growth factor-mediated activation of Akt was demonstrated to increase the association of hexokinase 2 with mitochondria in normal tissues and cells (
      • Majewski N.
      • Nogueira V.
      • Bhaskar P.
      • Coy P.E.
      • Skeen J.E.
      • Gottlob K.
      • Chandel N.S.
      • Thompson C.B.
      • Robey R.B.
      • Hay N.
      Hexokinase-mitochondria interaction mediated by Akt is required to inhibit apoptosis in the presence or absence of Bax and Bak.
      ). Akt activation inhibits the dissociation of hexokinase 2 from mitochondria and is the primary event in the induction of apoptosis (
      • Rajala A.
      • Gupta V.K.
      • Anderson R.E.
      • Rajala R.V.
      Light activation of the insulin receptor regulates mitochondrial hexokinase. A possible mechanism of retinal neuroprotection.
      ,
      • Majewski N.
      • Nogueira V.
      • Bhaskar P.
      • Coy P.E.
      • Skeen J.E.
      • Gottlob K.
      • Chandel N.S.
      • Thompson C.B.
      • Robey R.B.
      • Hay N.
      Hexokinase-mitochondria interaction mediated by Akt is required to inhibit apoptosis in the presence or absence of Bax and Bak.
      ,
      • Majewski N.
      • Nogueira V.
      • Robey R.B.
      • Hay N.
      Akt inhibits apoptosis downstream of BID cleavage via a glucose-dependent mechanism involving mitochondrial hexokinases.
      ,
      • Gottlob K.
      • Majewski N.
      • Kennedy S.
      • Kandel E.
      • Robey R.B.
      • Hay N.
      Inhibition of early apoptotic events by Akt/PKB is dependent on the first committed step of glycolysis and mitochondrial hexokinase.
      ). The mechanism of interaction between hexokinase 2 and mitochondria is that Akt phosphorylates glycogen synthase kinase 3β (GSK3β), which renders GSK3β inactive (
      • Feng J.
      • Lucchinetti E.
      • Ahuja P.
      • Pasch T.
      • Perriard J.C.
      • Zaugg M.
      Isoflurane postconditioning prevents opening of the mitochondrial permeability transition pore through inhibition of glycogen synthase kinase 3beta.
      ,
      • Pastorino J.G.
      • Hoek J.B.
      • Shulga N.
      Activation of glycogen synthase kinase 3beta disrupts the binding of hexokinase II to mitochondria by phosphorylating voltage-dependent anion channel and potentiates chemotherapy-induced cytotoxicity.
      ). In the absence of Akt activation, GSK3β is active and phosphorylates the voltage-dependent anion channel (VDAC) on serine 51 that disrupts the binding of hexokinase 2 to the VDAC (
      • Cross D.A.
      • Alessi D.R.
      • Cohen P.
      • Andjelkovich M.
      • Hemmings B.A.
      Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B.
      ). The PI3K-generated PIP-activated Akt prevents the release of cytochrome c from mitochondria and inhibits apoptosis (
      • Majewski N.
      • Nogueira V.
      • Bhaskar P.
      • Coy P.E.
      • Skeen J.E.
      • Gottlob K.
      • Chandel N.S.
      • Thompson C.B.
      • Robey R.B.
      • Hay N.
      Hexokinase-mitochondria interaction mediated by Akt is required to inhibit apoptosis in the presence or absence of Bax and Bak.
      ,
      • Robey R.B.
      • Hay N.
      Mitochondrial hexokinases, novel mediators of the antiapoptotic effects of growth factors and Akt.
      ). Retinal photoreceptors are postmitotic neurons and apoptosis is detrimental; therefore, the PIP-activated Akt promotes photoreceptor survival (
      • Li G.
      • Anderson R.E.
      • Tomita H.
      • Adler R.
      • Liu X.
      • Zack D.J.
      • Rajala R.V.
      Nonredundant role of Akt2 for neuroprotection of rod photoreceptor cells from light-induced cell death.
      ,
      • Rajala A.
      • Gupta V.K.
      • Anderson R.E.
      • Rajala R.V.
      Light activation of the insulin receptor regulates mitochondrial hexokinase. A possible mechanism of retinal neuroprotection.
      ).

      Interaction between the rod cng channel and PI3K in rod photoreceptor cells

      Class I PI3Kγ lacks a p85-binding motif. Hence, it interacts with p101 (
      • Stephens L.R.
      • Eguinoa A.
      • Erdjument-Bromage H.
      • Lui M.
      • Cooke F.
      • Coadwell J.
      • Smrcka A.S.
      • Thelen M.
      • Cadwallader K.
      • Tempst P.
      • et al.
      The G beta gamma sensitivity of a PI3K is dependent upon a tightly associated adaptor, p101.
      ) and p84/87 adaptor proteins for regulation (
      • Voigt P.
      • Brock C.
      • Nurnberg B.
      • Schaefer. M.
      Assigning functional domains within the p101 regulatory subunit of phosphoinositide 3-kinase gamma.
      ,
      • Suire S.
      • Coadwell J.
      • Ferguson G.J.
      • Davidson K.
      • Hawkins P.
      • Stephens L.
      p84, a new Gbetagamma-activated regulatory subunit of the type IB phosphoinositide 3-kinase p110gamma.
      ). Class I PI3Kγ is known to be regulated through Ras proteins (
      • Rubio I.
      • Rodriguez-Viciana P.
      • Downward J.
      • Wetzker R.
      Interaction of Ras with phosphoinositide 3-kinase gamma.
      ) (Fig. 3). Ras belongs to a family of related proteins called small GTPases (
      • Wennerberg K.
      • Rossman K.L.
      • Der C.J.
      The Ras superfamily at a glance.
      ). These proteins have a Ras-associating (RA) domain, and PI3K interacts with this RA domain (
      • Patel M.
      • Cote J.F.
      Ras GTPases’ interaction with effector domains: Breaking the families’ barrier.
      ). In photoreceptor cells, the C-terminal region of the rod CNG channel subunit α1 (CNGA1) has 50–70% tertiary structural homology with Ras proteins (
      • Rajala R.V.
      • Rajala A.
      • Gupta V.K.
      Conservation and divergence of Grb7 family of Ras-binding domains.
      ). This domain has been named the Ras-like domain (
      • Rajala R.V.
      • Rajala A.
      • Gupta V.K.
      Conservation and divergence of Grb7 family of Ras-binding domains.
      ). In rod photoreceptor cells, PI3Kγ activation occurs through the interaction of its RA -domain with the Ras-like domain of CNGA1 (
      • Gupta V.K.
      • Rajala A.
      • Rajala R.V.
      Non-canonical regulation of phosphatidylinositol 3-kinase gamma isoform activity in retinal rod photoreceptor cells.
      ). The interaction of PI3Kγ with CNGA1 does not affect the channel physiology. However, PI3Kγ uses CNGA1 as an anchor to achieve a close vicinity to its substrates to generate PIPs (
      • Gupta V.K.
      • Rajala A.
      • Rajala R.V.
      Non-canonical regulation of phosphatidylinositol 3-kinase gamma isoform activity in retinal rod photoreceptor cells.
      ).

      PI3K KO phenotypes in the retina and rpe

      In the retina, loss of p85α does not affect the overall morphology, but decreased PI3K activity associated with the IR is observed (
      • Rajala R.V.
      • McClellan M.E.
      • Ash J.D.
      • Anderson R.E.
      Regulation of retinal phosphoinositide 3-kinase activity in p85alpha-subunit knockout mice.
      ). Conditional deletion of the p85α (pik3r1) regulatory subunit in rod photoreceptor cells does not affect the structure of the retina (
      • Ivanovic I.
      • Allen D.T.
      • Dighe R.
      • Le Y.Z.
      • Anderson R.E.
      • Rajala R.V.
      Phosphoinositide 3-kinase signaling in retinal rod photoreceptors.
      ). Mice with conditional deletion of p85α exhibited a slight delay in recovery kinetics and a delay in the translocation of rod arrestin from the inner segments to the outer segments (
      • Ivanovic I.
      • Allen D.T.
      • Dighe R.
      • Le Y.Z.
      • Anderson R.E.
      • Rajala R.V.
      Phosphoinositide 3-kinase signaling in retinal rod photoreceptors.
      ). The absence of the disease phenotype in mouse rods lacking p85α could be explained by the expression of p85β in p85α KO mice (
      • Ivanovic I.
      • Allen D.T.
      • Dighe R.
      • Le Y.Z.
      • Anderson R.E.
      • Rajala R.V.
      Phosphoinositide 3-kinase signaling in retinal rod photoreceptors.
      ). Interestingly, the deletion of the p85α-subunit of PI3K in cones results in age-related cone degeneration (
      • Ivanovic I.
      • Anderson R.E.
      • Le Y.Z.
      • Fliesler S.J.
      • Sherry D.M.
      • Rajala R.V.
      Deletion of the p85alpha regulatory subunit of phosphoinositide 3-kinase in cone photoreceptor cells results in cone photoreceptor degeneration.
      ). The surviving cone-terminals of the cone-p85α KO mice exhibit progressive disorganization of synaptic structures (
      • Ivanovic I.
      • Anderson R.E.
      • Le Y.Z.
      • Fliesler S.J.
      • Sherry D.M.
      • Rajala R.V.
      Deletion of the p85alpha regulatory subunit of phosphoinositide 3-kinase in cone photoreceptor cells results in cone photoreceptor degeneration.
      ). The loss of p85α in cones does not affect rod structure and function (
      • Ivanovic I.
      • Anderson R.E.
      • Le Y.Z.
      • Fliesler S.J.
      • Sherry D.M.
      • Rajala R.V.
      Deletion of the p85alpha regulatory subunit of phosphoinositide 3-kinase in cone photoreceptor cells results in cone photoreceptor degeneration.
      ). Conditional deletion of the catalytic subunit of PI3K, p110α, in cones also resulted in cone degeneration (
      • Rajala R.V.
      • Ranjo-Bishop M.
      • Wang Y.
      • Rajala A.
      • Anderson R.E.
      The p110alpha isoform of phosphoinositide 3-kinase is essential for cone photoreceptor survival.
      ). These studies highlight that PI3K signaling is indispensable for cone photoreceptor survival. These findings also suggest that other PIP-signaling pathways may regulate rod survival.
      Conditional deletion of the class III PI3K Vps34 gene in rods results in a failure in the fusion of endosomal and autophagy-related membranes with lysosomes that prompts the buildup of anomalous membrane structures (
      • He F.
      • Agosto M.A.
      • Anastassov I.A.
      • Tse D.Y.
      • Wu S.M.
      • Wensel T.G.
      Phosphatidylinositol-3-phosphate is light-regulated and essential for survival in retinal rods.
      ). These mice have normal structure and function and trafficking of rhodopsin to the outer segments; however, the mice experience progressive rod degeneration by 12 weeks of age (
      • He F.
      • Agosto M.A.
      • Anastassov I.A.
      • Tse D.Y.
      • Wu S.M.
      • Wensel T.G.
      Phosphatidylinositol-3-phosphate is light-regulated and essential for survival in retinal rods.
      ). The rod degeneration accompanying Vps34 gene deletion is much faster than that of rods lacking autophagy genes, highlighting that PI(3)P is required for endosome recycling and other pathways that are necessary for rod photoreceptor survival (
      • He F.
      • Agosto M.A.
      • Anastassov I.A.
      • Tse D.Y.
      • Wu S.M.
      • Wensel T.G.
      Phosphatidylinositol-3-phosphate is light-regulated and essential for survival in retinal rods.
      ). Vps34 has recently been shown to be essential for on-bipolar cell survival. Loss of this enzyme in these cells results in a significant loss of structure and function (
      • He F.
      • Nichols R.M.
      • Kailasam L.
      • Wensel T.G.
      • Agosto M.A.
      Critical role for phosphatidylinositol-3 kinase Vps34/PIK3C3 in ON-bipolar cells.
      ). This study further highlights that PI(3)P is necessary for the fusion of autophagosomes with lysosomes and maturation of late endosomes, and PI(3)P is needed for the maintenance of on-bipolar cells health (
      • He F.
      • Nichols R.M.
      • Kailasam L.
      • Wensel T.G.
      • Agosto M.A.
      Critical role for phosphatidylinositol-3 kinase Vps34/PIK3C3 in ON-bipolar cells.
      ).
      In RPE, PI(3)P is essential for the fusion with autophagosomes, lysosomes, and phagosomes (
      • He F.
      • Agosto M.A.
      • Nichols R.M.
      • Wensel T.G.
      Multiple phosphatidylinositol(3)phosphate roles in retinal pigment epithelium membrane recycling.
      ). These findings highlight that PI(3)P is essential for RPE cell health. In cone photoreceptors, the ablation of Vps34 resulted in an age-related cone degeneration (R. V. S. Rajala, unpublished observations).

      Phosphatidylinositol phosphate kinases

      Phosphatidylinositol 4-kinases (PI4Ks) make PI(4)P from phosphatidylinositol (
      • Rusten T.E.
      • Stenmark H.
      Analyzing phosphoinositides and their interacting proteins.
      ). PI(4)P is an important molecule for the generation of other phosphoinositides involved in signaling, such as PI(4,5)P2, and is the substrate for the generation of second messengers, such as IP3 and DAG, through the action of PLC (
      • Streb H.
      • Irvine R.F.
      • Berridge M.J.
      • Schulz I.
      Release of Ca2+ from a nonmitochondrial intracellular store in pancreatic acinar cells by inositol-1,4,5-trisphosphate.
      ). PI(4,5)P2 also serves as a substrate for PI3K for the generation of PI(3,4,5)P3 (
      • Fruman D.A.
      • Meyers R.E.
      • Cantley L.C.
      Phosphoinositide kinases.
      ,
      • Rajala R.V.
      • Anderson R.E.
      Focus on molecules: phosphatidylinositol-4,5-bisphosphate (PIP2).
      ). PIPKs phosphorylate the D5 position of inositol on PI(4)P and the D4 position of PI(5)P to generate PI(4,5)P2 (
      • Anderson R.A.
      • Boronenkov I.V.
      • Doughman S.D.
      • Kunz J.
      • Loijens J.C.
      Phosphatidylinositol phosphate kinases, a multifaceted family of signaling enzymes.
      ). Numerous PIPKs have been identified, purified, and cloned (
      • Divecha N.
      • Brooksbank C.E.
      • Irvine R.F.
      Purification and characterization of phosphatidylinositol 4-phosphate 5-kinases.
      ,
      • Moritz A.
      • De Graan P.N.
      • Ekhart P.F.
      • Gispen W.H.
      • Wirtz K.W.
      Purification of a phosphatidylinositol 4-phosphate kinase from bovine brain membranes.
      ,
      • Boronenkov I.V.
      • Anderson R.A.
      The sequence of phosphatidylinositol-4-phosphate 5-kinase defines a novel family of lipid kinases.
      ), and are separated into type 1 and type II based on their biochemical and immunological characteristics (
      • Fruman D.A.
      • Meyers R.E.
      • Cantley L.C.
      Phosphoinositide kinases.
      ,
      • Loijens J.C.
      • Boronenkov I.V.
      • Parker G.J.
      • Anderson R.A.
      The phosphatidylinositol 4-phosphate 5-kinase family.
      ). Based on substrate specificity, PIPK1 (type 1) phosphorylates PI(4)P and PIPKII (type II) phosphorylates PI(5)P (
      • Rameh L.E.
      • Tolias K.F.
      • Duckworth B.C.
      • Cantley L.C.
      A new pathway for synthesis of phosphatidylinositol-4,5-bisphosphate.
      ). The mechanism of activation of PIPKs is not completely understood. Type I PIPK is activated by GTPγs (
      • Smith C.D.
      • Chang K.J.
      Regulation of brain phosphatidylinositol-4-phosphate kinase by GTP analogues. A potential role for guanine nucleotide regulatory proteins.
      ), small G proteins (
      • Martin A.
      • Brown F.D.
      • Hodgkin M.N.
      • Bradwell A.J.
      • Cook S.J.
      • Hart M.
      • Wakelam M.J.
      Activation of phospholipase D and phosphatidylinositol 4-phosphate 5-kinase in HL60 membranes is mediated by endogenous Arf but not Rho.
      ), PA (
      • Jenkins G.H.
      • Fisette P.L.
      • Anderson R.A.
      Type I phosphatidylinositol 4-phosphate 5-kinase isoforms are specifically stimulated by phosphatidic acid.
      ), heparin, and spermine (
      • Bazenet C.E.
      • Ruano A.R.
      • Brockman J.L.
      • Anderson R.A.
      The human erythrocyte contains two forms of phosphatidylinositol-4-phosphate 5-kinase which are differentially active toward membranes.
      ). Spermine and PA do not affect PIPKII activity, but heparin inhibits PIPKII activity (
      • Bazenet C.E.
      • Ruano A.R.
      • Brockman J.L.
      • Anderson R.A.
      The human erythrocyte contains two forms of phosphatidylinositol-4-phosphate 5-kinase which are differentially active toward membranes.
      ). Tyrosine phosphorylation has been shown to influence the activity of PIPKs (
      • Cochet C.
      • Filhol O.
      • Payrastre B.
      • Hunter T.
      • Gill G.N.
      Interaction between the epidermal growth factor receptor and phosphoinositide kinases.
      ).
      In most cells, the PI(4,5)P2 is generated through type I PIPKs and global KOs of the α, β, and γ isoforms of this kinase have been produced (
      • Choi S.
      • Thapa N.
      • Tan X.
      • Hedman A.C.
      • Anderson R.A.
      PIP kinases define PI4,5P2signaling specificity by association with effectors.
      ). This isoform is highly expressed in the retina (
      • Sakagami H.
      • Katsumata O.
      • Hara Y.
      • Tamaki H.
      • Fukaya M.
      Preferential localization of type I phosphatidylinositol 4-phosphate 5-kinase γ at the periactive zone of mouse photoreceptor ribbon synapses.
      ) and other neurons (
      • Wenk M.R.
      • Pellegrini L.
      • Klenchin V.A.
      • Di Paolo G.
      • Chang S.
      • Daniell L.
      • Arioka M.
      • Martin T.F.
      • De Camilli P.
      PIP kinase Igamma is the major PI(4,5)P(2) synthesizing enzyme at the synapse.
      ,
      • Wright B.D.
      • Loo L.
      • Street S.E.
      • Ma A.
      • Taylor-Blake B.
      • Stashko M.A.
      • Jin J.
      • Janzen W.P.
      • Frye S.V.
      • Zylka M.J.
      The lipid kinase PIP5K1C regulates pain signaling and sensitization.
      ). Early postnatal mortality was reported when this kinase was deleted (
      • Di Paolo G.
      • Moskowitz H.S.
      • Gipson K.
      • Wenk M.R.
      • Voronov S.
      • Obayashi M.
      • Flavell R.
      • Fitzsimonds R.M.
      • Ryan T.A.
      • De Camilli P.
      Impaired PtdIns(4,5)P2 synthesis in nerve terminals produces defects in synaptic vesicle trafficking.
      ). The mouse genes that encode PIPKs are Pip4k2a, Pip4k2b, Pip4k2c, Pip5k1a, and Pip5k1c; all of these were detected in the retinal mouse proteome (
      • Zhao L.
      • Chen Y.
      • Bajaj A.O.
      • Eblimit A.
      • Xu M.
      • Soens Z.T.
      • Wang F.
      • Ge Z.
      • Jung S.Y.
      • He F.
      • et al.
      Integrative subcellular proteomic analysis allows accurate prediction of human disease-causing genes.
      ).
      In bovine ROS membranes, type II PIPK activity is regulated by tyrosine phosphorylation (
      • Huang Z.
      • Guo X.X.
      • Chen S.X.
      • Alvarez K.M.
      • Bell M.W.
      • Anderson R.E.
      Regulation of type II phosphatidylinositol phosphate kinase by tyrosine phosphorylation in bovine rod outer segments.
      ), and PI(5)P serves as a substrate for the synthesis of PI(4,5)P2 (
      • Huang Z.
      • Guo X.X.
      • Chen S.X.
      • Alvarez K.M.
      • Bell M.W.
      • Anderson R.E.
      Regulation of type II phosphatidylinositol phosphate kinase by tyrosine phosphorylation in bovine rod outer segments.
      ). In rodents and transgenic frog retina, PIPKIIα activity is stimulated by light, and the membrane binding of PIPKIIα to ROS proteins is tyrosine phosphorylation-dependent (
      • Huang Z.
      • Anderson R.E.
      • Cao W.
      • Wiechmann A.F.
      • Rajala R.V.
      Light-induced tyrosine phosphorylation of rod outer segment membrane proteins regulate the translocation, membrane binding and activation of type ii alpha phosphatidylinositol-5-phosphate 4-kinase.
      ). PI(4,5)P2 is a critical PIP and regulates several key biological processes, such as actin cytoskeletal organization, endocytosis, exocytosis, modulation of ion channels, gene expression, angiogenesis, vesicular transport, cell migration, and nuclear functions (
      • Doughman R.L.
      • Firestone A.J.
      • Anderson R.A.
      Phosphatidylinositol phosphate kinases put PI4,5P(2) in its place.
      ). PI(4,5)P2 has been shown to activate cGMP-phosphodiesterase in ROS membranes, which results in the inhibition of ion influx through CNG channels (
      • He F.
      • Mao M.
      • Wensel T.G.
      Enhancement of phototransduction g protein-effector interactions by phosphoinositides.
      ,
      • Womack K.B.
      • Gordon S.E.
      • He F.
      • Wensel T.G.
      • Lu C.C.
      • Hilgemann D.W.
      Do phosphatidylinositides modulate vertebrate phototransduction?.
      ). In mammalian retina, the rod CNG channels, KCN1, TPR channels, and Na+-Ca2+ exchange are known to be regulated by PI(4,5)P2 (
      • Hille B.
      • Dickson E.J.
      • Kruse M.
      • Vivas O.
      • Suh B.C.
      Phosphoinositides regulate ion channels.
      ). In photoreceptor cells, PI(4,5)P2 regulates the biogenesis of light-sensing organelles and plays an important role in the delivery of rhodopsin-containing membranes to the ROS (
      • Deretic D.
      • Traverso V.
      • Parkins N.
      • Jackson F.
      • Rodriguez de Turco E.B.
      • Ransom N.
      Phosphoinositides, ezrin/moesin, and rac1 regulate fusion of rhodopsin transport carriers in retinal photoreceptors.
      ).
      One of the receptor tyrosine kinases, epidermal growth factor receptor, has a cluster of basic residues in the juxtamembrane domain. PI(4,5)P2 directly binds to this region and has been shown to activate EGFR (
      • Michailidis I.E.
      • Rusinova R.
      • Georgakopoulos A.
      • Chen Y.
      • Iyengar R.
      • Robakis N.K.
      • Logothetis D.E.
      • Baki L.
      Phosphatidylinositol-4,5-bisphosphate regulates epidermal growth factor receptor activation.
      ). These basic residues are also present in other growth factor receptors, such as IGF-1R, IR, FGFR1, PDGFR, VEGFR1, EPHB2, TRKA, and TRKB receptor (
      • Michailidis I.E.
      • Rusinova R.
      • Georgakopoulos A.
      • Chen Y.
      • Iyengar R.
      • Robakis N.K.
      • Logothetis D.E.
      • Baki L.
      Phosphatidylinositol-4,5-bisphosphate regulates epidermal growth factor receptor activation.
      ). However, the activation of these receptors by PI(4,5)P2 has not been studied.

      Phosphatidylinositol-3-phosphate 5-kinase or pikfyve

      In humans, FYVE finger-containing phosphoinositide kinase is encoded by the PIKFYVE gene (
      • Shisheva A.
      • Sbrissa D.
      • Ikonomov O.
      Cloning, characterization, and expression of a novel Zn2+-binding FYVE finger-containing phosphoinositide kinase in insulin-sensitive cells.
      ). PIKfyve phosphorylates PI(3)P to PI(3,5)P2 and PI to PI(5)P (
      • Vicinanza M.
      • Korolchuk V.I.
      • Ashkenazi A.
      • Puri C.
      • Menzies F.M.
      • Clarke J.H.
      • Rubinsztein D.C.
      PI(5)P regulates autophagosome biogenesis.
      ,
      • Cabezas A.
      • Pattni K.
      • Stenmark H.
      Cloning and subcellular localization of a human phosphatidylinositol 3-phosphate 5-kinase.
      ,
      • Hasegawa J.
      • Strunk B.S.
      • Weisman L.S.
      PI5P and PI(3,5)P2: minor, but essential phosphoinositides.
      ). The FYVE finger domain of PIKfyve tethers to membrane PI(3)P and is essential for membrane localization of PIKfyve to the cytosolic leaflet of endosomes (
      • Stenmark H.
      • Aasland R.
      • Driscoll P.C.
      The phosphatidylinositol 3-phosphate-binding FYVE finger.
      ). This binding is needed to phosphorylate PI to PI(5)P and PI(3)P to PI(3,5)P2 (
      • Vicinanza M.
      • Korolchuk V.I.
      • Ashkenazi A.
      • Puri C.
      • Menzies F.M.
      • Clarke J.H.
      • Rubinsztein D.C.
      PI(5)P regulates autophagosome biogenesis.
      ). Dysregulated enzyme activity of PIKfyve results in enlarged lysosomes due to defective synthesis of PI(3,5)P2, which results in defective lysosome fission events; thus, PIKfyve navigates all aspects of the vesicular and endocytic pathways (
      • Hasegawa J.
      • Strunk B.S.
      • Weisman L.S.
      PI5P and PI(3,5)P2: minor, but essential phosphoinositides.
      ,
      • Nicot A.S.
      • Laporte J.
      Endosomal phosphoinositides and human diseases.
      ). Mutations in one of the alleles of PIKfyve are linked to Francois-Neetens corneal fleck dystrophy (
      • Boisset G.
      • Polok B.K.
      • Schorderet D.F.
      Characterization of pip5k3 fleck corneal dystrophy-linked gene in zebrafish.
      ). The ablation of both alleles in the mouse is lethal (
      • Ikonomov O.C.
      • Sbrissa D.
      • Delvecchio K.
      • Xie Y.
      • Jin J.P.
      • Rappolee D.
      • Shisheva A.
      The phosphoinositide kinase PIKfyve is vital in early embryonic development: preimplantation lethality of PIKfyve-/= embryos but normality of PIKfyve+/- mice.
      ). Changes in PIKfyve have been shown to inhibit insulin-mediated glucose uptake (
      • Liu Y.
      • Lai Y.C.
      • Hill E.V.
      • Tyteca D.
      • Carpentier S.
      • Ingvaldsen A.
      • Vertommen D.
      • Lantier L.
      • Foretz M.
      • Dequiedt F.
      • et al.
      Phosphatidylinositol 3-phosphate 5-kinase (PIKfyve) is an AMPK target participating in contraction-stimulated glucose uptake in skeletal muscle.
      ). Mice with selective gene ablation of PIKfyve in skeletal muscle show prediabetic symptoms, which include insulin resistance, hyperinsulinemia, glucose intolerance, and increased adiposity (
      • Ikonomov O.C.
      • Sbrissa D.
      • Delvecchio K.
      • Feng H.Z.
      • Cartee G.D.
      • Jin J.P.
      • Shisheva A.
      Muscle-specific Pikfyve gene disruption causes glucose intolerance, insulin resistance, adiposity, and hyperinsulinemia but not muscle fiber-type switching.
      ). In one study, inhibition of PIKfyve resulted in the prevention of myocardial apoptosis and cardiac hypertrophy mediated through the activation of sirtuin-3 (SIRT3), a major mitochondria NAD+-dependent deacetylase in obese mice (
      • Tronchere H.
      • Cinato M.
      • Timotin A.
      • Guitou L.
      • Villedieu C.
      • Thibault H.
      • Baetz D.
      • Payrastre B.
      • Valet P.
      • Parini A.
      • et al.
      Inhibition of PIKfyve prevents myocardial apoptosis and hypertrophy through activation of SIRT3 in obese mice.
      ). Because photoreceptor cells are highly metabolic (
      • Rajala R.V.S.
      Aerobic glycolysis in the retina: functional roles of pyruvate kinase isoforms.
      ,
      • Rajala A.
      • Soni K.
      • Rajala R.V.S.
      Metabolic and non-metabolic roles of pyruvate kinase M2 isoform in diabetic retinopathy.
      ), the PIKfyve enzyme might regulate several important functions. However, there are no available studies of this enzyme in the retina.
      It was previously reported that Vps34-generated PI(3)P regulates the canonical autophagy (
      • Simonsen A.
      • Tooze S.A.
      Coordination of membrane events during autophagy by multiple class III PI3-kinase complexes.
      ). However, Vps34-independent noncanonical autophagy has been observed in sensory neurons from the PI(3)P-generated Vps34 enzyme, T-lymphocytes, and glucose-starved cells incubated with PI3K inhibitor, Wortmannin, which inhibits the Vps34 enzyme (
      • McAlpine F.
      • Williamson L.E.
      • Tooze S.A.
      • Chan E.Y.
      Regulation of nutrient-sensitive autophagy by uncoordinated 51-like kinases 1 and 2.
      ). PI(5)P-dependent noncanonical autophagy has been reported in cells depleted of PI(3)P (
      • Vicinanza M.
      • Korolchuk V.I.
      • Ashkenazi A.
      • Puri C.
      • Menzies F.M.
      • Clarke J.H.
      • Rubinsztein D.C.
      PI(5)P regulates autophagosome biogenesis.
      ). PIKfyve plays an important role in generating PI(5)P upon binding to PI(3)P through its FYVE domain (
      • Vicinanza M.
      • Korolchuk V.I.
      • Ashkenazi A.
      • Puri C.
      • Menzies F.M.
      • Clarke J.H.
      • Rubinsztein D.C.
      PI(5)P regulates autophagosome biogenesis.
      ). This PI(5)P can be converted to PI(4,5)P2 through the action of the type II phosphatidylinositol 5-phosphate 4-kinase (PIP4K) enzyme, which was previously shown to express in rod photoreceptor cells (
      • Huang Z.
      • Guo X.X.
      • Chen S.X.
      • Alvarez K.M.
      • Bell M.W.
      • Anderson R.E.
      Regulation of type II phosphatidylinositol phosphate kinase by tyrosine phosphorylation in bovine rod outer segments.
      ). The generated PI(4,5)P2 can perform various functions, including serving as a substrate for class I PI3K for the generation of PI(3,4,5)P3 (
      • Fruman D.A.
      • Meyers R.E.
      • Cantley L.C.
      Phosphoinositide kinases.
      ,
      • Rajala R.V.
      Phosphoinositide 3-kinase signaling in the vertebrate retina.
      ,
      • Rajala R.V.
      • Anderson R.E.
      Focus on molecules: phosphatidylinositol-4,5-bisphosphate (PIP2).
      ). In cone photoreceptor cells, the deletion of class I PI3K that makes PI(3,4,5)P3 resulted in age-related cone degeneration (
      • Ivanovic I.
      • Anderson R.E.
      • Le Y.Z.
      • Fliesler S.J.
      • Sherry D.M.
      • Rajala R.V.
      Deletion of the p85alpha regulatory subunit of phosphoinositide 3-kinase in cone photoreceptor cells results in cone photoreceptor degeneration.
      ,
      • Rajala R.V.
      • Ranjo-Bishop M.
      • Wang Y.
      • Rajala A.
      • Anderson R.E.
      The p110alpha isoform of phosphoinositide 3-kinase is essential for cone photoreceptor survival.
      ).

      Neuroprotective roles of PI3K in the retina

      Several growth factors, such as PDGF, brain-derived neurotrophic factor, insulin, IGF-1 and -2, basic fibroblast growth factor, erythropoietin (EPO), and ciliary neurotrophic factor (CNTF), promote retinal cell survival through PI3K/Akt activation. Insulin and IGF-1 have been shown to promote photoreceptor survival (
      • Reiter C.E.
      • Sandirasegarane L.
      • Wolpert E.B.
      • Klinger M.
      • Simpson I.A.
      • Barber A.J.
      • Antonetti D.A.
      • Kester M.
      • Gardner T.W.
      Characterization of insulin signaling in rat retina in vivo and ex vivo.
      ,
      • Biswas S.K.
      • Zhao Y.
      • Nagalingam A.
      • Gardner T.W.
      • Sandirasegarane L.
      PDGF- and insulin/IGF-1-specific distinct modes of class IA PI 3-kinase activation in normal rat retinas and RGC-5 retinal ganglion cells.
      ), whereas PDGF, CNTF, and EPO promote the survival of ganglion cells and RPE (
      • Hollborn M.
      • Bringmann A.
      • Faude F.
      • Wiedemann P.
      • Kohen L.
      Signaling pathways involved in PDGF-evoked cellular responses in human RPE cells.
      ). basic fibroblast growth factor-mediated activation of PI3K has been observed in Müller cells (
      • Hollborn M.
      • Jahn K.
      • Limb G.A.
      • Kohen L.
      • Wiedemann P.
      • Bringmann A.
      Characterization of the basic fibroblast growth factor-evoked proliferation of the human Muller cell line, MIO-M1.
      ). PI3K exerts its neuroprotective effect through its downstream effector, Akt (
      • Li G.
      • Rajala A.
      • Wiechmann A.F.
      • Anderson R.E.
      • Rajala R.V.
      Activation and membrane binding of retinal protein kinase Balpha/Akt1 is regulated through light-dependent generation of phosphoinositides.
      ,
      • Reiter C.E.
      • Sandirasegarane L.
      • Wolpert E.B.
      • Klinger M.
      • Simpson I.A.
      • Barber A.J.
      • Antonetti D.A.
      • Kester M.
      • Gardner T.W.
      Characterization of insulin signaling in rat retina in vivo and ex vivo.
      ,
      • Li G.
      • Anderson R.E.
      • Tomita H.
      • Adler R.
      • Liu X.
      • Zack D.J.
      • Rajala R.V.
      Nonredundant role of Akt2 for neuroprotection of rod photoreceptor cells from light-induced cell death.
      ). IR activity is important for PI3K/Akt activation (
      • Li G.
      • Rajala A.
      • Wiechmann A.F.
      • Anderson R.E.
      • Rajala R.V.
      Activation and membrane binding of retinal protein kinase Balpha/Akt1 is regulated through light-dependent generation of phosphoinositides.
      ,
      • Reiter C.E.
      • Sandirasegarane L.
      • Wolpert E.B.
      • Klinger M.
      • Simpson I.A.
      • Barber A.J.
      • Antonetti D.A.
      • Kester M.
      • Gardner T.W.
      Characterization of insulin signaling in rat retina in vivo and ex vivo.
      ). For the signal to be maintained for a longer period, the protein tyrosine phosphatase, PTP1B, must be inactivated. It has been shown that activated Akt phosphorylates PTP1B on serine 50 (
      • Ravichandran L.V.
      • Chen H.
      • Li Y.
      • Quon M.J.
      Phosphorylation of PTP1B at Ser(50) by Akt impairs its ability to dephosphorylate the insulin receptor.
      ), which inhibits the activity of PTP1B to facilitate a positive feedback mechanism for IR/PI3K/Akt signaling. In diabetic retinopathy, PI3K activity is downregulated (
      • Reiter C.E.
      • Wu X.
      • Sandirasegarane L.
      • Nakamura M.
      • Gilbert K.A.
      • Singh R.S.
      • Fort P.E.
      • Antonetti D.A.
      • Gardner T.W.
      Diabetes reduces basal retinal insulin receptor signaling: reversal with systemic and local insulin.
      ) due to increased PTP1B activity (
      • Rajala R.V.
      • Wiskur B.
      • Tanito M.
      • Callegan M.
      • Rajala A.
      Diabetes reduces autophosphorylation of retinal insulin receptor and increases protein-tyrosine phosphatase-1B activity.
      ). Furthermore, decreased activation of mTOR and p760S6K, and increased activation of GSK3β, the downstream effector of PI3K, are downregulated in diabetic retinopathy (
      • Reiter C.E.
      • Gardner T.W.
      Functions of insulin and insulin receptor signaling in retina: possible implications for diabetic retinopathy.
      ).
      PI3K activation through the IR inhibits caspase-mediated cell death of retinal neurons in culture (
      • Barber A.J.
      • Nakamura M.
      • Wolpert E.B.
      • Reiter C.E.
      • Seigel G.M.
      • Antonetti D.A.
      • Gardner T.W.
      Insulin rescues retinal neurons from apoptosis by a phosphatidylinositol 3-kinase/Akt-mediated mechanism that reduces the activation of caspase-3.
      ). 17β-Estradiol protects stressed retina through PI3K activation (
      • Yu X.
      • Rajala R.V.
      • McGinnis J.F.
      • Li F.
      • Anderson R.E.
      • Yan X.
      • Li S.
      • Elias R.V.
      • Knapp R.R.
      • Zhou X.
      • et al.
      Involvement of insulin/phosphoinositide 3-kinase/Akt signal pathway in 17 beta-estradiol-mediated neuroprotection.
      ,
      • Cao W.
      • Rajala R.V.
      • Li F.
      • Anderson R.E.
      • Wei N.
      • Soliman C.E.
      • McGinnis J.F.
      Neuroprotective effect of estrogen upon retinal neurons in vitro.
      ). The deletion of the phosphatase and tensin homolog (PTEN) results in an increased level of PI(3,4,5)P3, which protects ganglion cells (
      • Park K.K.
      • Liu K.
      • Hu Y.
      • Smith P.D.
      • Wang C.
      • Cai B.
      • Xu B.
      • Connolly L.
      • Kramvis I.
      • Sahin M.
      • et al.
      Promoting axon regeneration in the adult CNS by modulation of the PTEN/mTOR pathway.
      ). Activation of PI3K through PDGF stimulation protects retinal pericytes under diabetic conditions (
      • Stitt A.W.
      • Hughes S.J.
      • Canning P.
      • Lynch O.
      • Cox O.
      • Frizzell N.
      • Thorpe S.R.
      • Cotter T.G.
      • Curtis T.M.
      • Gardiner T.A.
      Substrates modified by advanced glycation end-products cause dysfunction and death in retinal pericytes by reducing survival signals mediated by platelet-derived growth factor.