Novel phosphoinositide-protein interactions and inhibition of Akt activation

The PI3K/Akt signaling pathway is critical for normal growth and development of a cell. The activation of Akt, while playing an important role in cell survival, has also been linked to cancer and diabetes. A specific Akt inhibitor is predicted to be of immense value in cancer treatment. Akt activation requires recruitment to the plasma membrane through interaction of its N-terminal pleckstrin homology (PH) domain with the phosphoinositide products of phosphatidylinositol-3-kinase (PI3K); phosphatidylinositol-3,4-bisphosphate (PI(3,4)P2) and (PI(3,4,5)P). Two synthe tic peptide libraries were screened for binding to the Akt PH domain using competitive displacement of PI(3,4)P2. One library consisted of random octamers and the second library was biased with alternate racemic glutamate or aspartate amino acids. Twenty-seven sequences were obtained and analyzed for Akt PH binding and the three sequences with highest affinity were chosen for further study. Each peptide demonstrated low micromolar <italic>in vitro

NOVEL PHOSPHOINOSITIDE-PROTEIN INTERACTIONS AND INHmITION OF AKT ACTIVATION by Randy Alan Booth A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Medicinal Chemistry The University of Utah May 2004 Copyright © Randy Alan Booth 2004 All Rights Reserved THE UNIVERSITY OF UTAH GRADUATE SCHOOL SUPERVISORY COMMITTEE APPROVAL of a dissertation submitted by Randy Alan Booth This dissertation has been read by each member of the following supervisory committee and by majority vote has been found to be satisfactory. ~SMCCIOSkey ( David J e Erik Jorgensen THE UNIVERSITY OF UTAH GRADUATE SCHOOL FINAL READING APPROVAL To the Graduate Council of the University of Utah: I have read the dissertation of Randy Alan Booth in its final form and have found that (l) its format, citations, and bibliographic style are consistent and acceptable; (2) its illustrative materials including figures, tables, and charts are in place; and (3) the final manuscript is satisfactory to the supervisory committee and is ready for submission to The Graduate School Date Chair: Supervisory Committee Approved for the Major Department f1JQ Chris Ireland ChairlDean Approved for the Graduate Council ABSTRACT The PI3K1Akt signaling pathway is critical for nonnal growth and development of a cell. The activation of Akt, while playing an important role in cell survival, has also been linked to cancer and diabetes. A specific Akt inhibitor is predicted to be of immense value in cancer treatment. Akt activation requires recruitment to the plasma membrane through interaction of its N-terminal pleckstrin homology (PH) domain with the phosphoinositide products of phosphatidylinositol- 3-kinase (PI3K); phosphatidylinositol-3,4-bisphosphate (PI(3,4 )P2) and (PI(3,4,5)P3). Two synthetic peptide libraries were screened for binding to the Akt PH domain using competitive displacement of PI(3,4 )P2. One library consisted of random octamers and the second library was biased with alternate racemic glutamate or aspartate amino acids. Twenty-seven sequences were obtained and analyzed for Akt PH binding and the three sequences with highest affinity were chosen for further study. Each peptide demonstrated low micromolar in vitro inhibition of Akt binding to PI(3,4)P2 and demonstrated Akt selectivity over other PH domains. However, the affinity was determined to be nonspecific and inhibition of phosphoinositide-binding was determined to be due to masking of the phosphoinositides, not binding the Akt PH domain. When attached to a highly basic membrane penneable sequence, two peptides were able to enter cells and delay membrane localization of an expressed EGFP-Akt PH construct. The basic peptides demonstrated minimal cellular toxicity and inhibition of Akt activation presumably through masking newly formed phosphoinositides at the cell membrane to prevent Akt activation. Analysis of protein-phosphoinositide interactions can indicate possible pathways in which a protein may be involved in cell signaling. Peroxiredoxin 1 (Prxl) was identified in a screen for high affinity phosphatidylinositol-3,4,5- trisphosphate (PI(3,4,5)P3) binding proteins with PI(3,4,5)P3 PIP Beads™. The murine Prx 1 gene was clone and characterized for phosphoinositide binding. Photoaffinity labeling was employed to examine phosphoinositide interactions with Rab5 effector proteins, Dnmlp, caspase 3, caspase 8, caspase 9, Uncl04, EVHl, and Db!. Novel tandem PH domain constructs were created in an attempt to 'create a more sensitive reporter protein of phosphoinositides with in vitro assays, though none exhibited any advantage over wild type PH domain. v To my family for their years of encouragement To my wife Caroline for her constant support and love TABLE OF CONTENTS ABSTRACT ................................................................................... iv LIST OF TABLES. .. ... ... . . .. ..... . .. . .. ... . . . . .... . . ... ...... .. . .. ... . . . . .. . . . . . . . . . . .. .. . IX LIST OF ABBREVIATIONS........ .......... ......... ......... ...... ... ....... ........... X ACKNOWLEDGMENTS ................................................................... xvi CHAPTER 1. PHOSPHOINOSITIDE SIGNAL TRANSDUCTION... ......... ... ..... ..... 1 Introduction..................................................................... ...... 1 Regulation of Phosphoinositides.................................................. 2 Phosphoinositide Binding Domains...................................... ......... 9 Signal Transduction of Phosphoinositides....................................... 18 Conclusion................................. ......... .................. ... ... ......... 27 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 28 2. AKT SIGNALING............... ............................................ ....... 46 Introduction........................ ................. ......... ....... ... ........ . ...... 46 Akt Structure and Activation... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 46 Akt Signaling. .. ......... .................. .................. ......... ...... .......... 49 Akt and Disease ................................. '. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 Conclusion..................... .............................. ....... . . ...... . .. ...... 56 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 57 3. THE SPECIFIC INHIBITION OF AKT ......................................... 64 Introduction ........................................................................... 64 Materials and Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 69 Results ................... '. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 78 Discussi on ............................................................................ 102 References ............................................................................ 107 4. PEROXIR.EDOXIN 1 .............................................................. 112 Introd1..!ction ........................................................................... 112 Materials and Methods ..... ' ......................................................... 115 Results ................................................................................. 118 Discussion ............................................................................ 121 References ............................................................................ 122 5. PHOTOAFFINITY LABELING AND TANDEM PH DOMAINS .......... 125 Introduction ........................................................................... 125 Materials and Methods .............................................................. 128 Results ................................................................................. 133 Discussion ............................................................................ 137 Referen.ces ............................................................................ 142 viii LIST OF TABLES 3.1. Sequences obtained from library screening... . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. 81 3.2. Visual alignment of the acid-biased sequences.................................... 82 3.3. Visual alignment of the random peptide library sequences ...................... 83 3.4. Sequences chosen for synthesis ...................................................... 88 3.5. Molecular weights of pep tides ...•................................................... 88 3.6. ICso values of peptides and natural ligands....... .......... ...... ............. .... 90 3.7. Percent binding due to ionic interactions ........................................... 94 4E-BPl ARF ANTH ATP BCIP BODIPY-FL BSA Btk BZDC CALM CCV CMTP COPI COPIT DAG DAPPl DMEM LIST OF ABBREVIATIONS 4E binding protein 1 ADP-ribosylation factor AP180 N-tenninal homology adenosine triphosphate 5-bromo-4-chloro-3-indolyl phosphate D( + )-sn-I-O-[I-[6-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a­diaza- s-indacene-3-pentanoyl)amino]hexanoyl]-2- hexanoylglyceryl bovine serum albumin Bruton's tyrosine kinase benzoyldihydrocinnamoy I clathrin assembly lyphoid myloid clathrin-coated vesicles carboxy-terminal modulator protein coat protein I coat protein IT diacylglycerol dual adaptor for phospho tyrosine and 3-phosphoinositides Dulbecco's modified eagle medium DMF DNA DNAlPeptide RF DPI DTT EDTA EGF EGFP EGFR EGTA eNOS ENTH ER PBS FP GAP GEF GFP Grpl GSK3~ GST GTP N ,N -dimethyl formamide deoxyribonucleic acid DNAlPeptide Resource Facility D-3-deoxy-phosphatidyl-myo-inositol dithiothreitol ethy lenediaminetetraacetic acid epidermal growth factor enhanced green fluorescent protein endothelial growth factor receptor ethylene glycol-bis(p-aminoethyl ether)-N,N,N' ,N' -tetraacetic acid endothelial nitric oxide synthase epsin N-terminal homology endoplasmic reticulum fetal bovine serum fluorescence polarization GTPase-activating protein guanine nucleotide exchange factor green fluorescent protein general receptor for 3-phosphoinositides glycogen synthase kinase 3 ~ glutathione S-transferase guanosine triphosphate xi H20 2 HM hr I(I,3)P2 I( 1,3A,5)P 4 I(I,3,4,5,6)P5 I(IA,5)P3 IKK ILK IP3 IP6 IPTG kDa LMV LPA MAPK MARCKS MBP MDM2 min MMDB mTOR hydrogen peroxide hydrophobic motif hour, hours inositol-I,3-bisphosphate inositol-I,3 A,5-tetrakisphosphate inositol-I,3 A,5 ,6-pentakisphosphate inositol-l A,5-trisphosphate IlCB kinase integrin-linked kinase inositol trisphosphate inositol hexakisphosphate Isopropyl ~-D-l-thiogalactopyranoside kiloDaltons large multilamellar vesicles lysophosphatidic acid mitogen activated protein kinase myristoylated alanine-rich C kinase substrate maltose-binding protein murine double minute-2 minute, minutes molecular modeling database mammalian target of rapamycin xii MTT NADPH NCBI NEM NHS NO O2-- PA PBS PC PET PDB PDGP PDKI PE PH PI PI kinases PIP PI(3)P PI(3,4)P2 PI(3,4,5)P3 3-(4 ,5-dimethylthiazol-2-y 1)-2,5-diphenyl-2H -tetrazolium bromide methylthiazolyldiphenyl-tetrazolium bromide ~-nicotinamide adenine dinucleotide phosphated National Center for Biotechnology Information N -ethylmaleimide N -succinimidyl nitric oxide superoxide phophatidic acid phosphate-buffered saline phosphatidy lcholine polyelectrolyte theory protein database platelet-derived growth factor phosphoinositide-dependent kinase 1 phosphatidylethanolamine pleckstrin homology phosphatidylinositol phosphoinositide kinase phosphoinositide phosphatidylinositol-3-phosphate phosphatidylinositol-3,4-bisphosphate phosphatidy linositol-3,4,5 -trisphosphate xiii PI(3,5)P2 PI(4)P PI(4,5)P2 PI(5)P PI3K PI4K PITP PKB PKC PLC PLD PP2A Prx PTB PTEN RAC RNA ROS RTk SDS SH2 Domain SH3 Domain SHIP phosphatidylinositol-3,5-bisphosphate phosphatidylinositol-4-phosphate phosphatidylinositol-4,5-phosphate phosphatidylinositol-5-phosphate phosphatidylinositol 3-kinase phosphatidylinositol 4-kinase phosphatidylinositol transfer protein protein kinase B protein kinase C phospholipase C phospholipase D protein phosphatase 2A peroxiredoxin phospho-tyrosine binding domain phosphatase and tensin homolog deleted on chromosome ten related to protein kinases A and C ribonucleic acid reactive oxygen species receptor tyrosine kinase sodium lauryl sulfate Src homology 2 domain Src homology 3 domain SH2-domain-containing inositol 5-phosphatase xiv SOD TEAB TON VeA region VEOF VPS WASP XIAP superoxide dismutase triethylamine bicarbonate trans-Oolgi network verprolin homology, cofilin-like region, and acidic region vesicular epidermal growth factor vacuolar protein sorting Wiskott -Aldrich syndrome protein X -inhibitor of apoptosis xv ACKNOWLEDGMENTS I thank my research advisor, Professor Glenn D. Prestwich, for the help, support and encouragement for the past five years. He has given me the opportunity to grow intellectually and helped me to gain the confidence and skills necessary for future scientific research. I thank him for his example of unending optimism in the face of adversity and specifically his patience and guidance. I also acknowledge Professors James McCloskey, David Jones, Thomas McIntyre, and Erik Jorgensen who have served on my committee. I thank them for their guidance over the past five years, as they have been very helpful and supportive in my graduate career. I want to thank Ms. Angie Branch and Robyn James, and Drs. Paul Neilsen, Colin Fergusen, Beth Drees, Leena Chakravarty of Echelon Biosciences Inc. for their invaluable time and help with my graduate work. I thank Dr. Daryll DeWald and Ms. Kelly Manabe at Utah State University for their excellent assistance with confocal microscopy. I thank Dr. Robert Schackmann for his patience and perseverance in synthesizing and sequencing peptides for me. I thank Dr. Christian Rommel and Christian Pasquali of Serono Pharmaceutical Research Institute in Geneva Switzerland for a wonderful collaboration. I thank Drs. Jerry Hinshaw for his consistent chemical and instrumental assistance, Nicole Bernshaw for her advice and direction in cell culture, and Li Feng for her direction in my early graduate years. I also thank Drs. Koen Vercruysse, Dae­Yeon Suh, Philippe Garnier, Josef Lazar, Kelly Kirker, Michael Ziebell for assistance and many stimulating conversations. I must also thank. Lian Qian, Tyler Rose, Shenshen Cai, Hao Li, and Drs. Shoichiro Ozaki, Bharat Mehrotra, Yi Luo, Y ong Xu, Yanchun Liu, and Xiao-zheng Shu for their consistent help and input. A special thank you to Marie Dippolito for her enthusiasm for life outside of the lab and excellent direction and assistance in it. Additionally, I thank Anna Scott, Dustin Updike, Hongfang Wang, John Johnston, and Lei Wang for their assistance as rotation students and Matthew Borrowman for his excellent technical assistance. I must also thank my parents who have always encouraged me to do my best, my grandparents for great examples and their belief in my potential. I thank my wife Caroline, for all her love and support without which this work would not have been possible. xvii CHAPTER! PHOSPHOINOSITIDE SIGNAL TRANSDUCTION Introduction Cellular membranes serve to enfold the cell, compartmentalize cellular components and functions, and direct nl0vement within the cell (1). While membrane lipids are fluid and allow for movement of cells in response to stimuli (1), specific lipids also mediate cellular function. Phosphoinositides (PIPs) only make up about 10% of the total membrane lipids and perform specific functions within the cell such as regulation of membrane traffic and the actin cytoskeleton, and act as second messengers in signal transduction pathways (2-5). The unique structure of phosphoinositides enables the many functions of this lipid (Figure 1.1). All phosphoinositides are created from phosphatidylinositol (PI), the structure of which consists of two fatty acid chains usually attached through an ether linkage to two sequential alcohol groups of a glycerol molecule. The fatty acid in the C-1 position is generally a saturated fatty acid, while the C-2 position usually contains an unsaturated or polyunsaturated fatty acid with common chain lengths of 16-24 carbons each (6). Commonly the acyl chains consist of sn-1 stearoyl, and sn-2 arachidonyl (2). The third alcohol group of the glycerol molecule is bound to the inositol group via a phosphate diester bond. 2 PIP-K I l HO OH PI3K ~ 3H 2 ./ HO 1¥ o· 16 I H OH o-p=o HO H I o PIP-KII CO~ lO-'1I o Figure 1.1. The structure of phosphatidylinositol with the kinases that target each hydroxyl position. The phosphodiester bond is designated the D-l carbon of the inositol ring. Reversible phosphorylation at the 0-3, 0-4, and 0-5 carbons of the inositol ring enables the creation of eight unique second messenger molecules (4,7). The eight naturally occurring phosphoinositides include PI, phosphatidylinositol-3-phosphate (PI(3)P), phosphatidylinositol-4-phosphate (PI( 4 )P), phosphatidylinositol-5- phosphate (PI(5)P), phosphatidylinositol-3,4-bisphosphate (PI(3,4)P2), phosphatidylinositol-4,5-phosphate (PI(4,5)P2), phosphatidylinositol-3,5- bisphosphate (PI(3,5)P2), and phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3) (2,4,7). Regulation of Phosphoinositides Phosphoinositides are regulated by lipid kinases, phosphatases, and lipases to direct many cellular functions. Phosphoinositides are well suited as signaling molecules because even though there is relative low overall abundance in the cell, phosphoinositides can be temporarily highly concentrated within membrane 3 microdomains (7). The roles of phosphoinositides important in membrane trafficking, cytoskeletal remodeling, metabolic control, membrane ruffling, secretion, cell adhesion, chemotaxis, deoxyribonucleic acid (DNA) synthesis, and mitogenic signaling will be discussed later (7-9). Phosphatases Phosphoinositide phosphatases are named after the position of the phosphate removed (Figure 1.2). The 5-phosphatases remove the phosphate from the D-5 position of the substrates inositol-1 ,4,5-trisphosphate (I( 1 ,4,5)P3), inositol-1 ,3,4,5- tetrakisphosphate (I(1,3,4,5)P4),PI(4,5)P3, and PI(3,4,5)P3, and are made up of four classes (2). The first class of phosphatases dephosphorylates only I(1,4,5)P3 and I(1,3,4,5)P4 and are believed to be membrane bound through isoprenylation and play a role in calcium signaling (345). The second class of 5-phosphatases is able to dephosphorylate all substrates mentioned with varying efficiency (2). Synaptojanin is an example of a class II 5-phosphatase with a role in synaptic vesicle trafficking (10). Other examples are the merrlbrane associated phosphatases OCRL-1 and platelet 5-phosphatase II (11). Class m phosphatases only hydrolyze the 5- phosphates in PI(4,5)P3, and PI(3,4,5)P3 (11). Examples include type I and type II src homology 2 (SH2)-containing inositol5-phosphatase (SHIP1 and SHIP2) and are both important regulators of PI(3,4,5)P3 and PI(4,5)P2 (2). SHIP2 activity is linked to PI3K and Ras/mitogen-activated protein kinase activity and loss of SHIP2leads to increased insulin sensitivity (12,13). SHIP mutations have been implicated in acute myloid leukemia (14). Little is known about the class N 5-phosphatases, but they appear to be specific for PI(3,4,5)P3 and form complexes with PI3K (2). The 4- PI(4)P Type IIIl P13K PI(3,4)P2 ~ PI ~Kinase 13K Type III PI(5)P PIC 4,5)P2 Kinase PTENi P13K 1 Type I PI(3,4,5)P3 lType I PIP Kinase Figure 1.2. The anabolic and catabolic pathways of phosphoinositides with regulatory kinases and phosphatases (2). phosphatases and 3-phosphatases both participate in PI3K signaling (2). Although the 4-phosphatases dephosphorylate PI(3,4 )P2 to yield PI(3)P, the 3-phosphatases 4 regulate PI(3,4,5)P3 to produce PI(4,5)P2 (2). The tumor suppressor phosphatase and tensin homolog deleted on chromosome ten (PTEN)IMMAC 1 is a member of the 3- phosphatases that converts PI(3,4,5)P3 to PI(4,5)P2 (2,15). Several mutations have been identified in PTEN that link it to mUltiple forms of cancer (16,17). Loss of PTEN function results in increased cell motility and tumor cell invasion (18). PTEN is also important for B cell development and homeostasis (19). 5 Lipases Phospholipases represent another class of enzymes important in phosphoinositide signaling. Specifically the families of phospholipase C (PLC) enzymes hydrolyze PI( 4,5)P2 to form the second messengers inositol-1 ,4,5- trisphosphate (I( 1 ,4,5)P3) and diacylglycerol (DAG) (20). The three isofoms of PLC, ~, '¥, and 0, appear to have differential activation. GTP-binding proteins activate PLC~ enzymes (21). Research has shown that binding ofPI(4,5)P2, calcium, and GTP-binding proteins contribute to activation of PLCo isoforms, although the mechanism is still unclear (22-24). Fine-tuned regulation of PLCy activity is accomplished through protein-protein interactions via two SH2 domains and a src homology 3 (SH3) domain, and through phosphorylation at three key sites in response to receptor-tyrosine kinases activation, and through lipid-protein interactions with PI(3,4,5)P3 (25,26). Kinases PI serves as a substrate for phosphoinositide kinases (PI kinases) to form the monophosphorylated phosphoinositides, PI(3)P, PI(4)P, and PI(5)P (Figure 1.2). Cellular levels of PI(3)P are not believed to fluctuate in response to stimuli although increased levels have been observed in platelets (27,28). The PI 5 kinase is responsible for the synthesis of both PI(5)P and PI(3,5)P2 in response to hypo­osmotic shock (29,30). Formation of PIC 4)P is most understood of the three monophosphorylated phosphoinositides. PI( 4)P is synthesized by phosphorylation at the D-4 position by type II and type ill PI 4-kinases (PI4K). Type I PI4Ks were later determined to be PI 3-kinases (PI3K) and are discussed later. Type II PI4Ks are membrane- associated and range between 45-55 kDa in size (31-33). The type ill PI4K exist as both a 110 and a 210-kDa protein that are soluble, membrane associated, and sensitive, to the PI3K inhibitors wortmannin and LY294002 (34,35). PI4K type II is present on the earl y endosome and the plasma membrane, whereas the (l isoform of type III kinase is linked to the endoplasmic reticulum (ER) and the ~ isoform is associated with the Golgi (36,37). 6 The creation of PIC 4)P is a regulated and is involved in many cellular processes. PI(4)P levels quickly drop, believed to be due to PLC activation, with a slow return to basal levels in response to calcium-mobilizing agonist (38). Activation of receptor tyrosine kinases has been shown to result in activation of PI 4-kinases (32,38-41). PI 4-kinases are activated in response to G protein-coupled receptors. The calcium-sensing receptor activated PI4K in a Rho-dependent and PLC independent manner (42). Balla et al. report rapid increase in PI4-kinase activity in response to angiotensin II stimulation (43). Cardiotoxin and mastoparan have also been shown to stimulate PI4K activity (43). Two types of PIP kinases create higher phosphorylated phosphoinositides. The PIP kinases are overall promiscuous and can use all naturally occurring PIPs as substrates with the exception ofPI(3,5)P2 and PI(3,4,5)P3 (44). Type I PIP kinases use PI(4)P to generate PI(4,5)P2 and can also use PI(3)P to form PI(3,4)P2 and PI(3,5)P2 in vitro in an isoform dependent manner (45-47). Type II kinases appear to prefer PI(5)P as a substrate for the formation of PI(4,5)P2, but can also produce PI(3,4)P2 from PI(3)P (45). Observed masses of type I kinases range from 68 to 90 kDa, while type n kinases are smaller in the range of 46 to 57 kDa (44). The X-ray structure of PIP kinase type np revealed a homodimeric protein complex that contains a highly basic flat surface that interacts with the negative phosphoinositides in the membrane (48). The PIP kinases are regulated by factors that influence their subcellular distribution. At the membrane, phosphatidic acid (P A) is implicated in PIP type I kinase stimulation. However, the type II kinases and a putative type I PIP kinase in Swiss 3T3 cells is found to be insensitive to PA (49-53). PIP kinase activity is reported to increase in response to epidermal growth factor (EGF), phorbol esters, and vascular endothelial cell attachment to fibronectin (40,44,54-58). Inhibition of PIP kinases was observed by cyclic adenosine monophosphate (cAMP) in platelets (44). Type I kinases show activation in response to small G proteins. Rho kinase regulates the activity of PIC 4)P 5-kinase and PIC 4,5)P2 formation leading to actin filament uncapping in cytoskeletal remodeling (59,60). While there is no evidence that the type n kinases are regulated by G proteins, type II PIP kinases are translocated to the cytoskeleton remodeling in response to integrin-mediated signals and the type IIp PIP kinase associates with the TNFa receptor (61,62). The type II kinases are also regulated by their phosphorylation state of serine residues (63,64). 7 Originally classified as a type I PIP kinase, PI 3-kinase was later reclassified as a family of kinases responsible for phosphorylating the D-3 position (9,65). Class I PI3Ks are divided into two groups, both of which are heterodimeric protein complexes with a 110-120 kDa catalytic subunit and a 50-100 kDa regulatory subunit (9). The p85 subunit of group IA containing both the a and ~ isoforms associate with phosphotyrosine motifs, whereas the p85 subunit of group IIA, consisting of the 'Y isoform, associates with G proteins and the plOI protein for activation (66). Although the preferred substrate is PI(4,5)P2, class I PI3Ks will also phosphorylated PI and PI(4)P (34,67). All PI3Ks have two SH2 domains separated by an inter-SH2 domain (68-70). The plIO catalytic subunit binds the p85 regulatory subunit at the iSH2 domain (71-73). Due to the many protein-interacting domains, the p85 subunit is able to integrate different signaling pathways. PI3K can be inhibited by wortmannin forming a Schiff base with a conserved lysine residue in the p 110 or equivalent subunit to inhibit catalytic activity, (74). The quercetin derivative, L Y294002, is a potent inhibitor of PI3K via the adenosine triphosphate (A TP) binding site (75). 8 The structure of the class IA p85 subunit enables many possible means of activation. The p85 subunit contains an SH3 domain, a breakpoint-cluster-region homology domain, two proline-rich domains, and two Src homology 2 different signaling molecules for the integration of several pathways. This signaling integration results in the tight regulation of PI3K activity (9). In a resting state, the subunits of class I PI3Ks are not membrane bound, but upon stimulation the kinase is recruited to the membrane. Four cooperating processes in response to a stimulus can result in recruitment and activation of PI3K at the plasma nlembrane. The first way is translocation via the SH2 domains of the p85 subunits to the plasma membrane in response to phosphorylation YXXM motif substrates of receptor tyrosine kinases (76). The second way is through the plIO subunit binding of guanosine triphosphate (GTP)-bound (activated) Ras (9), The third way is through the bi-directional binding of the p85 subunit to proteins like Shc, Cbl, or dynamin via SH3 domain binding to proline-rich domains. The fourth way is through p85 mediated proline-rich domain binding to the SH3 domains of proteins like Lyn, Fyn, Grb-2, v-Src, or Lck (77-86). The PI3K'Y/p120, or class m PI3K, does not contain an N-terminal p85-binding site (66,87). Instead, the p101 protein was reported to bind and activate PI3K'Yvia G protein-mediated seven transmembrane receptors (66). PI3K'Yactivity is important for anti-apoptotic signals in neutrophils (88). 9 Class IT PI3Ks consist of 0:, ~, and 'Y isoforms, are 170-210 kDa proteins that contain a C2 domain, and have an in vitro specificity for PI and PI(4)P (9,89). While the method of regulation remains unknown, these enzymes are reported to have Ca ++ dependent catalytic function, to associate with the trans-Golgi network (TGN), to be present in clathrin-coated vesicles (CCV), and to be the primary source of PI(3,4 )P2 production in cells (2,90-92). Class ill PI 3-kinases are homologues of S. cerevisiae Vps34p and phosphorylate exclusively PI (93). Regulation of the class ill enzymes is associated with serine/threonine protein kinase activity and their primary role appears to be in both endocytic and exocytic vesicle sorting (94,95). Phosphoinositide Binding Domains Phosphoinositides are able to mediate cellular signals through protein recognition domains. Selective binding of phosphoinositides is critical for the sequestration of proteins important in discreet cellular function and signaling events. To date, PIP binding proteins are divided into seven types that include the FYVE, PX, pleckstrin homology (PH), epsin N-terminal homology (ENTH), AP-180 N­terminal homology (ANTH), Tubby, and FERM domains (96). Other domains such 10 as the C 1 and C2 domains also interact with membrane lipids, but are not specific for phosphoinositides. One of two protein domains specific for PI(3)P is the FYVE domain. FYVE domains are typically 60-70 amino acids and are named after the first four proteins that contain this domain, Fab1p, YOTB, Vaclp, and EEA1 (97). Proteins with the FYVE domain localize to endosomal membranes to help regulate early endosomal and Golgi to lysosome/vacuole trafficking (7,98). The crystal structure of the EEAl FYVE domain contains two anti parallel P sheets and a membrane interaction loop held in position by two zinc-binding clusters with a small 0: helix packed against the second P sheet made up of the p3 and P4 strands (Figure 1.3) (97). A basic motif in the PI strand combined with the P2 strand make up the PI(3)P binding site (96). Based on the EEA1 structure, the FYVE domain makes only nine hydrogen bonds with the phosphates of the PI(3)P (99). Fewer bonds may account for the lower affinity (KD = 24flM) compared to other phosphoinositide binding domains. Hydrogen bonds were also found with the D-4, D-5, and D-6 hydroxyls that create the specificity for PI(3)P over other phosphorylated phosphoinositides (96). Although a membrane interaction loop does penetrate the membrane to moderately increase PI(3)P affinity, an N-terminal coiled-coil motif mediates dimerization of most FVYE domains resulting in high avidity binding (99). Other FYVE domains such as SARA and FENS 1 can efficiently be targeted to membranes without dimerization (96). A second domain specific for PI(3)P is the PX domain, although the PX domain of p47phox is reported to also have affinity for PI(3,4)P2 (100). PX domains 11 PX FYVE Figure 1.3. The structures of the PX and FYVE domains. The FYVE domains are a homodimer ofEEA1 proteins binding I(1,3)P2 (99). The PX domain is from the p40PHOX protein and shown bound to I(1,3)P2 (101). The protein database (PDB) files 1H6H and 1JDC were obtained from the National Center for Biotechnology Information (NCBI) Molecular Modeling Database (MMDB) and visualized using CD3D 4.1 software from NCBI (http://www.ncbi.nlm.nih.govD. are named after the first proteins found to have this domain, the p40phox and p47phox subunits ofNADPH oxidase (100). The size of the PX domain is about 140amino acids, and it is involved in lipid modification, vesicle trafficking, and protein sorting (96,100). The crystal structure of p40Phox shows a three-stranded ~ sheet adjacent to an a helical subdomain with the phosphoinositide binding cleft between the two (Figure 1.3) (101). Many PX domains also contain a C-terminal 12 SH3 domain, which in the case of p47Phox has no effect on enzyme activity but serves as a phosphorylation dependent regulator of phosphoinositide binding (102). As with the FYVE domain, the PX domain forms only nine hydrogen bonds between side chains and the phosphates ofPI(3)P (101). In addition to hydrogen bonding, a van der Waal' s contact is made between a conserved tyrosine side chain and the glycerol backbone of the phosphoinositide (103). PI(3)P binding is prerequisite for insertion of a hydrophobic loop into the membrane which stabilizes the membrane-protein complex (102). Although the few higher affinity PX domains are believed to independently target to PI(3)P containing membranes, most PX domains bind with a lower affinity and appear to play roles in multiprotein complexes (96). Examples of the low affinity PX domains are the nexins involved in endosomal targeting (104,105). It should also be noted that a few proteins with PX domains - e.g. p47phox , Bemlp, and class IT PI3Ka - preferentially bind PI(3,4)P2, PI(4)P, and PI(4,5)P2 respectively, indicating that binding variation exists in this domain (100,106-108). Another phosphoinositide binding motif is the ANTH domain. The ANTH domain is completely (l helical and binds phosphoinositides through surface contacts instead of a binding cleft, which may explain the promiscuity of this domain (96). Structure analyses show phosphoinositide binding by way of a similar basic motif that is described as holding the phosphoinositide head group like "a ball balanced on the fingertips" (Figure 1.4) (109,110). The predominate ligand of the ANTH domain is PI(4,5)P2 and the low affinity (KD = 5 JlM for AP180) is overcome by multivalent interactions as in the case of AP180/clathrin complexes (111). Other proteins with this domain include AP2-a and clathrin assembly lymphoid myeloid (CALM) ANTH ENTH Figure 1.4. The ANTH and ENTH domains. The ANTH domain is from the CALM protein and is shown bound to I(I,4,5)P3 (110). The ENTH domain is also bound to I(1,4,5)P3 and is from the epsin protein (115). The PDB files 1HG2 and 1HOA were obtained from the NCBI MMDB and visualized using CD3D 4.1 software from NCBI (http://www.ncbi.nlm.nih.govD. leukemia protein, all of which are implicated in receptor mediated endocytosis (110,112,113). The ENTH domain binds specifically to PIC 4,5)P2. ENTH domains are structurally homologous but distinct from ANTH domains and have a high affinity 13 for PI(4,5)P2 that facilitates independent targeting to membranes (114). The general structure is a superhelix of seven helices with another askew helix (Figure 1.4). However, the crystal structure of the epsin ENTH domain revealed an entirely (l helical domain containing eight (l helices, with a ninth helix that formed at the N-terminus upon binding PIC 4,5)P2 (114-116). The formation of this new helix creates a 14 deep basic groove by folding back on helix 1 and the loop between helix 1 and helix 2 (114). The ENTH domain is able to convert liposomes containing 10% phosphoinositides into tubules (114). The formation of the amphipathic ninth a helix upon PIC 4,5)P2 binding induces membrane curvature important in clathrin polymerization and endocytosis (114). It is interesting to observe that all phosphoinositide recognition domains that penetrate mernbranes play roles in vesiculation (96). Tubby, TULP 1, TULP2, and TULP3 make up a homologous family of protein that contain the characteristic "tubby" domain. The tubby domain is made up of about 260 amino acids that form a helix-filled barrel structure (117). Four additional a-helices exist on the outside of the ~-barrel and the phosphoinositide-binding pocket is between the ~-barrel and helix 6A (Figure 1.5) (117). Despite in vitro binding data showing affinity for PI(3,4)P2 and PI(3,4,5)P3, the functional ligand in vivo is reported to be PIC 4,5)P2 (117). All members of the tubby family are believed to bind similarl y due to conserved residues that form hydrogen bonds with the phosphoinositide head group (117). The tubby domain serves to sequester these transcription factors at the plasma membrane until activated G protein-coupled receptors stimulate PLC-~ mediating hydrolysis of PI(4,5)P2 and releasing the I( 1 ,4,5)P3-bound tubby domain into the cytosol (117). Once in the cytosol, the tubby proteins are localized to the nucleus where they regulate gene transcription. The PERM domain derives its name from 4.1 plus ERM, where ERM represents the ezrinlradixinlmoesin family of proteins (118). The crystal structure of radixin shows the PERM domain actually consists of three subdomains with the IS tubby FERM Figure 1.5. The tubby and PERM domains. The tubby domain is shown bound to I( 1 ,4,S)P3 from the tubby protein (117). The PERM domain, also shown bound to I(1,4,S)P3, is from the radixin protein (118). The PDB files 1I7E and 1GC6 were obtained from the NCBI MMDB and visualized using CD3D 4.1 software from NCBI (http://www.ncbi.nlm.nih.govl). PI(4,S)P2 binding site located between the A and C subdomains (Figure 1.S) (118). A positively charged cleft is formed between the C-terminal a helix of the C subdomain and the loops of the ~3 and ~4 strands of subdomain A (118). The overall structure resembles that of the pleckstrin homology domain to be described later. However the phosphoinositide binding sites are quite distinct from one another (118). PI(4,S)P2 binding induces structural changes in radixin that are believed to promote additional protein-protein interactions (118). The promiscuity in phosphoinositide specificity of the PERM domain is explained by fewer hydrogen bonds formed with the D- 4phosphate (96). The ERM proteins function to couple actin filaments to the plasma membrane in structures such as microvilli, membrane ruffles, and cell-adhesion sites via binding both the FERM domain and a C-terrninal domain that binds F-actin (96,118). PERM domains may also exist in nonreceptor tyrosine kinases based 16 onsequence analysis (119,120). The PH domain superfamily is the largest of all phosphoinositide-binding domain-containing proteins with over 250 recognized in the human proteome (96), The main determinant in the PH domain super family is the structure rather than the primary sequence, demonstrating stability of this protein scaffold (121), Three categories of PH domains including the PTB, Ran-binding, and EVH 1 domains are all structurally related, but have no phosphoinositide binding ability and will not be reviewed here (121), The Pleckstrin Homology domain, first identified in 1993, is a ~-sandwich motif with two antiparallel ~-sheets held together by a C-terminal a-helix containing about 120 amino acids (Figure 1.6) (122-124), Of the four resulting four comers, two are closed, one contains the a-helix, and the fourth open comer containing three interstrand loops is the phosphoinositide-binding site (96,125), The entire domain is electrostatically polarized with the negative charge at the C-terminal and the positive charge at the binding cleft (121). Despite the high sequence homology, there are three loops (~1/~2, ~3/~4, and ~6'/~7) that are variable and in certain proteins contain SH2, SH3, or even BDZ domains (126-131). The PH domains that exhibit the strongest univalent phosphoinositide binding known for PI(4,5)P2, PI(3,4)P2 and PI(3,4,5)P3 enable direct membrane localization in response to stimuli (96,121). The group of PI(3,4,5)P3 specific PH domains consists of about 20 proteins and have the highest phosphoinositide affinity (~ = 27 nM for I(1,3,4,5)P4) (128). A basic sequence motif between the ~1 and ~2 strand maximizes hydrogen bonding between the protein and the phosphoinositide head 17 PH Figure 1.6. The PH domain structure of Grpl bound to I(1,3,4,5)P4 (132). The PDB file IFHX was obtained from the NCBI MMDB and visualized using CD3D 4.1 software from NCB! (http://www.ncbi.nlm.nih.govD. group forming 17-19 bonds (132). The PI(3,4 )P2 binding PH domains are similar to the PI(3,4,5)Pg binding domains with more hydrogen bonds made with the D-4 phosphate and fewer with the D-5 phosphate (132). The PI(3,4)P2 specific PH domains still have comparable affinity for the preferred ligand despite the loss of hydrogen bonds. For example, the Kct of the dual adaptor for phosphotyrosine and 3- phosphoinositides (DAPPl) PH domain is 43 nM for I(1,3,4)P3, (133). The PH domain ofPLC~l and related proteins have high affinity PI(4,5)P2 binding (134). PLC~l binding ofPI(4,5)P2 forms only 12 hydrogen bonds (11 of which directly bind the D-4 and D-5 phosphates) and the basic sequence of the ~ 1/~2 loop is reduced to one lysine residue, which may explain the relatively weaker affinity (~ = 210 nM for 18 I(I,4,5)P3) (133,135). Most PH domains have similar electrostatic polarization, are promiscuous, and exhibit weak affinities for phosphoinositides (133). Although many domains do not have established functional importance, for some, the importance appears to be in the ability to undergo signal dependent oligomerization to increase the avidity for membranes (128). An example of this is the GTPase, Dynamin-l, required for endocytic vesicle scission from the plasma membrane (136,137). Monomeric Dynamin-l binds I( 1,4,5)P3 with a ~ of 1.4 mM to 4 mM, but increases to 9 J..LM upon dimerization (138-140). An additional group of proteins is associated with PIC 4,5)P2. The MARCKS (myristoylated alanine-rich C kinase substrate) proteins, CAP23, and GAP43 are all anchored to the membrane and contain basic motifs with a high affinity for PI(4,5)P2 (141). There is very little headgroup specificity and the proteins are believed to regulate the distribution and availability of phospho in os it ides (141,142). Actin­binding proteins such as WASP (Wiskott-Aldrich syndrome protein), cofilin, gelsolin, and profilin also bind PIC 4,5)P2 through short sequences of basic residues (143,144). Signal Transduction of Phosphoinositides PI(3)PIPI(3,5)P2 Signaling PI(3)P, and PI(3,5)P2 appear to have roles in directing anterograde and retrograde vesicle trafficking from the late Goigi to the vacuolellysosome. Studies of carboxypeptidase Y delivery to the vacuole in yeast demonstrated how PI(3)P directs anterograde movement (95). In yeast, there are more than 40 gene products involved 19 in delivery from the late Golgi to the early endosome or vacuole (145-148). One of these products, Vps (vacuolar protein sorting) 34, is a wortmannin sensitive PI3K specific for PI (93,149,150). When another gene product is activated, the membrane associated serine/threonine kinase Vpsl5p, Vps34p is recruited to the late Golgi and activated to form PI(3)P on the cytosolic leaflet (94,151,152). PI(3)P directs vesicles leaving the Golgi toward the vacuole/lysosome through the binding of effector proteins such as the FYVE domain containing EEAl(95). EEA1 and Rab5 facilitate the fusion of vesicles with the early endosome (153). The FYVE domain of V ps27p binds PI(3)P and facilitates the formation of vesicular invaginations leading to multivesicular bodies with PI(3)P on the inner leaflet (154,155). The multivesicularbodies are transported to the vacuole/lysosome where the t-SNARE Vam7p binds PI(3)P via a PX domain and facilitates fusion with the vacuole/lysosome (103). Internalized by the vacuolellysosome, the multivesicular bodies are degraded by phosphatases and hydrolases (95). Fab 1 p converts the PI(3)P that remains on the surface of the endosome and vacuole/lysosome in response to Vac 1 p activation (95,156). PI(3,5)P2 is required for invagination and formation of multi vesicular bodies in the endosome and for retrograde vesicular transport from the vacuolellysosome to earlier compartments (157,158). Termination of the PI(3,5)P2 signal has been linked to the phosphatase Fig4 (156). Currently no PI(3,5)P2 binding proteins have been identified (2,156). PIC 4,5)P2 Signaling PIC 4)P as a precursor, and PIC 4,5)P2 are second messengers in secretory vesicles, calcium signaling, cytoskeletal rearrangement, and vesicular trafficking. 20 The PH domain ofPLC binds specifically to PI(4,5)P2 to localize it to the membrane (21). In response to G-protein coupled receptor and receptor tyrosine kinase activation, PLC is activated and hydrolyzes PI(4)P and PI(4,5)P2 to form I(1,4)P2, I(1,4,5)P3), and DAG in an isozyme dependent manner (2,25,159-161). Inositol triphosphate (IP3) negatively regulates PLC and binds to the IP3 receptors in the ER, the outer membrane of the nuclear envelope, secretory granules, and the plasma membrane resulting in a biphasic release of intracellular calcium (162,163). Increases in DAG and Ca ++ concentrations result in the activation and translocation of members of the protein kinase C (PKC) family to the plasma and other cellular membranes (20,164,165). Phospholipase D (PLD) is activated by both protein­protein interactions with PKC and upon binding PI(3,4,5)P3 and PIC 4,5)P2 with PH and PX domains (164-166). Although this is not the historical activation pathway, members of the ARF and Rho families can also activate PLD (164,167). Activated PLD hydrolyzes phophatidylcholine to form free choline and PA, which is known to stimulate PI(4)P 5-kinase activity (44,168,169). PLD has two isoforms that are differentially located. PLD1 is found on intracellular membranes and PLD2 is mainly found at the plasma membrane (166,170). One of the most potent inhibitors ofPLD is the phosphatase synaptojanin (171-173). The formation of PIC 4)P is an important step in the release of secretory and presynaptic vesicles. PIC 4)P 5-kinase activity was found in membrane fractions of PC12 and PI(4,5)P2 formation was shown to be important for the priming step necessary for Ca ++ induced exocytosis (174,175). Also implicated in the priming step of exocytosis is phosphatidylinositol transfer protein (PITP) (176). Synaptotagmin 21 and dynamin bind PI(4,5)P2 in neurotransmitter release and the concomitant membrane internalization events in presynaptic membranes (177). Synaptojanin, a type IT 5-phosphatase, associates with amphiphysin to localize on synaptic vesicles (171). Phosphatidylinositol4-kinase is believed to oppose the phosphatase action of synaptojanin in synaptic vesicle transport (10). Calcium regulated exocytosis is mediated by ARF6, ARNO, and PLOI. ARF6, a member of the ARF family GTPases, has been linked to cell motility, vesicle recycling, Fc-mediated phagocytosis, insulin-regulated secretion, and Glut-4 translocation (178-183). Associated with secretory granules at the plasma membrane, ARF6 is activated by ARNO when translocated to the membrane in response to PI(3,4,5)P3 formation (184,185). ARF6 then activates PLOI to promote membrane curvature through the formation ofPA and PI(4)P 5-kinase to form PI(4,5)P2 necessary for the rearrangement of the actin cytoskeleton to enable vesicle fusion (184,186-190). PI4KlIa is also shown to associate with synaptic vesicles and is believed to playa primary role in producing the PI(4)P necessary for PI(4,5)P2 synthesis (10). Synaptotagmin binds PIC 4,5)P2 in a Ca ++ -dependent manner to regulate SNARE complexes in the fusion event of synaptic vesicles and plasma membrane in neurotransmitter release (191-193). Phosphoinositides play many important roles in the regulation of the actin cytoskeleton. The Rho family of GTPases governs the assembly of actin filaments (f­actin) in response to extracellular stimuli (4). Specifically, RhoA regulates assembly of stress fibers, Rac controls formation of lamellipodia, and Cdc42 directs the formation of filopodia and microspikes (4). ~uring chemotaxis Rac and Cdc42 22 stimulate actin polymerization at the leading edge, while RhoA controls the retraction at the lagging edge (4). Activated Rac interacts with the PI(4)P 5-kinase to increase PI(4,5)P2 Ievels (60). In a resting state, actin monomers are sequestered in the cytosol by profilin (194). Profilin releases the actin monomers upon binding PI(4,5)P2• Although studies have also shown that profilin binds PI(3,4)P2 and PI(3,4,5)P3 indicating a role ofPI3K in actin cytoskeletal rearrangement (4,195). The release of actin increases the concentration of monomeric actin and promotes polymerization (195). Actin filaments can grow from both ends; however, the barbed end polymerizes faster than the pointed end (2). Gelsolin and CapZ proteins bind the barbed end of actin filaments to inhibit further polymerization (196). CapZ binding of PIC 4,5)P2 and gelsolin binding of PI(3,4,5)P3 and PI(3,4)P2 result in the uncapping of f-actin to increase polymerization (60,197). Gelsolin and profilin are also reported to stimulate PI3K activity (198). Additionally, activation ofPI(4)P 5-kinase induces actin polymerization while synaptojanin inhibits it (4,194). The mechanism of Cdc42-mediated actin polymerization has been well characterized. N-WASP, upon binding both PI(4,5)P2 and activated Cdc42, undergoes a conforIl?ational change to expose a verprolin homology region, cofilin­like region, and acidic region (VCA) (144). The VCA region recruits and activates the Arp2/3 complex to facilitate de novo actin nucleation and polymerization (144). The N-WASP-Arp2/3 complex system is also implicated in endocytosis. Actin tails have been observed attached to endosomes, pinosomes, and CCV s (199- 201). Syndapin is shown to associate with synaptojanin, dynamin I, synapsin I, and N-WASP linking the actin cytoskeleton to endocytosis (202-204). The Arp2/3 complex has also been shown through genetic analysis to be important for endocytosis at the plasma membrane (205,206). PIC 4,5)P2 actin filament assembly has also been linked to focal adhesions. 23 Proteins such as vinculin, a-actinin, and talin connect the extracellular matrix to the cytoskeleton. Binding of PIC 4,5)P2 regulates the function of these proteins in focal adhesion formation (207-210). Actin polymerization and cross-linking is enhanced by a-actinin binding PI(4,5)P2 (209). PI(4,5)P2 also enhances the f-actin affinity of vinculin (207). The movement of proteins from the endoplasmic reticulum (ER), where they are synthesized, to the location of function requires directed movement through exocytic and endocytic pathways. Vesicular coat proteins mediate vesicle formation in these pathways (211,212). Three types of coat proteins include coat protein I (COP 1), coat protein II (COP II), and clathrin (213). Vesicles are coated with COPI and COPII proteins to mediate trafficking within the ER and Golgi apparatus, whereas beginning at the TGN, clathrin-coated vesicles are formed (212,214). COP II coated vesicles involved in anterograde flow from the ER to an intermediate compartment between the Golgi and ER where the coats are replaced with a COP I coat (215). Vesicles coated with COP I are found in the intra-Golgi network (212). The recruitment of the COP I and COP II coat proteins is directed by small GTP­binding proteins, members of the ADP-ribosylation factor (ARP) family of Golgi­associated GTPases, and Sarlp respectively (212,214). A protein complex of Sarlp, Sec13/31p, and the GAP Sec 23/24p binding to PI(4)P and PI(4,5)P2 in the ER 24 membrane mediates COPII coating (216). In COPI formation, activation of the ARF members is dependent upon the corresponding guanine nucleotide exchange factors (GEFs) such as ARNO, also known by the mouse homolog general receptor for 3- phosphoinositides (Grp1), and cytohesin (217). ARNO, but not cytohesin, is activated in a Pl3K dependent manner by binding PI(3,4,5)P3 (218). Interestingly, one of the COPl subunits a-COP, has also demonstrated affinity for PI(3,4,5)P3 (219). ARF activation, however, has also been reported with other PI(4,5)P2 dependent GTPas activating proteins (GAPs) (220,221). PI( 4,5)P2 is an important regulator of endocytosis which serves to internalize both membrane receptors with bound ligands and budding vesicles from the TGN (212,222). Clathrin coated pits, the initial process of clathrin-mediated endocytosis, form by proteins such as epsin, CALM, and AP180 binding to PI(4,5)P2 in the plasma membrane and inducing curvature (110,111,222,223). Clathrin binds to these proteins and forms a lattice around the pits (11 0). ~-arrestin and the ANTH domain containing adapter protein 2 (AP-2) bind both phosphoinositides and the structural proteins of the clathrin-coated pits to sequester receptors and transmembrane proteins for endocytosis (4,224,225). Amphiphysin I and II also recruit the GTPase dynamin to clathrin-coated pits (226). The PH domain binding PI( 4,5)P2 stimulates the GTPase activity of dynamin, which activates endophilin to convert lysophosphatidic acid (LPA) to PA. Formation ofPA is important for vesicle scission and the formation of CCVs (138,139,227). PI(4,5)P2levels, important for CCV formation, are maintained through PLD inhibition by AP 180, amphiphysin I and II, and synaptojanin (222,226,228,229). After the formation of CCV s, synaptojanin binds 25 clathrin and AP-2, and hydrolyzes PI(4,5)P2 to PI(4)P resulting in the disassembly of the clathrin lattice (230). The uncoated vesicles move to the endosome where they are sorted and return to the plasma membrane or continue to the lysosome for destruction (4). Inositol phosphates are also implicated in signal transduction, although little is currently known about their specific roles. 1(1,4,5)P3, formed by PLC activation and responsible for Ca++ release, is metabolized by 1(1,4,5)P3 3-kinase to 1(1,3,4,5)P4, which also aids in Ca++ release into cells upon binding the receptors GAplP4BP and GAPm located at the plasma membrane and ER respectively (231-236). 1(2,4,5)P3 is also reported to augment 1(1,3,4,5)P2, although no known metabolic route produces this metabolite (237). The 1(1,4,5)P31I(1,3,4,5)P4 signal is terminated by type 1 phosphatases (238). The metabolite of 1(1 ,4,5)P31I( 1 ,3,4,5)P 4 5-phosptase, 1(1,3,4)P3, is shown to inhibit 1(3,4,5,6)P41-kinase, which links PLC activation with observations of concurrent increases in 1(3,4,5,6)P4 Ievels (239,240). 1(3,4,5,6)P4 is reported to inhibit Ca++-dependent cr release by the Clca cr channel (241). Inositol hexaphosphate (IP 6) is also observed to coat cellular membranes and interact with vinculin, coatomer, and synaptotagmin suggesting a role in endocytosis (242-246). IP6 also inhibits AktINFKB signaling and stimulates apoptosis in tumor cells (247). The diphophorylated inositol phosphates [PPh-IP4 and PP-IPs are linked to cAMP­dependent signaling and Ca ++ release respectively (248,249). PI(3,4 )P2IPI(3,4,5)P3 Signaling PI(3,4 )P2 is usually associated with PI(3,4,5)P3 signaling. SHIP is generally thought to act on PI(3,4,5)P3 to form PI(3,4)P2 after PI3K activation (2,44). 26 However, PKC mediated PI(3) 4-kinase stimulation to form PI(3,4)P2 is observed in platelets. PI(3,4)P2 is a stimulus for membrane localization and activation of PKCo, PKCe, PKC~, and AktlProtein Kinase B (PKB) (27,28,250-254) DAPP1 is also shown to bind both PI(3,4)P2 and PI(3,4,5)P3 (132). PI(3,4,5)P3 is a second messenger in the regulation of actin cytoskeleton and cell motility, insulin signaling, glucose transport, cell growth, cell proliferation, and cell death (2). Members of the RaslRho family of small GTPases are activated downstream of PI3K (2). Specifically, Vav is activated upon binding PI(3,4,5)P3 and stimulates the guanine nucleotide exchange of Rac, RhoA, and Cdc42, all of which are important proteins in cytoskeletal remodeling (255,256). In contrast, binding PIC 4,5)P2 inhibits Vav activity (2). Cellular treatment of Ras results in membrane ruffling and both PI(3,4)P2 and PI(3,4,5)P3 cause stimulated cell motility (257,258). Additionally, both PI(3,4)P2 and PI(3,4,5)P3, as well as PI(4,5)P2, initiate uncapping of actin filaments (197). The process of insulin signaling and glucose uptake is PI3K dependent. An essential step for insulin stimulated glucose uptake is translocation of the Glut4 transporter to the cell membrane is inhibited by both SHIP expression and the PI3K inhibitor wortmannin (259,260). Inhibition of membrane translocation is reversed by treatment with endogenous PI(3,4,5)P3 (261). Genetic studies have validated these observations to directly link PI3K and Akt activity to gluconeogenesis and glycolysis (2,262). PI3K regulates cell growth, proliferation, and death. PI(3,4,5)P3 levels in resting cells are almost nonexistent until stimulation with anyone of many ligands, 27 such as growth factors and hormones (2). PI3K activity is required for cell cycle progression from Gl to S phase (263,264). Interestingly, two peaks of PI3K activity are observed in response to platelet-derived growth factor (PDGF) stimulation. The first peak appears about 15 minute (min) poststimulation, while the second and equal peak: occurs 3 to 5 hour (hr) poststimulation. The second peak is necessary for cell cycle progression (265). One of the most studied pathways of PI3K effects on growth, proliferation, and death is through the Akt signaling pathways reviewed in chapter 2. However, PI(3,4,5)P3 also activates members of the PKC family through both direct binding of the lipid and PI(3,4,5)P3 mediated phosphorylation by phosphoinositol-dependent kinase 1 (PDKl) (252,266-269). PKC~ activity leads to activation of the mitogen activated protein kinase (MAPK) pathway (270,271). The PI3K signal is abrogated by both PTEN and SHIP (18,272). Conclusion The study of signal transduction would not be complete without the understanding of phosphoinositides and their regulatory proteins. 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Activation of this 57 -kDa cytosolic serine/threonine kinase plays central role in cell survival, proliferation, angiogenesis, and growth (2,3). However, aberrant signaling is implicated in many types of cancer and diabetes (4-6). Akt Structure and Activation Akt expression is seen in a broad range of tissues. The three human isoforms are identified as AktlIPKBcx, Akt2IPKB~, and Akt3IPKByand contain 81 and 83% amino acid identity respectively (7,8). Two splice variants, Akt~l/PKB~l, and Akty1lPKBy1, of Akt also exist (7). The Akt~lIPKB~l splice variant contains a C­terminal 40 amino acid extension and the Akty1IPKByl splice variant contains a C­terminal truncation (9,10). All forms of Akt contain an N-terrninal PH domain of 100 amino acids that 47 specifically binds PI(3,4)P2 and PI(3,4,S)P3, a central kinase domain, and a C­tenninal hydrophobic or regulatory domain (8,11). The Akt PH domain is somewhat different from other PH domains with similar specificity (12). First, the protein is rotated 45° in the plane of the inositol ring as compared to structures of Bruton's tyrosine kinase (BTK), DAPP1, and Grpl. Second, the inositol ring sits 4 A deeper in the domain, which results in a difference in the number and type of bonds formed (12). Structure analysis verifies the importance of protein contacts with the D-3 and D-4 phosphates for specificity, although there are fewer total protein-ligand interactions explaining the observed lower affinity compared to similar PH domains (12). Akt belongs to the AGC family of protein kinases that share sequence similarity in the catalytic domain (11). Part of the catalytic domain is an activation T­loop whose phosphorylation state regulates the activity of the kinase (11). Residue Thr308 of Akt is the site of phosphorylation within the T-Ioop and is phosphorylated by PDK1 in a PI(3,4,S)P3-dependent manner (S,13-15). The catalytic domain contains an additional phosphorylation site at Ser-124 that is basally phosphorylated, and not required for kinase activity (16). Thr308 phosphorylation alone causes only partial activation (16). The active site of Akt phosphorylates substrates with the consensus sequence of R-X-R-X-X-Srr-bulky hydrophobic (S). Characteristic of the AGC kinases, the C-terrninal regulatory or hydrophobic motif (HM) (F-X-X-F-Srr-F) serves as a docking motif to enable phosphorylation by PDK1 (IS). Binding of PDK1 to the HM requires phosphorylation of Ser473 to bring the active site of PDK1 into proximity with Thr308 of Akt, and increases the catalytic 48 activity of PDKI (11,15). Phosphorylation of Ser473 in the C-terminal hydrophobic or regulatory motif is necessary for complete Akt activation, although the kinase responsible has yet to be determined (11). Evidence suggests kinases such as PDKl, PRK-2, Integrin-linked kinase (ILK), or a yet unidentified PDK2 may phosphorylate Ser473, (17-20). Another hypothesis includes a mechanism of autophosphorylation (21). Thr-450 also appears to be basally phosphorylated and not required for kinase activity (16), Akt activation is a multistep process. The PI3K1 Akt signaling pathway is activated in response to over 43 known activators including growth factors and hormones (4,5). Growth factors bind the cognate receptor to induce a conformational change activating the receptor tyrosine kinase activity of the cytosolic domain of the receptor. The phosphorylation of tyrosine residues of the cytosolic domain of the receptor recruits adaptor proteins via SH2 domains that in tum recruit and activate PI3K at the plasma membrane. PI3K can also be activated in a ras-dependent manner. Seven transmelubrane receptors also activate PI3K in response to ligand binding. Activated PI3K forms PI(3,4,5)P3 from PIC 4,5)P2, which is then partially converted to PI(3,4)P2 by SHIP (4,5,22). Although the PI3K pathway is the primary route, Akt activation is also reported in response to activated Rac 1, heat shock, increases in intracellular levels of Ca2 +, and cAMP (23-28). Akt is recruited to the inner leaflet of the cell membrane via the N -terminal PH domain, which has an affinity for both PI(3,4)Pz and PI(3,4,5)P3 (5). Research in SHIP-1 - cells indicates that PI(3,4 )P2 is required for Akt activation, but that PI(3,4,5)P3 is also necessary to colocalize PDKI (22). Upon binding PI(3,4)P2, Akt undergoes a conformational 49 change that induces the formation of homomultimers (29-31). Ser473 is phosphorylated and the HM of Akt binds PDK1 that has also localized to the plasma membrane in response to the phosphoinositide formation (Figure 2.1) (11). PDK1 contains a PH domain specific for PI(3,4,5)P3 (5). The catalytic site of PDK1 is brought into proximity of Thr308, which is subsequently phosphorylated (11). Phosphorylation of Akt Thr308 induces a conformational change that reduces the affinity of the.HM for PDK1 and increases the affinity of the HM for the phosphate­binding pocket of the Akt kinase domain (11). Akt is now fully activated and translocates away from PDK1 and the plasma membrane to the cytosol to phosphorylate downstream substrates (5,11,32). Observations that PTEN expression alters Akt Ser473 and not Thr308 phosphorylation support this mechanism of activation (33). Akt has several modes of inactivation. Direct inactivation is seen through dephosphorylation by protein phosphatase 2A (PP2A). PP2A is antagonized by the 90-kDa heat-shock protein (Hsp90), which stabilizes Akt in an active conformation (34,35). Akt activity is also reduced by the lipid phosphatase PTEN, which converts PI(3,4 )P2 and PI(3,4,5)P3 to PIC 4)P and PIC 4,5)P2 (36,37). PKC negatively regulates Akt activity (38). The carboxy-terminal modulator protein (CMTP) is shown to bind Akt and reduce Ser-472 phosphorylation (39). Akt Signaling Activated Akt phosphorylates cytosolic and nuclear substrates in the cell (Figure 2.2). Akt regulates cell cycle progression by modulating the activity of proteins such as glycogen synthase kinase 3 (GSK-3), cyclin Dl, p21 Cipl, and p27Kipl Plasma Membrane A. ,/" Inactive Akt ~E. Active Akt c. " 50 Figure 2.1. The process of Akt activation. (A) Akt in resting state in the cytosol. (B) Akt is recruited to the membrane upon formation of PIP3 and Ser473 is phosphorylated. (C) PDKI is localized to the plasma membrane in response to PIP3 formation. (D) Akt and PDKI colocalize at the plasma membrane where PDKI phosphorylates Akt at Thr308. (D) The Akt HM dissociates from PDKI and binds to the Akt kinase domain producing a fully active kinase that phosphorylates downstream substrates (11). (40). GSK-3 inhibits the activity of proteins such as glycogen synthase, cyclin D, p21Cipt, and c-Myc (6). GSK-3-dependent phosphorylation of cyclin D and p21Cipi targets these proteins for proteosome-mediated degradation (40). Inhibition of GSK- 3 by Akt through phosphorylation maintains proper levels of cyclin D and p21 Cipl that are important for cell cycle progression in the Gl/S phase transition (4). GSK-3 mediated activation of ~-catenil1 is also inhibited by Akt resulting in ~-catenin 51 Plasma Membrane -+ PIP2 + PIP3 t Figure 2.2. The Akt signaling pathway. PI3K is activated in response to receptor tyrosine kinase (RTK) activation, or other agonist such as G-coupled receptors not shown. Akt is activated in response to PI(3,4)P2 and PI(3,4,5)P3 formed by PI3K activation and regulates proteins in the cell through phosphorylation (11). 52 remaining in the cytosol where it·is unable to initiate transcription of apoptotic genes (40). Akt signaling promotes proliferative effects by controlling the activity of transcriptions factors such as p21 Cipl and p27Kipl • The transcription factor p21 Cipl normally induces cyclin-dependent kinase (Cdk)4/cyclin D and Cdk6/cyclin D activity in the early G1 through the middle of S phase and then inhibits Cdk2/cyclin A and Cdk2/cyclin E from late G 1 through S phase (41-43). A complex of p21 Cipl, a cyclin, a Cdk, the proliferating cell nuclear antigen inhibits DNA polo holoenzyme activity and is disrupted by Akt-dependent phosphorylation of p21 Cipl resulting in DNA synthesis (44-47). Although it may be cell type specific, Akt phosphorylates p27KiPl to keep it from translocating to the nucleus where it inhibits Cdk2 and is associated with cell cycle arrest (48-50). Interestingly, Akt inhibits RaflMEKlERK signaling, also important in cell growth, proliferation, and apoptosis, through phosphorylation of Raf (40). Phosphorylated Rafis then sequestered in the cytosol 14-3-3 proteins (7,51). Unphosphorylated Raf increases levels of p21 Cipl and decreases p27Kip1 levels associated with G1 progression (40). NF-KB is another downstream effector of Akt. Phosphorylation by Atk activates IKK, which subsequently phosphorylates I-KB bound to NF-KB in the cytosol (52). Phosphorylation of I-KB targets it for proteosome degradation resulting in the release of NF-KB to translocates to the nucleus and initiate transcription of anti­apoptotic genes such as Bcl-XL and inhibitor-of-apoptosis 1 and 2 (5,52-55). Another substrates of Akt includes Cot, a member of the MAP3K family, that then also 53 phosphorylates the IKB kinases (IKK) to release the transcription factor NF-KB (53). Akt plays further roles in cell cycle control and initiating protein synthesis. Akt activates mammalian target of rapamycin (mTOR), which then phosphorylates and activates p70 S6 kinase (p70S6K) (56). mTOR inhibits by phosphorylation 4E binding protein 1 (4E-BPl) to allow protein translation to proceed (57). Phosphorylation of 4E-BPl results in dissociation from eIF-4E allowing eIF-4E to associate with the p220 subunit of the mRNA CAP binding protein complex (58). p70S6K activates the transcription factor E2F, important for expression of genes important in cell cycle regulation in the G1 phase (59,60). The ribosomal protein S6 is also phosphorylated by p70S6K to activate 5 'TOP RNA translation for biogenesis of translational components (61). Akt also stimulates the transcriptions factor CREB to initiate transcription of the antiapoptotic protein Mcl-l (53). Additional anti-apoptotic targets of Akt include mitogen-activated protein kinase-I, apoptosis signal-regulating kinase-I, and the murine double min-2 (MDM2) protein that is shown to promote degradation of the tumor suppressor p53 (62-65). Akt mediates cellular survival through inhibition of apoptosis. The pro­apoptotic proteins BAD and Bax contribute to the formation of the apoptosome by stimulating the release of cytochrome C from the mitochondria (5,66,67). To inhibit apoptosis, Akt phosphorylates BAD at Ser-136 causing dissociation from the anti­apoptotic protein Bcl-X (67). Bcl-X is then free to bind and inhibit Bax while BAD is sequestered in the cytosol by 14-3-3 proteins (5,67). A second way Akt directly inhibits apoptosis is through inhibiting caspases 9 from combining with Apaf-l and 54 cytochrome c to form the apoptosome (66,68). Members of the forkhead box factor (FOX) family of transcription factors that initiate transcription of proapoptotic genes such as Fas are also inactivated by Akt phosphorylation (69-71), Akt phosphorylation mediates nuclear export and sequestering in the cytosol by 14-3-3 proteins of FOX transcription factors (7,8,72). Akt promotes angiogenesis in response to vascular endothelial growth factor (VEGF) released from tumor cells (7,73). Endothelial nitric-oxide synthase (eNOS) is activated by Akt to produce nitric oxide (NO), which regulates vascular tone, arterial pressure, platelet and leukocyte adhesion to endothelial surface, and vascular smooth muscle proliferation (2,73). Akt and Disease Akt plays an important role in both insulin signaling and protection from diabetes. Both Akt 1, predominant in skeletal muscle, and Akt2, predominant in adipocytes, are essential for glucose uptake into the cell (74). As previously reported, Akt inhibits GSK-3 activity to prevent inhibition of glycogen synthase, important in glucose storage (74). Insulin stimulation activates PI3K and subsequently Akt, which promotes the translation of the insulin-regulated glucose transporter, GLUT-4 containing vesicles to the plasma membrane and initiates glucose uptake (74). Supporting evidence includes observations that mice lacking Akt2 demonstrate impaired insulin sensitivity, illustrating a necessary role in normal glucose metabolism (75). Furthermore, overexpression of Aktl in mouse ~-cel1s led to improved glucose tolerance and resistance to chemically induced diabetes mellitus ,,~ 55 (76). Reduction in glucose transport stimulation is linked to decreased Akt activity (77). Akt is implicated in many types of cancer, which is not surprising due to the many proliferative effects of activation. Cells with elevated Akt activity are less sensitive to apoptotic signals and Akt upregulation is associated with chemotherapeutic resistance (8,78,79). Akt is implicated in gastric, thyroid, ovarian, breast, prostate, and pancreatic cancers with ties to glioblastoma, endometrial, hepatocellular, melanoma, lung, renal-cell, and lymphoid cancers (4,8). "'" ", '~ Current evidence also suggests Akt promotes invasiveness. Akt activity has been li~ to increased matrix metalloproteinase production, suppression of matrix \ \ detachment-in~uced apoptosis, and activation of sphingosine-l-phosphate-induced chemotaxis (62~~O-82). Akt also stimulates angiogenesis, providing necessary blood to a tumor (7). Overexpression of Akt2 resulted in increased invasiveness and metastasis in human ovarian and breast cancer cells (83). Although Akt activity is directly linked to cancer, many connections are a consequence of other cellular abnormalities. Gene amplification of PI3K occurs in ovarian and colon cancer, while overexpression of Akt is seen in ovarian, breast, and colon cancer (4). Additionally, overexpression of the HER21neu receptor in breast cancer cells leads to increased Akt activation and Akt-dependent induction of the breast cancer susceptibility gene, BRCAI (84,85). The oncogene TCLI binds Akt and initiates dimerization, which results in activation (86,87). Truncations and mutations of proteins such as PI3K, epidermal growth factor receptor (EGFR), MET receptor, and the tumor suppressors, SHIP, also lead to Akt activation (4,88,89). 56 Mutations in PTEN, another tumor suppressor, are common to many types of cancers (90). The Hepatitis B virus X also down regulates PTEN expression to increase Akt activity (91). IL-4 abnormally activates Akt in prostate cancer cells via the androgen receptor (92). Cyclooxygenase-2 is overexpressed in liver cancer cells and caveolin- 1 is abnormally expressed in multiple myloma cells, both resulting in Akt activation (93,94). All of these cellular abnormalities result in excessive Akt signaling. Constitutive Akt activation results in cells that lose failsafe mechanisms that initiate apoptosis and senescence in normal cells as a result of nutrient and growth factor withdrawal (3,95). Conclusion The serine/threonine kinase, Akt, is important for normal cell growth, proliferation, and survival. PI3K-dependent formation of in response to Growth factors, cytokines, and hormones stimulate PI3K to form phosphoinositides (PI(3,4,5)P3, and PI(3,4)P2) at the cell membrane that results in Akt signaling (6). Akt activation results in direct and indirect inhibition of apoptosis, stimulation of protein synthesis, signals to progress in the cell cycle, initiation of glucose transport, and transcription of genes involved in these processes (40). 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CHAPTER 3 THE SPECIFIC INHIBITION OF AKT Introduction During cell stimulation, hormones and growth factors bind to the appropriate receptor on the cell membrane. The binding of receptors initiates conformational changes resulting in activation of intracellular complexes on the inner leaflet of the cell membrane. PI3K is activated by direct binding of the receptor, or through adaptor proteins, and PI(3,4,S)P3 is formed from PIC 4,5)P2 at the plasma membrane (1). The phosphatase, SHIP, hydrolyzes a portion ofPI(3,4,S)P3 to PI(3,4)P2 (2). Alet is recruited to the cell membrane via a PI-I domain specific for PI(3,4)P2 and PI(3,4,5)P3 where it is phosphorylated at Thr308 and Ser473 resulting in an active kinase (3). Alet leaves the cell membrane and phosphorylates downstream substrates such as mTOR, GSK-3, eNOS, BAD, and transcription factors such as p21 Cipl, p27Kipl , and FOX proteins (3). The result of these phosphorylation events is a strong growth, proliferation, and survival signal to the cell. Although Alet is important for normal cell functions, unregulated Akt activity leads to disease. Akt is the cellular homolog of v-Akt, an important protein of a transforming murine leukemia virus discovered in 1977, demonstrating the first link between Alet and cancer (4,5). Since then Akt has been linked to at least eleven 65 different types of cancer (5). The evidence that Akt is important in tumorigenesis and tumor growth include: (i) overexpressed and constitutively active Akt in connection with other genetic alterations contribute to tumor formation; (ii) Akt activation is included in the early response to carcinogens; and (iii) in vivo detection of constitutively active Akt in breast, colon, ovarian, prostate, and pancreatic cancers (6), Cells with elevated Akt activity are less sensitive to apoptotic signals and Akt upregulation is also associated with chemotherapeutic resistance (6-8). See Chapter 2 for a more detailed explanation of the effects of unregulated Akt activity in cells. Studies using the combination of PI3K1Akt inhibition with standard chemotherapy reveal decreased chemotherapeutic resistance (6). Figure 3.1 illustrates promising targets of therapeutic inhibition in the PI3K1 Akt pathway. PI3K inhibitors have demonstrated the ability to inhibit tumor growth and sensitize cells to other chemotherapeutic reagents (9,10). Current drugs such as Gleevec and Herceptin inhibition upstream of the PI3K1Akt pathway by inhibiting the cell receptors (9). The PI3K plIO subunit inhibitor, wortmannin, is a fungal metabolite with nanomolar inhibition of PI3K (11). Use of wortmannin in combination with chemotherapeutics such as paclitaxel and cisplatin has shown encouraging results (12,13). The compound L Y294002, an inhibitor of the ATP binding site of PI3K, are effective in combination with both chemotherapy and radiation treatment (14), Both wortmannin and L Y294002 treatment increase HL-60 sensitivity to apoptotic inducing compounds such as etoposide, doxorubicin, mitoxantrone and camptothecin (10). However, the drug potential of both wortmannin and L Y294002 is reduced due to minimal aqueous solubility (6). Famesyltransferase inhibitors block the post- Plasma Membrane Figure 3.1. Therapeutic targets of PI3K1Akt signaling. The receptor tyrosine kinase is activated resulting in activation of PI3K through Ras or adaptor proteins. PI3K forms PI(3,4)P2 and PI(3,4,5)P3 at the cell membrane. Akt is activated in response to the formation of phosphoinositides and colocalization with PDKI. Active Akt then phosphorylates to regulate downstream. Promising therapeutic protein targets are identified with stars (9). 66 67 translational modification Ras necessary for activation and have shown efficacy in inhibiting PI3K activation (15). There are currently four farnesyltransferase inhibitors in clinical trials for cancer therapy (16,17). Small peptidomimetics of the SH2 domains of the p85 subunit of PI3K are observed to be effective inhibitors of kinase activation (18). Analogs of rapamycin inhibit mTOR downstream of PI3K and Akt and several analogs are in clinical trials as cancer therapeutics (9). Targeted Akt inhibition may be an effective cancer treatment. Due to the important role Akt plays in normal cell homeostasis, there exists a debate on the efficacy of such a drug (7,9). However, hyper activated Akt may be more sensitive to inhibitors than normal cells (9). Antisense ribonucleic acids (RNAs) for Akt demonstrated reduced ability of tumor growth, induction of apoptosis, and enhanced sensitivity to chemotherapeutic reagents in several cell lines (19). Akt inhibitors have been developed in the past few years. Studies of the Akt A TP binding site has not led to inhibitors with high specificity, although an inhibitor with selectivity for Akt over PKA has been reported (6,20). Staurosporine and analogs inhibit Akt, but lack specificity for Akt. The analog, UCN-Ol, demonstrates promise as a PDK1 inhibitor (21). N-ethylmaleimide (NEM) is reported to inhibit Akt by activating PP2A resulting in rapid inactivation of Akt once activated (22). Overexpression of dominant negative alleles such as "kinase dead" or "just PH domain" forms of Akt is also reported to inhibit Akt activity in cells (23-26). Another reported specific Akt inhibitor was developed through an understanding of the mechanism of Akt activation (27-30). The D-3-deoxy­phosphatidyl- myo-inositol analogues (DPls), such as 1L-6-hydroxymethyl-chiro- 68 inositol2-(R)-2-0-methyl-3-0-octadecylcarbonate from Calbiochem, are reported to have an IC50 as low as 5 ~M for Akt, which is much less than the IC50 of 95 ~M for inhibition of PI3K by this analog (28). DPls demonstrated reduced resistance upon treatment of HL-60 leukemia cells with constitutively active Akt to etoposide, cytarabine, TRAIL, ATRA and ionizing radiation (31). The mode of inhibition by these inhibitors is to block the formation of PI(3,4,5)P3 and PI(3,4)P2 at the plasma membrane. The analogues do not contain a functional hydroxyl on the D-3 carbon of the inositol ring and therefore cannot be phosphorylated by PI3K (28,32). Cellular Akt inhibition is reported by inhibiting the initial step of activation, that is, membrane localization. DPI analogs are taken up by the myo-inositol transporter and incorporated into cellular phosphoinositol pools (28). The DPls cannot be phosphorylated at the D-3 carbon by PI3K, thus inhibiting PH domain-mediated Akt translocation to the plasma membrane (33). Membrane localization is a key event in Akt activation. In response to a diverse array of stimuli, Akt kinase activity can be induced up to 40-fold, principally in a PI3K-dependent manner (34). PI3K catalyzes the formation of PI(3,4,5)P3 and PI(3,4)P2 on the inner leaflet of the plasma membrane in order to recruit the positive end of the polarized Akt PH domain to the newly-formed phosphoinositides (34,35). At the plasma membrane, Akt is believed to undergo a conformational change and homodimerization, which enables the PDK1-dependent phosphorylation of Thr-308 and subsequent Ser-473 phosphorylation for full kinase activity (36,37). An Akt mutant containing an N-terminal membrane targeting sequence for myristoylation or palmitylation is activated 60-fold above that of resting wild type Akt, signifying the importance of membrane localization in activation (38). The R25C mutation of the Akt PH domain abrogated lipid binding and resulted in the inhibition of Akt in response to insulin activation (37,39). 69 DPls provide a proof of concept that Akt inhibition can be achieved through interfering with membrane localization and may lead to novel anticancer drugs. Whereas DPls functioned at the plasma membrane to inhibit Akt translocation, a possible alternative would be to bind the PH domain of Akt to inhibit membrane localization. An approach based on these observations was devised for the development of a specific Akt inhibitor. We hypothesized that the PH domain of Akt could be used to screen two synthetic peptide libraries in order to identify peptide sequences that bind specifically to the phosphoinositide-binding cleft of the Akt PH domain to inhibit the lipid binding ability of Akt (Figure 3.2). We further hypothesized that a cellular Akt inhibitor could be developed by synthesizing the sequences found from screening the libraries in tandem with a Tat-derived membrane-permeable sequence. Materials and Methods Materials D( + )-sn-l-O-[ 1-[6-(4,4-difluoro-5,7 -dimethyl-4-bora-3a,4a-diaza-s-indacene- 3-pentanoyl)amino]hexanoyl]-2-hexanoylglyceryl (BODIPY-FL) D-myo­phosphatidylinositol- 3,4,5-trisphosphate (PI(3,4,5)P3-BODIPY-FL) and D( + )-sn-l-O­[ 1-[6-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3- pentanoyl ) amino ]hexanoyl]-2-hexanoylgl yceryl D-myo-phosphatidylinositol-3,4- bisphosphate (PI(3,4)P2-BODIPY-FL) were a gift from Echelon Biosciences Inc Plasma Merrlbrane -+ PIP2 + PIP3 Figure 3.2. Hypothesized model of Akt inhibition by cell-permeable peptides. The inhibitory peptides block the Akt PH domain from binding membrane phosphoinositides to inhibit activation. (Echelon). All chemicals used were commercial available of reagent grade. All 70 peptides and oligonucleotides were synthesized at the DNNPeptide Resource Facility (DNAlPeptide RF) at the University of Utah (UUtah). . Methods Subcloning of the MBP-Akt PH R25C mutant. The Maltose-Binding Protein (MBP)-Akt PH R25C mutant was created by site-directed mutagenesis changing the arginine codon from CGC to the cysteine condon TGC using the following primers: FOR (5"-ACCTGGCGGCCATGCTACTTCCTCCTCAAG-3') and REV (5'- CTTGAGGAGGAAGTAGCATGGCCGGGAGGT-,]'). Mutations were verified by sequence analysis. Expression of MBP proteins. Both vectors containing the MBP gene alone 71 and the MBP-Akt PH domain construct were a gift provided by Dr. Joan S. Brugge (Harvard Medical School). The identity of each construct was confinned by dideoxynucleotide sequencing. All proteins were grown in LB media under 50 J.lg/mL ampicillin selection and protein expression was induced by isopropyl P-D-1- thiogalactopyranoside (IPTG) for 4 hr. The MBP, MBP-Akt PH, and MBP-Akt R25C proteins were expressed in BL21 (DE3) cells (Novagen) grown at 30°C in media supplemented with 10 mM glucose and purified using amylose resin as outlined by New England Biolabs. Glutathione-S transferase (GST)-PH domain fusion proteins were also expressed in BL21 (DE3) cells (Novagen) grown at 37°C and purified using Glutathione Sepharose 4B following the batch method of the supplied protocol (Amersham Biosciences). The Bradford assay was conducted in a 96-well plate with a bovine serum albumin (BSA) standard curve of 0 Jlg/ml to 10 J.lg/mL in 100 JlL with protein samples serially diluted 1:10 in water. Bradford reagent was added at 100 JlL per well to all wells and incubated for at least 5 min a