Alexandra C Newton
University Of California San Diego, 9500 Gilman Dr, Dept 0934, La Jolla, Ca 92093-0934
Abstract: 1 The long-term goal of this proposal is to understand how specific proteinprotein interactions regulate the localization and function of protein kinase C (PKC) and protein kinase D (PKD). These two kinase families play pivotal roles in transducing the myriad of extracellular signals that promote phospholipid hydrolysis or, in the case of atypical PKC isozymes, 3´-phosphoinositide generation. We will focus on 1] the C-terminus of PKC as a paradigm for a molecular switch that controls kinase function through specific protein interactions, 2] identifying scaffolds for the atypical PKC isozymes and PKD isozymes, testing the hypothesis that these kinases have PDZ-binding motifs, and 3] uncovering mechanisms for lipid second messenger signaling in the matrix of mitochondria. Specifically, the following Aims are proposed 1. The C-terminus of PKC as a molecular switch -The goal of this section is to understand how the interaction of specific binding partners with the C-terminal tail of PKC controls the lifecycle of this kinase. In the current funding period, and in collaboration with Pat Jennings (Project 4), we discovered that the prolyl isomerase Pin1 converts PKC into a species that can be degraded following activation. This Aim focuses on understanding the structural (with Project 4), biochemical, and cellular mechanisms underlying the molecular switch in the C-terminus of PKC. 2. Regulation of atypical PKC and PKD signaling by protein scaffolds - The goal of this section is to understand how protein interactions confer specificity in signaling by atypical PKC isozymes and PKD isozymes. We propose to identify PDZ domain scaffolds for these kinases and test how disruption of scaffolding interactions impact signaling. 3. Signaling at intracellular organelles the mitochondria! matrix - The hypothesis driving this aim is that lipid second messenger signaling is a key component in mitochondria! function. Taking advantage of the novel genetically-encoded kinase activity reporters we developed in the current funding period, we aim to unambiguously demonstrate kinase activity in the matrix of mitochondria, an unexplored signaling terrain. We ask how disrupting kinase function at this location compromises mitochondria! function
Keywords: Adipocytes; Adipose Cell; Affect; Automobile Driving; Beta Cell; Binding; Binding (Molecular Function); Biochemical; C protein; C-terminal; CBP protein (citrate-binding); Calcium Phospholipid-Dependent Protein Kinase; Calcium-Activated Phospholipid-Dependent Kinase; Cell Communication and Signaling; Cell Signaling; Cells; Chick Embryo; Collaborations; D-Glucose; Dextrose; Drivings, Automobile; EC 2.7; Enzymes; Family; Fat Cells; Funding; Generations; Glucose; Goals; Humulin R; Hydrolysis; Inositide Phospholipids; Inositol Phosphoglycerides; Inositol Phospholipids; Insulin; Insulin (ox), 8A-L-threonine-10A-L-isoleucine-30B-L-threonine-; Insulin Cell; Insulin Secreting Cell; Insulin, Regular; Intracellular Communication and Signaling; Intracellular Second Messengers; Isoenzymes; Isozymes; Kinases; Life Cycle; Life Cycle Stages; Lipids; Lipocytes; Location; Mature Lipocyte; Mature fat cell; Mitochondria; Mitochondrial Matrix; Molecular; Molecular Configuration; Molecular Conformation; Molecular Interaction; Molecular Stereochemistry; Neural Development; Novolin R; Organelles; PKC; PKD protein; PPIase; Peptidyl-Prolyl cis-trans-Isomerase; Peptidylproline cis-trans-isomerase; Peptidylprolyl Isomerase; Phosphatides; Phosphatidyl Inositol; Phosphatidylinositols; Phosphoinositides; Phospholipid-Sensitive Calcium-Dependent Protein Kinase; Phospholipids; Phosphotransferases; Play; Proline Isomerase; Proline Rotamase; Prolyl Isomerase; Protein Kinase C; Proteins; Proteomics; PtdIns; Regulation; Reporter; Role; Second Messenger Systems; Second Messengers; Signal Transduction; Signal Transduction Systems; Signaling; Signaling Protein; Specificity; Tail; Testing; Transphosphorylases; atypical protein kinase C; biological signal transduction; citrate carrier; citrate periplasmic carrier protein; citrate transporter; citrate-binding transport protein; conformation; conformational state; cyclophilin; driving; extracellular; gene product; immunophilin; insulin secretion; life course; mitochondrial; neurodevelopment; novel; protein kinase D; protein protein interaction; scaffold; scaffolding; second messenger; social role; tricarboxylate carrier; tricarboxylate transporter; tricarboxylate-binding C protein
Budget start date: 1-JUL-2010
Budget end date: 30-JUN-2011
5P01DK054441-12_8221 (2010): $374740
Sponsored Links Excellgen http://Excellgen.com
Alexandra C Newton
University Of California San Diego, 9500 Gilman Dr, Dept 0934, La Jolla, Ca 92093-0934
Keywords: Adenosine Cyclic Monophosphate-Dependent Protein Kinases; Adipocytes; Adipose Cell; Affect; Automobile Driving; Beta Cell; Binding; Binding (Molecular Function); Biochemical; C protein; C-terminal; CBP protein (citrate-binding); Calcium Phospholipid-Dependent Protein Kinase; Calcium-Activated Phospholipid-Dependent Kinase; Cell Communication and Signaling; Cell Signaling; Cells; Chick Embryo; Collaborations; Cyclic AMP-Dependent Protein Kinases; D-Glucose; Dextrose; Drivings, Automobile; EC 2.7; Enzymes; Family; Fat Cells; Funding; Generations; Glucose; Goals; Humulin R; Hydrolysis; Inositide Phospholipids; Inositol Phosphoglycerides; Inositol Phospholipids; Insulin; Insulin (ox), 8A-L-threonine-10A-L-isoleucine-30B-L-threonine-; Insulin Cell; Insulin Secreting Cell; Insulin, Regular; Intracellular Communication and Signaling; Intracellular Second Messengers; Isoenzymes; Isozymes; Kinases; Life Cycle; Life Cycle Stages; Lipids; Lipocytes; Location; Mature Lipocyte; Mature fat cell; Mitochondria; Mitochondrial Matrix; Molecular; Molecular Configuration; Molecular Conformation; Molecular Interaction; Molecular Stereochemistry; Neural Development; Novolin R; Organelles; PKA; PKC; PKD protein; PPIase; Peptidyl-Prolyl cis-trans-Isomerase; Peptidylproline cis-trans-isomerase; Peptidylprolyl Isomerase; Phosphatides; Phosphatidyl Inositol; Phosphatidylinositols; Phosphoinositides; Phospholipid-Sensitive Calcium-Dependent Protein Kinase; Phospholipids; Phosphotransferases; Play; Proline Isomerase; Proline Rotamase; Prolyl Isomerase; Protein Kinase A; Protein Kinase C; Proteins; Proteomics; PtdIns; Regulation; Reporter; Role; Second Messenger Systems; Second Messengers; Signal Transduction; Signal Transduction Systems; Signaling; Signaling Protein; Specificity; Tail; Testing; Transphosphorylases; atypical protein kinase C; biological signal transduction; cAMP-Dependent Protein Kinases; citrate carrier; citrate periplasmic carrier protein; citrate transporter; citrate-binding transport protein; conformation; conformational state; cyclophilin; driving; extracellular; gene product; immunophilin; insulin secretion; life course; mitochondrial; neurodevelopment; novel; protein kinase D; protein protein interaction; scaffold; scaffolding; second messenger; social role; tricarboxylate carrier; tricarboxylate transporter; tricarboxylate-binding C protein
Budget start date: 1-JUL-2009
Budget end date: 30-JUN-2010
5P01DK054441-11_8221 (2009): $378525
Grants awarded to Alexandra C Newton
PROTEIN-LIPID INTERACTIONS IN SIGNAL TRANSDUCTION
Alexandra C Newton, Professor
Indiana University Bloomington P.o. Box 1847 Bloomington, In 474021847
Grant 5R01GM043154-05 from National Institute Of General Medical Sciences IRG: PC
Abstract: The long-term objective of this research is to understand how information is transferred from the outside to the inside of the cell and how it is processed within the cell. Specifically, the role of proteinlipid interactions in the processing of information by the lipid-dependent protein kinase C and the insulin receptor will be investigated. Proteinlipid interactions and protein conformation will be examined by fluorescence spectroscopy of reconstituted proteins in model membranes. In addition, the stoichiometry and specificity of proteinlipid interactions will be determined with detergentlipid mixed micelles, where the species and number of lipids interacting with one protein can be systematically varied. The effect of phosphorylation on proteinlipid interactions will be examined, testing the hypothesis that phosphorylation alters the protein s interaction with the membrane in such a way as to transduce the extracellular signal. Three specific aims will be addressed 1] Regulation of Protein Kinase C by Lipid The mechanism of the protein kinase Cmembrane interaction will be investigated. The hypotheses to be tested are a] protein kinase C cooperatively sequesters phosphatidylserine, b] diacylglycerol targets the kinase to the plasma membrane, and c] autophosphorylation releases the kinase from the membrane. 2]Regulation of Insulin Receptor by Lipid Specificity in the insulin receptorlipid interaction will be examined, with the goal of elucidating the role of lipid in the molecular control of the insulin receptor. The hypothesis that receptor autophosphorylation alters the receptor s interaction with specific phospholipids, and thus effector molecules such as protein kinase C, will be explored. 3] Regulation of Insulin Receptor Function by Protein Kinase C The role of membrane environment in insulin receptor phosphorylation by protein kinase C will be determined. The hypothesis that specific lipids serve as a matrix to bring together substrate and kinase will be addressed. In addition, the possibility that alterations in the receptorlipid interaction occur after phosphorylation by protein kinase C and induce down-regulation and receptor internalization will be investigated. Unregulated protein kinase C has been proposed to play a role in both carcinogenesis and some forms of diabetes, where its uncontrolled phosphorylation of the insulin receptor may reduce the receptor s sensitivity to incoming signals. Elucidating the role of the membrane in the mechanism of action of both protein kinase C and the insulin receptor is central to understanding how these two signalling components transduce chemical information.
Keywords: biological signal transduction, insulin receptor, membrane lipid, protein kinase C, protein structure function, biological information processing, conformation, diacylglycerol, enzyme substrate complex, phosphatidylserine, phosphorylation, fluorescent dye /probe, laboratory rat
Project start date: 1989-12-01
Project end date: 1994-11-30
5R01GM043154-05 (1994): $182084
5R01GM043154-04 (1993): $174530
5R01GM043154-03 (1992): $134712
STRUCTURE, FUNCTION AND REGULATION OF PROTEIN KINASE C
Alexandra C Newton, Professor
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 5R01GM043154-14 from National Institute Of General Medical Sciences IRG: BIO
Abstract: The long-term objective of this research is to elucidate the molecular mechanism of protein kinase C s regulation. Isozymes of this ubiquitous family of proteins transduce the plethora of signals that promote lipid hydrolysis. The proposed research is aimed at elucidating how lipid regulates the structure and function of this key signal transducer. A combination of biochemical, biophysical, molecular biological, and molecular modeling approaches are proposed in order to analyze the contribution of specify protein determinants in 1] targeting protein kinase C to acidic membranes, 2] inducing specific binding to diacylglycerol and phosphatidylserine, and 3] promoting release of the autoinhibitory pseudosubstrate from the active site allowing catalysis. In addition, the regulatory role of phosphorylation will be addressed. Four specific aims are described below The first aim addresses the question how do diacylglycerol and phosphatidylserine, in concert, induce the high-affinity membrane interaction that is required for pseudosubstrate release and activation? Experiments will address whether diacylglycerol and phosphatidylserine binding sites interact allosterically, whether phosphatidylserine is required to structure the diacylglycerol binding site, whether phorbol esters regulate protein kinase C by the same mechanism as diacylglycerol, and whether atypical protein kinase Cs display the same regulation by diacylglycerol and phosphatidylserine as the other isozymes. Second, the hypothesis that all isozymes of protein kinase C bind to acidic membranes by a common mechanism that involves recognition of acidic lipids by a conserved domain will be tested. The possibility that this domain is already structured for acidic lipid-recognition by the Ca2+- independent isozymes, but requires binding of Ca2+ in order to be structured in the conventional isozymes will be explored. The third aim tests the role of specific residues in controlling the pseudosubstrateactive site interactions, and in regulating interfacial contacts between the catalytic and regulatory domains. Lastly, the role of phosphorylation on the structural stability, lipid interaction, and function of protein kinase C will be addressed. Experiments are proposed to determine whether the intramolecular autophosphorylation prolongs or inhibits the activated state of protein kinase C, and to test the hypothesis that phosphorylation by another kinase converts protein kinase C from an inactive precursor to a form that is activatable by lipid. Uncontrolled activation of protein kinase C results in malignant transformation; by understanding the enzyme s regulation, new insights into the prevention and treatment of cancer and other diseases resulting from faulty signalling will be possible.
Keywords: conformation, enzyme activity, enzyme mechanism, enzyme structure, phosphorylation, protein kinase C, active site, biological signal transduction, cell membrane, diacylglycerol, intermolecular interaction, membrane lipid, phosphatidylserine, immunologic assay /test
Project start date: 1989-12-01
Project end date: 2003-02-28
5R01GM043154-14 (2002): $288420
5R01GM043154-13 (2001): $280108
5R01GM043154-12 (2000): $272036
STRUCTURE/FUNCTION AND REGULATION OF PROTEIN KINASE C
Alexandra C Newton, Professor
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 5R01GM043154-10 from National Institute Of General Medical Sciences IRG: PC
Abstract: The long-term objective of this research is to elucidate the molecular mechanism of protein kinase C s regulation. Isozymes of this ubiquitous family of proteins transduce the plethora of signals that promote lipid hydrolysis. The proposed research is aimed at elucidating how lipid regulates the structure and function of this key signal transducer. A combination of biochemical, biophysical, molecular biological, and molecular modeling approaches are proposed in order to analyze the contribution of specify protein determinants in 1] targeting protein kinase C to acidic membranes, 2] inducing specific binding to diacylglycerol and phosphatidylserine, and 3] promoting release of the autoinhibitory pseudosubstrate from the active site allowing catalysis. In addition, the regulatory role of phosphorylation will be addressed. Four specific aims are described below The first aim addresses the question how do diacylglycerol and phosphatidylserine, in concert, induce the high-affinity membrane interaction that is required for pseudosubstrate release and activation? Experiments will address whether diacylglycerol and phosphatidylserine binding sites interact allosterically, whether phosphatidylserine is required to structure the diacylglycerol binding site, whether phorbol esters regulate protein kinase C by the same mechanism as diacylglycerol, and whether atypical protein kinase Cs display the same regulation by diacylglycerol and phosphatidylserine as the other isozymes. Second, the hypothesis that all isozymes of protein kinase C bind to acidic membranes by a common mechanism that involves recognition of acidic lipids by a conserved domain will be tested. The possibility that this domain is already structured for acidic lipid-recognition by the Ca2+- independent isozymes, but requires binding of Ca2+ in order to be structured in the conventional isozymes will be explored. The third aim tests the role of specific residues in controlling the pseudosubstrateactive site interactions, and in regulating interfacial contacts between the catalytic and regulatory domains. Lastly, the role of phosphorylation on the structural stability, lipid interaction, and function of protein kinase C will be addressed. Experiments are proposed to determine whether the intramolecular autophosphorylation prolongs or inhibits the activated state of protein kinase C, and to test the hypothesis that phosphorylation by another kinase converts protein kinase C from an inactive precursor to a form that is activatable by lipid. Uncontrolled activation of protein kinase C results in malignant transformation; by understanding the enzyme s regulation, new insights into the prevention and treatment of cancer and other diseases resulting from faulty signalling will be possible.
Keywords: biological signal transduction, membrane lipid, protein kinase C, protein structure /function, active site, allosteric site, conformation, diacylglycerol, enzyme activity, enzyme induction /repression, enzyme structure, enzyme substrate complex, intermolecular interaction, isozyme, phosphatidylserine, phosphorylation, immunologic assay /test, point mutation, transfection
Project start date: 1989-12-01
Project end date: 1999-02-28
5R01GM043154-10 (1998): $222754
5R01GM043154-09 (1997): $214422
ROLE OF PROTEIN KINASE C IN PHOTORECEPTORS
Alexandra C Newton, Professor
Indiana University Bloomington
p.o. Box 1847
bloomington, In 474021847
Grant 5R01EY008820-03 from National Eye Institute IRG: VISA
Abstract: The long-term objective of the proposed research is to elucidate the role of the Ca2+/lipid-dependent protein kinase C in photoreceptors. Specifically, the role of protein kinase C in rod outer segment structure and function will be addressed. Protein kinase C plays a critical role in the processing of extracellular information in a wide variety of signalling systems. Phosphorylation of target proteins results in alteration of both cell structure and function, while phosphorylation of receptors results in desensitization to incoming signals. The proposed research will examine the function and mechanism of action of this ubiquitous effector molecule in rod outer segments. Three specific aims will be addressed 1] Function of Protein Kinase C in Rod Outer Segments The hypothesis that protein kinase C is involved in phototransduction by phosphorylating rhodopsin will be tested. Preliminary results have revealed that treatment of retinas with phorbol esters, protein kinase C activators, results in a marked change in the light-dependent phosphorylation of rhodopsin. The proposed research will explore the mechanism by which protein kinase C affects the phosphorylation state of rhodopsin. In addition, a combination of in situ and in vitro phosphorylation experiments will be undertaken to identify other physiological substrates of protein kinase C in rod outer segments. 2] Characterization of Protein Kinase C from Bovine Retina Biochemical and biophysical studies will address the mechanism of action of retinal protein kinase C. The hypothesis that retinal protein kinase C represents a unique isozyme will be investigated. Additionally, monoclonal antibodies to retinal protein kinase C will be generated. 3] Subcellular Distribution of Protein Kinase C in Dark- and Light- Adapted Rod Outer Segments The distribution of protein kinase C isozymes in the retina (especially subcellularly in the photoreceptors) will be determined. The possibility that protein kinase C translocates between inner and outer segments, or from the cytosol to the membrane, in response to light, will be explored. Immunochemistry and subcellular fractionation studies will be undertaken on dark and light-adapted retinas and rod outer segments. The abundance of protein kinase C in rod outer segments, its ability to phosphorylate rhodopsin and the ubiquitous role of the enzyme in both signal transduction and receptor desensitization suggest that it may play a central role in photoreceptor function. Elucidating the role of protein kinase C in rod outer segments is central to a complete understanding of phototransduction and the cell biology of photoreceptors
Keywords: enzyme mechanism, phosphorylation, protein kinase C, rod cell, visual photoreceptor, visual phototransduction cytoskeletal protein, enzyme inhibitor, intracellular transport, isozyme, light adaptation, phorbol, phospholipid, protein kinase, protein structure, protein transport, receptor sensitivity, retina, rhodopsin animal tissue, fluorescence microscopy, immunocytochemistry, immunoelectron microscopy, immunoprecipitation, laboratory mouse, laboratory rat, monoclonal antibody, radiotracer, western blotting
Project start date: 1991-08-01
Project end date: 1994-07-31
5R01EY008820-03 (1993): $83970
Sponsored Links Excellgen http://Excellgen.com
SIGNAL TERMINATION BY PHLPP, PH DOMAIN LEUCINE-RICH REPEAT PROTEIN PHOSPHATASE
Alexandra C Newton
University Of California San Diego, 9500 Gilman Dr, Dept 0934, La Jolla, Ca 92093-0934
Grant 5R01GM067946-08 from National Institute Of General Medical Sciences
Abstract: The long-term goal of this proposal is to understand the molecular and cellular mechanisms of signal termination mediated by the novel phosphatase PHLPP (PH domain Leucine-rich repeat Protein Phosphatase; pronounced ´flip´) that we discovered in the preceding funding period. The central hypothesis driving this proposal is that PHLPP terminates signaling pathways that are turned on by PDK- 1, notably Akt signaling pathways, and that specificity in termination is achieved by subcellular location and macromolecular interactions. 1. Molecular Mechanisms of PHLPP The goal of this section is to understand the enzymology and biochemistry of PHLPP, a new PP2C family member. Specifically, we will examine the kinetics and substrate specificity of the three PHLPP isozymes, the alternatively spliced PHLPP1 (a and and PHLPP2, and develop tools for cellular studies in Aims 2 and 3. 2. Cellular Mechanisms of PHLPP The goal of this section is to understand the mechanisms that control the function of PHLPP in cells. We will test the hypothesis that the PDZ binding motifs of the PHLPP isoforms selectively tether them to specific NHERF PDZ domain proteins, forming a scaffolding network that allows effective control of the amplitude and duration of Akt signaling. We will characterize additional PHLPP binding partners in cells and, using novel imaging technologies, we will measure PHLPP activity in real time in live cells. 3. PHLPP in disease This aim addresses the role of PHLPP in human cancers. The PHLPP1 and PHLPP2 genes are located on the chromosomal loci reported to be the most commonly deleted in colon cancer and breast cancer, respectively. The function of PHLPP isozymes in controlling the amplitude of Akt signaling poises them as prime candidates to be the elusive tumor suppressors harbored on these loci. To test this, we will screen human tumors for mutations in PHLPP, address the role of PHLPP in cell migration in breast cancer cells, and develop a mouse model to address how genetic deletion of PHLPP could lead to abnormalities in cell survival and proliferation
Keywords: Address; Automobile Driving; Binding; Binding (Molecular Function); Biochemistry; Breast Cancer Cell; Cancer of Breast; Cancers; Cell Communication and Signaling; Cell Locomotion; Cell Migration; Cell Movement; Cell Signaling; Cell Survival; Cell Viability; Cells; Cellular Migration; Chemistry, Biological; Colon Cancer; Colon Carcinoma; Colonic Carcinoma; Disease; Disease Progression; Disorder; Drivings, Automobile; Enzymatic Biochemistry; Enzymology; Family member; Funding; Genes; Genetic; Genetic Alteration; Genetic Change; Genetic defect; Goals; Human; Human Breast Cancer Cell; Human, General; Imaging technology; Intracellular Communication and Signaling; Investigators; Isoenzymes; Isoforms; Isozymes; Kinetic; Kinetics; LRR protein; Lead; Life; Location; Malignant Neoplasms; Malignant Tumor; Malignant Tumor of the Breast; Malignant neoplasm of breast; Man (Taxonomy); Man, Modern; Measures; Mediating; Mg(2+)-dependent serine-threonine protein phosphatase; Molecular; Molecular Interaction; Motility; Motility, Cellular; Mutation; NHE-RF protein; NHERF; Na+-H+ exchanger-regulatory factor; PH Domain; PP2C; Pb element; Peptide Domain; Phosphatases; Phosphohydrolases; Phosphomonoesterases; Phosphoprotein Phosphatase; Phosphoprotein Phosphatase-2C; Phosphoprotein Phosphohydrolase; Phosphoric Monoester Hydrolases; Pleckstrin-Homology Domain; Programs (PT); Programs [Publication Type]; Protein Domains; Protein Isoforms; Protein Phosphatase C; Protein Phosphatase-1; Protein Phosphatase-2A; Protein phosphatase; Ptc2 phosphatase; RNA Splicing; Reporting; Research Personnel; Researchers; Role; Signal Pathway; Signal Transduction; Signal Transduction Systems; Signaling; Specificity; Splicing; Substrate Specificity; Tertiary Protein Structure; Testing; Time; Tumor Suppressor Proteins; biological signal transduction; cell motility; disease/disorder; driving; genome mutation; heavy metal Pb; heavy metal lead; leucine-rich repeat protein; malignancy; malignant breast neoplasm; mouse model; neoplasm/cancer; novel; programs; protein phosphatase 2C; scaffold; scaffolding; social role; sodium-hydrogen exchanger regulatory factor; tool; tumor; tumor suppressor
Project start date: 2003-05-01
Project end date: 2011-07-31
Budget start date: 1-AUG-2010
Budget end date: 31-JUL-2011
5R01GM067946-08 (2010): $298262
5R01GM067946-07 (2009): $301275
3R01GM067946-06S1 (2009): $278100
5R01GM067946-06 (2008): $301275
2R01GM067946-05A1 (2007): $301275
Phosphoinositide-dependent Kinase: Molecular Mechanisms
Alexandra C Newton, Professor
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 5R01GM067946-04 from National Institute Of General Medical Sciences IRG: BIO
Abstract: The long-term goal of the proposed research is to understand the molecular mechanisms that regulate the phosphoinositide-dependent kinase, PDK-1. The discovery of PDK-1 in 1997 unveiled a linchpin of cellular signaling. This kinase, alone, provides the on switch for the catalytic function of diverse members of the AGC superfamily of kinases, including Akt (protein kinase B), protein kinase C family members, p70S6 kinase, among many others. Although the function of PDK-1 has been known since its discovery, the molecular mechanisms of this pivotal kinase are largely unexplored. The goal of the proposed research is to understand the molecular mechanisms governing the regulation, function, and subcellular location of PDK-1. The central hypothesis guiding this proposal is that the subcellular location and macromolecular interactions of PDK-1 control its physiological function. Three Specific Aims are 1. Regulation of PDK-1 by Membrane Interactions The goal of this section is to understand the spatial and temporal regulation of PDK-1 in live cells. Specifically, we delve into the central questions of 1] where in the cell is PDK-1 active, 2] what are the cellular inputs that control this activity, and 3] how sustained is substrate phosphorylation? We will take advantage of novel fluorescence methodologies to monitor both the activity and subcellular location of PDK- 1 in real time in response to cellular stimuli. 2. Biochemical and Structural Analysis of PDK-1 The goal of this section is to understand the biochemical mechanisms controlling PDK-1 activity. Specifically, we ask the questions 1] what is the mechanism of membrane binding of PDK-1? 2] what mechanisms govern the catalytic activity of PDK-1, and 3] what is the structure of PDK-1? 3. Regulation of PDK-1 by Protein Protein Interactions The central hypothesis of this aim is that protein protein interactions are key regulators of PDK-1 function in vivo. We 1] address the mechanisms by which PDK-1 recognizes Akt, testing the hypothesis that the interaction is regulated by the PH domain of each kinase, 2] propose to screen for binding partners of the PH domain and linker region of PDK-1, 3] test the hypothesis that PDK-1 is in a complex with a phosphatase, and 4] test the hypothesis that PDK-1 is tethered at the centrosome in a signaling complex with AKAP 350.
Keywords: enzyme mechanism, enzyme structure, protein localization, serine threonine protein kinase, centrosome, enzyme activity, enzyme complex, enzyme substrate, intracellular, membrane structure, phosphatidylinositol, phosphorylation, protein binding, protein protein interaction, fluorescent dye /probe
Project start date: 2003-05-01
Project end date: 2007-08-14
5R01GM067946-04 (2006): $270399
5R01GM067946-03 (2005): $277110
5R01GM067946-02 (2004): $277305
1R01GM067946-01 (2003): $297241
Structure, Function And Regulation Of Protein Kinase C
Alexandra C Newton, Professor
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 5R37GM043154-19 from National Institute Of General Medical Sciences IRG: PC
Abstract: The long-term goal of the proposed research is to understand the molecular mechanisms and biological function of protein kinase C (PKC). PKC has been in the spotlight since the discovery 25 years ago that it is activated by the lipid second messenger, diacylglycerol. Despite PKC s enduring stage presence and tremendous advances in understanding the enzymology and regulation of this key protein, understanding the function of PKC in biology is still under intense pursuit. In the preceding funding period we focused on understanding the molecular mechanisms of PKC as a first step in understanding how this key protein functions in the cell. The goal of this proposal is to both continue elucidating the molecular mechanisms of PKC regulation and to use this knowledge to address the cellular function of PKC. Specific Aims are 1. Mechanism of Protein Kinase C s Membrane Interaction. The goal of this Specific Aim is to understand how the C1 and C2 domains target and retain PKC on the membrane. These studies continue our long-standing research in understanding the molecular mechanisms of how lipid mediators regulate PKC. Biophysical and cellular studies will be coupled to gain greater insight into the mechanism of translocation of PKC in vivo. 2. Regulation of Protein Kinase C by dephosphorylation. In the previous funding period, we made major advances in understanding how phosphorylation controls PKC. As part of this work, it became apparent that dephosphorylation, rather than phosphorylation, may be the agonist-dependent regulator of the phosphorylation state of PKC. The goal of this Specific Aim is to understand the mechanism and role of dephosphorylation of PKC in regulating its function. 3. Protein Kinase C Function In Vivo. Using chemical genetics and novel fluorescence technologies, we will test the hypothesis that a major role of PKC is to activate cellular phosphatase activity. This hypothcsis is based on evidence in the previous funding period that PKC activation increases phosphatase activity in cells. Upregulation of phosphatase activity could account for the pleiotropic effects of PKC activation.
Keywords: conformation, enzyme activity, enzyme mechanism, enzyme structure, phosphorylation, protein kinase C, active site, biological signal transduction, cell membrane, diacylglycerol, intermolecular interaction, membrane activity, membrane lipid, phosphatidylserine, 3T3 cell, fluorescence resonance energy transfer, immunologic assay /test, stop flow technique, two dimensional gel electrophoresis
Project start date: 1989-12-01
Project end date: 2008-02-29
5R37GM043154-19 (2007): $328976
Sponsored Links Excellgen http://Excellgen.com
5R37GM043154-18 (2006): $387673
5R37GM043154-17 (2005): $396078
3R37GM043154-16S1 (2004): $23434
5R37GM043154-16 (2004): $348349
2R37GM043154-15 (2003): $348770
PROTEIN KINASE C AND PHOTORECEPTORS
Alexandra C Newton, Professor
Indiana University Bloomington P.o. Box 1847 Bloomington, In 474021847
Grant 2R01EY008820-04 from National Eye Institute IRG: VISC
Abstract: The long term goal of this research is to understand the role of protein kinase C in rod outer segments of photoreceptors. Specifically, we will explore the phosphorylation of rhodopsin by protein kinase C. We will also characterize the structure and regulation of the rod outer segment protein kinase C. Protein kinase C is a ubiquitous signal transducer that processes the plethora of signals promoting lipid hydrolysis. The enzyme is known to desensitize a number of transduction pathways, generally by phosphorylating and deactivating unliganded and liganded receptors. Its relative abundance in rod outer segments and the identification of rhodopsin as its major substrate in situ lead us to test the hypothesis that protein kinase C provides a second messenger-regulated desensitization pathway in phototransduction, analogous to the second messenger-regulated, heterologous desensitization pathways in other G protein-coupled transduction pathways. The first two aims below address the function of protein kinase C in rod outer segments, and the third addresses the structure and regulation of rod outer segment protein kinase C. I. Phosphorylation of Rhodopsin by Protein Kinase C conditions for and sites of phosphorylation. Biochemical and cell biological approaches are proposed to determine which conditions promote the protein kinase C- catalyzed phosphorylation of rhodopsin in the intact retina. Inhibitors and activators of protein kinase C will be used to dissect out the contribution of protein kinase C to the phosphorylation of rhodopsin in situ. We will also test the hypothesis that protein kinase C plays a role in heterologous desensitization by asking the question do feedback pathways in the retina result in protein kinase C activation and rhodopsin phosphorylation? Lastly, we will identify the site(s) of phosphorylation of rhodopsin by protein kinase C and determine the sensitivity of this phosphorylated site to phosphatases. II. Phosphorylation of Rhodopsin by Protein Kinase C functional consequences. The goal of this specific aim is to determine the consequences of the protein kinase C-catalyzed phosphorylation of rhodopsin. Specifically, we will address how this phosphorylation alters three macromolecular interactions rhodopsin s interaction with rhodopsin kinase, arrestin, and transducin. We will test the hypothesis that the phosphorylation of rhodopsin by protein kinase C deactivates the receptor. III. Structure and Regulation of Protein Kinase C in Rod Outer Segments. This aim addresses the possibility that the protein kinase C in rod outer segments is a previously undescribed isozyme of the protein kinase C family. The goal of this aim is to prepare isozymically pure rod outer segment protein kinase C, to investigate its biochemical and molecular properties, and to determine its subcellular distribution.
Keywords: enzyme mechanism, phosphorylation, protein kinase C, visual photoreceptor, visual phototransduction, cytoskeletal protein, dopamine receptor, enzyme activity, enzyme inhibitor, intracellular transport, molecular site, protein kinase, protein structure, protein transport, retina, rhodopsin, stereochemistry, transducin, immunocytochemistry, immunoprecipitation, laboratory mouse, laboratory rabbit, laboratory rat, western blotting
Project start date: 1991-08-01
Project end date: 1994-12-31
2R01EY008820-04 (1994): $212306
5R01EY008820-12 (2002): $245934
5R01EY008820-11 (2001): $234121
5R01EY008820-10 (2000): $227301
2R01EY008820-08A1 (1998): $220253
Sponsored Links Excellgen http://Excellgen.com
PROTEIN KINASE C: STRUCTURE/REGULATION/CELLULAR FUNCTION
Alexandra C Newton, Professor
Keystone Symposia
silverthorne, Co 80498
Grant 1R13CA085161-01 from National Cancer Institute IRG: ZCA1
Abstract: The field of protein kinase C catapulted to the forefront of signal transduction with the discovery two decades ago that this enzyme transduces the myriad of extracellular signals that promote phospholipid hydrolysis. Since then, intense efforts have been devoted to understanding the structure, function, and regulation of this key enzyme. Yet, it is only in the past few years that major discoveries have started unveiling the complex regulation and function of the enzyme. In particular, the past year has been marked by the unanticipated discovery that protein kinase C is, itself, regulated by the phosphoinositide-dependent protein kinases, providing a new mechanism of regulation by lipid that is independent of the known regulation by diacylglycerol. This past year was also marked by the demonstration that disruption of a scaffold protein disrupts an entire signalling cascade, thus underscoring the importance of anchoring proteins. The development of techniques to study the activation of protein kinase C in real time has also poised the field to understand cellular signalling by this enzyme. The goals of this meeting are to address the recent advances in protein kinase C´s structure, regulation, and cellular function. This meeting will have an added impact by synergizing with the Lipid Second Messenger Meeting organized by George Carman. Three key aspects of the proposed meetings are 1. It is unique in that a) it will integrate cell biological, biochemical, structural, and biophysical advances in understanding protein kinase C and b) will synergize with the joint Lipid Second Messenger Meeting, providing new insight into the role of 2 independent lipid signalling pathways, the established phospholipase C one and the newly discovered phosphoinositide-3 kinase one, in regulating protein kinase C. This meeting is most similar to the 1994 Keystone Meeting on protein kinase C that was held jointly with one on lipid second messengers. 2. It is multidisciplinary in that it interfaces structural, mechanistic, and cell biological aspects of protein kinase C signalling. The leading researchers in these fields will be invited to participate. 3. It will set the agenda for future directions in the blossoming field of understanding the in vivo regulation and function of protein kinase C, which will be invaluable to graduate students and postdoctoral fellows in embarking upon their own careers
Keywords: biological signal transduction, meeting /conference /symposium, protein kinase C, protein structure /function phosphatidylinositol 3 kinase, phospholipase C travel
Project start date: 2000-02-07
Project end date: 2001-02-06
1R13CA085161-01 (2000): $4000
ON SIGNAL TRANSDUCTION AND LIPID SECOND MESSENGERS
Alexandra C Newton, Professor
Keystone Symposia Silverthorne, Co 80498
Grant 1R13GM057207-01 from National Institute Of General Medical Sciences IRG: BIO
Abstract: The field of lipid second messengers has catapulted to the forefront of signaling pathways in the past decade. Since the discovery that diacylglycerol serves as a ubiquitous second messenger that regulates the function of the protein kinase C family, the involvement of a vast repertoire of lipids and their breakdown products in transducing diverse signals has been clearly established. Now, pivotal questions are to identify and characterize 1) the enzymes that produce the lipid second messengers; and 2) the protein targets of these second messengers. A flurry of recent advances in the characterization of lipid kinases and phospholipases that regulate the levels of these second messengers, as well as identification of novel lipid second messengers, indicate that this is a prime time to evaluate lipid signaling. Th goal of this meeting is to address recent advances in elucidating the structure, function and regulation of proteins that produce lipid second messengers, and proteins that are targets for second messengers. Three key aspects of the proposed meeting are1) It is unique in that it will focus on structure, function and regulation of both the enzymes that produce lipid second messengers and the enzymes regulated by lipid second messengers. This meeting is most similar to the 1994 Keystone Meeting and will provide a timely update in this rapidly growing field. Other meetings have focused more on biophysical properties of lipids (Biophysics subgroup in 1995) or the membrane enzymology of lipases (ASBMB satellite in 1996). 2) It is multidisciplinary in that it interfaces structural, mechanistic and cell biological aspects of lipid signaling. The leading researchers in these fields will be invited to participate. 3) It will set the agenda for future direction in the blossoming field of lipid signaling, which will be invaluable to graduate students and postdoctoral fellows in embarking upon their own careers
Keywords: biological signal transduction, diacylglycerol, lipid metabolism, meeting /conference /symposium, second messenger, travel
Project start date: 1998-03-01
Project end date: 1999-02-28
1R13GM057207-01 (1998): $4000
Alexandra C Newton
University Of California San Diego
Project start date: 1989-12-01
Project end date: 2013-02-28
STRUCTURE, FUNCTION AND REGULATION OF PROTEIN KINASE C
Alexandra C Newton
University Of California San Diego, 9500 Gilman Dr, Dept 0934, La Jolla, Ca 92093-0934
Grant 5R37GM043154-22 from National Institute Of General Medical Sciences
Keywords: 1, 2-diacylglycerol; Active Sites; Address; Agonist; Automobile Driving; Award; Binding; Binding (Molecular Function); Biochemical; Biochemistry; Biology; C protein; C1 Domain; C2 Domain; CBP protein (citrate-binding); Calcium Phospholipid-Dependent Protein Kinase; Calcium-Activated Phospholipid-Dependent Kinase; Cell Communication and Signaling; Cell Signaling; Cells; Chaperone; Chemistry, Biological; Collaborations; Columbidae; Coupled; DAG; DAG Binding Domain; DAG/PE-Binding Domain; Diacylglycerol Binding Domain; Diacylglycerols; Diglycerides; Doves; Drivings, Automobile; E3 Ligase; E3 Ubiquitin Ligase; EC 2.7; Enzymatic Biochemistry; Enzymes; Enzymology; Event; Family; Family member; Feedback; Fluorescence; Funding; Goals; Grant; Hydrolysis; Image; In Vitro; Intracellular Communication and Signaling; Intracellular Second Messengers; Isoenzymes; Isozymes; Kinases; Laboratories; Learning; Life; Ligands; Lipids; Membrane; Membrane Transport; Memory; Modeling; Molecular; Molecular Chaperones; Molecular Interaction; PKC; Phorbol Ester Binding Domain; Phorbol Esters; Phosphatases; Phosphohydrolases; Phospholipid-Sensitive Calcium-Dependent Protein Kinase; Phosphomonoesterases; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Pigeons; Position; Positioning Attribute; Principal Investigator; Process; Production; Programs (PT); Programs [Publication Type]; Progress Reports; Property; Property, LOINC Axis 2; Protein Kinase C; Protein Kinase C Conserved Region 1 Domain; Protein Phosphorylation; Protein-Serine Kinase; Protein-Serine-Threonine Kinases; Protein-Threonine Kinase; Proteins; Regulation; Reporter; Reports, Progress; Research; Rewards; Role; Second Messenger Systems; Second Messengers; Serine Kinase; Serine-Threonine Kinases; Signal Pathway; Signal Transduction; Signal Transduction Systems; Signaling; Signaling Protein; Sites, Active; Staging; Stimulus; Stream; Structure; Substrate Specificity; Technology; Threonine Kinase; Time; Transmembrane Transport; Transphosphorylases; Ubiquitin-Protein Ligase E3; analog; atypical protein kinase C; biological signal transduction; citrate carrier; citrate periplasmic carrier protein; citrate transporter; citrate-binding transport protein; diacylglycerol; diglyceride; driving; gene product; imaging; membrane structure; new technology; novel; programs; protein function; response; second messenger; social role; tricarboxylate carrier; tricarboxylate transporter; tricarboxylate-binding C protein; tumor; ubiquitin-protein ligase
Project start date: 1989-12-01
Project end date: 2013-02-28
Budget start date: 1-MAR-2010
Budget end date: 28-FEB-2011
5R37GM043154-22 (2010): $416288
3R37GM043154-19S1 (2007): $43086
Phosphoinositide-dependent Kinase: Molecular Mechanisms
Alexandra C Newton
University Of California San Diego
3R01GM067946-04S1 (2006): $38197
Structure, Function And Regulation Of Protein Kinase C
Alexandra C Newton, Professor
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 3R37GM043154-15S1 from National Institute Of General Medical Sciences IRG: PC
Project start date: 1989-12-01
Project end date: 2008-02-29
3R37GM043154-15S1 (2003): $77000
PROTEIN KINASE C AND PHOTORECEPTORS
Alexandra C Newton, Professor
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 5R01EY008820-07 from National Eye Institute IRG: VISC
Project start date: 1991-08-01
Project end date: 1998-02-28
5R01EY008820-07 (1996): $222098
STRUCTURE/FUNCTION AND REGULATION OF PROTEIN KINASE C
Alexandra C Newton, Professor
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 5R01GM043154-08 from National Institute Of General Medical Sciences IRG: PC
Project start date: 1989-12-01
Project end date: 1999-02-28
5R01GM043154-08 (1996): $206399
Sponsored Links Excellgen http://Excellgen.com