Akt IN CELL GROWTH AND PATHOLOGY

Akt IN CELL GROWTH AND PATHOLOGY

25 YEARS OF AKT AN EVALUATION OF ITS FUNCTION IN CELL SURVIVAL AND CANCER PROF. V. LAKSHMIPATHI PROF (Retd.) CELL BIOLOGY &ENZYMOLOGY KAKATIYA UNIVERSITY WARANGAL-506009 Email: [email protected] MY FORAYS INTO CELL SIGNALING STARTED TWO YEARS AFTER MY RETIREMENT (2006) Akt (PROTEIN KINASE B) DISCOVERY AS A NOVEL S/T KINASE 1991: Three independent groups, headed by Hemmings, Woodgett and Tsichlis, discovered a novel serine threonine (ser/thr) protein kinase Screening cDNA libraries for novel ser/thr kinases: Jones PF, Jakubowicz T, Pitossi FJ, Maurer F, Hemmings BA(1991 May 15); Proc Natl Acad Sci U S A. Named it as rac protein kinase (rac, for related to the A and C protein kinases; RAC-PK) Coffer PJ, Woodgett JR. (1991 Oct 15) Eur J Biochem. Human epithelial (HeLa) cDNA Named it as Protein kinase B (between protein kinase A and C) Isolated from oncogenic provirus, AKT-8 Bellacosa A, Testa JR, Staal SP, Tsichlis PN. (1991 Oct 11) Science. named it as Akt (designated after the oncogenic provirus, AKT-8). Ever since its discovery, Akt remained the epicenter of cell survival pathways. It is at the center of all metabolic disorders, diabetes, obesity and cancer. Its aberrant activation has been documented well in cancer. A coincidence: the target of rapamycin (TOR) was discovered in 1991 as a regulator of cell cycle. 02/13/2020 9th Indo Global Summit on Cancer therapy/ November 02-04, 2015 Hyderabad 3

25 YEARS OF Akt, 93 YEARS OF INSULIN HAVE WE MOVED BEYOND ROSENS CARTOON? 1987 65 YEARS AFTER INSULIN DISCOVERY 02/13/2020 Macleod (Nobel lecture on Polonsky, K S. insulin discovery) 1925 -At N Engl J Med we 2012; the present are entirely 367:1332-40 at loss to account for the disappearing glucose (from if one views diabetes from blood). a public health and overall societal standpoint, --------Ironically, we still do not during the past 200 years, have answer to this!worse -----we are arguably off now than we were in 1812. 9th Indo Global Summit on Cancer therapy/ November 02-04, 2015 Hyderabad 4 Akt DOMAIN STRUCTURE, PHOSPHORYLATION AND REGLUATION IIS

PHOSPHOINOSITIDES (OVER ALL REGULATORS OF THE PATHWAY) Nutrients, PLD/PA mTORC2 ? PI3K PIP2 PIP3 UPON T308 PHOSPHORYLATION Akt DISSOCIATES FROM THE MEMB-TBK1RANE INTO THE CYTOPLASM ACTIVATES mTORC1 T/308 TARGETS OF T308 TSC2; mTORC1 GLUT1 (GSK3?) (ANAB-TBK1OLIC ACTIVITY) 02/13/2020 (INFLAMMATION) DNA PK (DNA DAMAGE) PHOSPHORYLATION IN CYTOPLASM? PTEN PDK1 IKKB-TBK1-TB-TBK1K1 COTRANSLATIONAL PHOSPHORYLATION [PROTECTIVE AGAINST UB-TBK1IQUITINATION] T/450 MEMB-TBK1RANE

S/473 Akt TARGETS OF S473 TRANSLOCATES TO THE FoxO; BCl2 FAMILY MEMBRANE AND mTORC2 PHOSPHORYLATES PROTEINS Akt ON S473 WHICH GSK3, AS160(GLUT4) PROMOTES GLUCOSE UPTAKE 9th Indo Global Summit on Cancer therapy/ November 02-04, 2015 Hyderabad PHLPP 5 CEREALS: BEYOND FIBER! PHOSPHOINOSITIDES IN MEMBRANE DYNAMICS (LESSONS FROM RURAL INDIA AND COLON CANCERS) KOORAADU KUDITHI WHOLE-GRAIN CEREALS?!?! IS THERE A THING BEYOND FIBRE? P SUMA, MUKESH, LAKSHMIPATHI V; World Journal of Microbiology & Biotechnology 20: 531534, 2004. 9th Indo Global Summit on Cancer therapy/ 02/13/2020 November 02-04, 2015 Hyderabad 6

Akt IN THE MIDST OF NUTRIENT UPTAKE AND UTILIZATION On T308 Phosphorylation, Akt dissociates from the membrane, Localizes into cytoplasm phosphorylates TSC2 & inhibits it TSC1/2 protein mTORC1 activation TRANSLOCATION TO MEMB-TBK1RANE T 308 Nutrients S 473 uptake mTORC2 Nutrients inside the cell synthesis PDPK1 (PDK1) ENERGY FOR Nutrient utilization IIS Pi GLYCOLYSIS, KREB-TBK1S CYCLE & OXPHOS (ATP PRODUCTION) 02/13/2020 9th Indo Global Summit on Cancer therapy/ November 02-04, 2015 Hyderabad

O2 7 mTORC1 AND mTORC2 HAVE DIFFERENT PROTEIN COMPONENTS AND FUNCTIONS PKC SGK Akt S473 Akt T308 DEPTOR mLST8 mLST8 COOPERATIVE ACTION OF BOTH mTOR PRA PRO mTOR mTORC1 AND mTORC2 IS REQUIRED RAPTOR TOR RICTOR S 40 mSIN1 FOR TRANSLATION OF SEVERAL HOUSE mTORC1 mTORC2 KEEPING PROTEINS, WHICH INCLUDE Akt INSULIN SIGNALING MOLECULES, RICTOR, mSIN1, PROTOR DEFINE THE C2 ,COMPLEX RAPTOR, PRAS40 DEFINE THE C1 COMPLEX LIKE IGF, AND PI3K REGULATOR PTEN SENSITIVE RAPAMYCIN INSENSITIVE RAPAMYCIN

DEPTOR UPSTREAM OF Akt; ACTIVATES KREB-TBK1S CYCLE DOWN STREAM OF Akt; ACTIVATES GLYCOLYSIS CYTOSKELETON REORGANIZATION (PROMOTES NUTRIENT, IONS UPTAKE) ACTIVATES NUTRIENT UTILIZATION ATP ACTIVATED WHEN ASSOCIATED WITH RIB-TBK1OSOMES GENERATING INHIB-TBK1ITED B-TBK1Y S6K PHOSPHORYLATION OF RICTOR/ ACTIONS mSIN1 02/13/2020 ATP CONSUMING INHIB-TBK1ITS AUTOPHAGY, MITOPHAGY; PROMOTES ACTIONS B-TBK1IOSYNTHESIS ACTIVATED B-TBK1Y ATP; PHOSPHORYLATES S6K AND 4E-B-TBK1P1/2; ACTIVATES RIB-TBK1OSOME B-TBK1IOGENESIS, EIF4 DEPENDENT PROTEIN SYNTHESIS 9th Indo Global Summit on Cancer therapy/ November 02-04, 2015 Hyderabad 8 FEEDBACK LOOPS IN Akt FUNCTION ON T308 PHOSPHORYLATION Akt ENTERS INTO CYTOPLASM AND PHOSPHORYLATES TSC2 RELEASING ITS INHIB-TBK1ITORY EFFECT ON RHEB-TBK1- mTORC1 COMPLEX Rictor phosphorylation impairs mTORC2 function and glucose

uptake; but protects IRS from ubiquitination mTORC1 / S6K Serine phosphorylation of IRS by mTORC1/ S6K inhibits insulin signaling IRS dissociates from the receptor mTORC2 promotes ubiquitination of serine phosphorylated IRS INSULIN/ IGF YP IRS 1/2 SP Tyr phosphorylation promotes IRS, Receptor complex mTORC1/ S6K 02/13/2020 mTORC2 T/450 T/308 PDPK1 (PDK1) P TSC2 PIP3 TSC1 mTORC1 REGULATOR

NUTRIENTS ACTIVATE mTORC2 AND Akt TRANSLOCATION TO THE MEMB-TBK1RANE S/473 AMINOACIDS KEEP mTORC1 ACTIVE MEMB-TBK1RANE INDEPENDENT OF IIS, WHICH DISRUPTS mTORC2 & CAUSES INSULIN RESISTANCE RHEB-TBK1 ANAB-TBK1OLIC PROGRAMMING RIB-TBK1OSOME B-TBK1IOGENESIS mTORC1 PIP2 AMINO ACIDS (AA) PROTEIN SYNTESIS CYTOPLASM MITOPHAGY 9th Indo Global Summit on Cancer therapy/November 02-04, 2015 Hyderabad AUTOPHAGY Dvl LIPID SYNTHESIS Wnt PATHWAY CELL PROLIFERA TION 9 INVERSE RELATION

B-TBK1ETWEEN CONSUMPTION OF GLUCOSE & O2 NITROGEN AND PHOSPHATE TRIGGER CELL GROWTH AEROB-TBK1IC GLYCOLYSIS (GLUCOSE CENTRC VIEW: ENERGY SUPPLIED BY CARBOHYDRATE MAIN TRIGGER FOR GROWTH) CANCER CELLS HAVE DAMAGED RESPIRATION & DEPEND ON FERMENTATION FOR ENERGY FOR CELL PROLIFERATION. IGNORED NITROGEN; PASTEUR WAS CATEGORICAL IN HIS DEMONSTRATION THAT NITROGEN TRIGGERS GROWTH. CANCER CELLS CONSUME MORE OF GLUCOSE AND PRODUCE LACTIC ACID. THIS THEORY IGNORES THE ROLE OF AMINO ACIDS AND FATTY ACIDS IN ENERGY PRODUCTION. MORE IMPORTANTLY, THE HYPOTHESIS LIMITS B-TBK1ASIC FUNCTION OF MITOCHONDRIA & KREB-TBK1S CYCLE TO ATP GENERATION AND IGNORES SUB-TBK1STRATE SHUTTLES AND ROLE IN B-TBK1IOSYNTHESIS. ? B-TBK1arnett, J. A. (2000). A history of research on yeasts 2 02/13/2020 9th Indo Global Summit on Cancer therapy/ November 02-04, 2015 Hyderabad 10

LACTIC ACID AT THE CENTER OF CELLULAR METABOLISM Berzelius (1808) -muscles of hunted stags - carbohydrates are split to lactic acid during active muscular activity. Fletcher and Hopkins (1907) Frog muscles produce lactic acid steadily both during activity and rest and it disappears when O2 is admitted. LACTIC Meyerhof (1920-30)- Muscle glycogen ACID is cleaved to lactic acid. Cori cycle- Muscle lactic acid converted to glucose in liver (inter organ transport) The notion prevailed in spite of the fact that it was shown that heavy muscular exercise can be performed on pure fat diet, and the respiratory quotient (RQ) supports the complete combustion of fat (Lundsgaard11). Warburg (1927) proliferative cells produce lactic acid. Pasteur reaction - damaged respiration leads to aerobic glycolysis Respiratory uncouplers - glycolyse the cells Pasteuriana- glucose transport, metabolism, proliferation - lactic acid It projects ignores role of amino acids, fatty acids, and inorganic phosphate in cell energy production 02/13/2020 GLUCOSE: THE ONLY SOURCE OF LACTATE & ENERGY 9th Indo Global Summit on Cancer therapy/ November 02-04, 2015 Hyderabad 11 INORGANIC PHOSPHATE (Pi) IN INSULIN SIGNALING?

Macleods Nobel lecture Inorganic phosphate (Pi) : Insulin reduces circulating levels of both glucose and Pi. During recovery period Pi reappears in blood at a faster rate and is excreted in urine. Fate of glucose in cells: Glucose is neither converted into glycogen nor oxidized, but recycled as some unknown hexose phosphates. Excessive insulin action: Excessive insulin action results in hypoglycemic symptoms, which promote increased intake of O2 in insulin deprived experimental animals and man. It is controlled, when sugar is administered along with insulin. 02/13/2020 9th Indo Global Summit on Cancer therapy/ November 02-04, 2015 Hyderabad 12 1a 1b OXIDATIVE PHOSPHORYLATION A C T I V I T Y BIOSYNTHESIS CRAB-TBK1TREE EFFECT O2 WARB-TBK1URG EFFECT MITOCHONDRIAL

RESPIRATION O2 Pi GLYCOLYSIS Nrf2 OXIDATIVE PHOSPHORYLATION ATP PRODUCTION B-TBK1IOSYNTHESIS SERINE, FOLATES NUCLEOTIDES, etc. 2 MINUTES 2 Min IB-TBK1SEN; RACKER GLUT4 AktS473 mTORC2 RELATION B-TBK1ETWEEN GLUCOSE, Pi, O2 CONSUMPTION GLUT1 mTORC1 Akt T308 PI3K INSULIN SPATIO - TEMPORAL RELATION B-TBK1ETWEEN SIGNAL PATHWAYS, O2,PI & GLUCOSE UPTAKE TIME COURSE (MIN) mTORC2 REGULATES ION, NUTRIENT TRANSPORTERS & OXYGEN,ENERGY DYNAMICS 02/13/2020 9th Indo Global Summit on Cancer therapy/November 02-04, 2015 Hyderabad

14 LIPID SYNTHESIS OXYGEN PROMOTES BOTH ENERGY PRODUCION AND BIOSYNTHESIS CITRATE (CYTOPLASM) ISOCITRATE EXPORT CITRATE CO2 NH4+ REDUCTIVE CARB-TBK1OXYLATION / NITRIFICATION NADPH+H+ - KETONADP+ GLUTARATE -S-S+ SH ANAB-TBK1OLISM AH2 AROS NNT REGULAE FLUX B-TBK1ETWEEN MALATE ENERGY AND B-TBK1IOSYNTHESIS CATAB-TBK1OLISM NAD+ PYRUVATE B-TBK1CA METAB-TBK1OLISM LACTATE

8/7/2015 ALANINE FAD+ NADH+H+ OXIDATION (H++H-) DENITRIFICATION VL_KU_WARANGAL ELECTRON TRANSPORT SYSTEM CO2 NH4+ FADH2 ? ATP TRANS AND DEAMINATION 15 FEEDBAK LOOPS REGULATE GLUCOSE TRANSPORTERS, GLUT4 AND GLUT1 ENVIRONMENTAL SIGNALS, NUTRIENTS (AMINO AIDS)/ GROWTH FACTORS, TRANSFORMING SIGNALS INSULIN SIGNALING MOLECULES (AKT, IGF, PTEN & HOUSE KEEPING PROTEINS/ ENZYMES ?) mTORC1 mTORC2 Cooperative action HYDROPHOB-TBK1IC MOTIF PHOSPHORYLATIONS AKT S473 SGK3 TB-TBK1C1D4

TB-TBK1C1D4 AMINO ACIDS TSC1/2 PKC AMINO ACIDS,IONS & O2 UPTAKE ? GLUT4 MEDIATED GLUCOSE UPTAKE INSULIN SIGNALING (PI3K Akt T308) ENHANCED ACTIVATION OF mTORC1 Rag GTPases Rheb GLUT1 MEDIATED GLUCOSE UPTAKE mTORC1 KREB-TBK1S CYCLE, ENERGY & ROS PRODUCTION, CATAPLEROSIS TB-TBK1C1D1 AMPK NUTRIENTS UTILIZATION GLYCOLYSIS (B-TBK1IOSYNTHETIC ACTIVITY; ENERGY CONSUMPTION) ER/ NUTRIENT STRESS Akt IN REGULATION OF MITOCHONDIAL RESPIRATION

AMINO ACIDS mTORC2 mTORC1 COOPERATIVE ACTION (TRANSLATION) 4E-B-TBK1P1 PROTEIN SYNTHESIS LOW GLUCOSE S6K FYN S 473 Akt GSK3 INSULIN SIGNALS ? PHLPP Nrf2 INCREASED GLUCOSE UPTAKE HIGH GLUCOSE ATP /ADP RATIO ANTIOXIDANT & ROS MITOCHONDRIAL RESPIRATORY GENES

INSULIN AND GLUCOSE REGULATES PROTEIN SYNTHESIS Downstream of insulin activated and mTORC1 AMPK and in energy spent situations, AMPK promotes AMINO ACIDS Akt S473glucose uptake by ?phosphorylation of AS160; PLD/ PA TB-TBK1C1D1 appears to play critical role on this. AS160 Rab GTPase I N S U L I N G L U T INSULIN 4 PI3K mTORC2 AktT308 RECEPTOR/ IRS COMPLEX B-TBK1ASAL MECHANISM OF REGULATION OF PROTEIN SYNTHESIS Rheb TSC1/2 GLUCOSE RAPAMYCIN

GLUCOSE ? GLYCOGEN G L U T 1 ATP Pi 02/13/2020 mTORC1 B-TBK1ASAL RIB-TBK1OSOME B-TBK1IOGENESIS PROTEIN SYNTHESIS 4E-B-TBK1P1 GAPDH GLYCOLYSIS OXPHOS P70S6K LOW GLUCOSE INSULIN RESISTANCE RELEASES REGULATION OF TSC1/2, INHIB-TBK1ITS GLUCOSE AND PI UPTAKE 9th Indo Global Summit on Cancer therapy/ November 02-04, 2015 Hyderabad 18

mTORC1 is activated by nutrient & energy enriched state AMPK inhibits mTORC1 Down regulation of AMPK activates mTORC1& promotes glucose induced protein synthesis Causes insulin resistance Ribosomal association of mTORC2 promotes assembly of IIS signals; activation of IIS leads to activation of mTORC1 Activated by energy spent state Activated by nutrients, growth signals Activated mTORC1 inhibits mTORC2; mTORC2 inhibition regulates nutrient uptake but Its disruption causes Insulin resistance WORK IN HCU (BINDU MADHAVA REDDYS LAB) 02/13/2020 9th Indo Global Summit on Cancer therapy/ November 02-04, 2015 Hyderabad 20 BIOGRAPHICAL MEMOIRS OF THE ROYAL

SOCIETY, VOL. 41 (NOV., 1995), -----Himsworth stands out ------------------a great variety of worthwhile careers------------------------------------------ could not, of course, command success in all his endeavours; but like Cato in Addison's tragedy, he generally deserved it; nor could he have foreseen ---------------------- THANK YOU FOR YOUR academic philistinism which was to overtake PATIENCE -------------in 1980s SIR DOUGLAS B-TBK1LACK 02/13/2020 9th Indo Global Summit on Cancer therapy/ November 02-04, 2015 Hyderabad 21

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