Chemico-Biological Interactions

Transcription

IT'S MY GREAT PLEASURE TO INTRODUCE TODAY'S SPEAKER. IN PREPARING FOR INTRODUCTORY COMMENTS, THE NIH STAFF REACHED OUT TO SEVERAL OF JERRY'S FORMER TRAINEES AND COMMON THEMES EMERGED, WHICH I'D LIKE TO SHARE WITH YOU AND I'LL ONLY SHARE THE ONES I CAN SHARE IN A PUBLIC FORUM. THEY COMMENTED ON HIS TREMENDOUS PASSION FOR SCIENCE IN GENERAL AND OF COURSE FOR HIS WORK ON JAMES OLESKE IN PARTICULAR. I LEARNED JERRY IS A SCIENTIST WHO THROUGHOUT HIS CAREER HAS BEEN ENORMOUSLY GENEROUS WITH HIS TIME AND JUST ON A PERSONAL NOTE, I CALLED JERRY IN THE LATE 1980s WHEN WE WERE TRYING TO PURIFY THIS TRANSFERASE, POLYPEPTIDE TRANSFERASE THAT INITIATE BIOSYNTHESIS AND JERRY TALKED US THROUGH ALL THE NUANCES OF A GENERAL APPROACH THAT ONE WOULD TAKE TO PURIFY THIS TRANSFERASE AND HE SENT ME A XEROX COPY OF HIS TECHNICIAN'S LAB NOTES WHICH INCLUDED EVERY TRICK TO SYNTHESIZING A KEY AFFINITY MATRIX. IN OUR CASE IT WAS UDP GLUCOSE MEAN BIOPROPIL SEVER ROWS AND AS A RESULT OF THAT GENEROSITY, OF JUST GIVING ME THE LAB NOTEBOOK, THAT STARTED US ON AN EFFORT THAT l HAS NOW LASTED OVER&UñAGñ 20 YEA5Yu ON WHAT TURNED OUT TO BE A FAMILY OF POLYPEPTIDE TRANSFERASES WHICH WE CONTINUE TO THIS50 DAY. AND I NEVER THANKED YOU FOR THAT. SO THANK YOU. JERRY CONTRIBUTED W( TEMLY TO ALL FIELDSK OF FLAKEO ;8 AND ITS IMPORTANCE TO HUMAN HEALTH. IT'S TRULIY GREAT PLEASURE I WELCOME HIM TODAY AS THE WALS HIS PRESENTATION BITTER SWEET MZ OF O-GLCNAC IN DIABETES A DISEASE AND CANCER OF THE f2W TO SHOW THAT IN FACT THESE WERE THESE PROTEINS. SHOW SHE USED ENZYMATIC TAGGING WITH CLICK IT CHEMISTRY AND ENZYMATIC LABELING WITH UDP GALLON TOP OF THE SUGAR AND AUTO RAIDOGRAPHY AND ANTIBODIES AGAINST O-GLCNAC, OVER EXPRESSED PROTEINS WITH FLAG TAGS AND THEN PROBING FOR O-GLCNAC. AND IN THESE 2D GELS SHE SHOWED HYPERACETYLATED CELLS BECOME HYPERACETYLATEOD HISTONES AND THEY ARE ALSO MODIFIED BY O-GLCNAC. THIS STUDY BECAME PARTICULARLY INTERESTING BECAUSE WHEN WE DID THE SITE MAPPING, WE ASKED WHERE ARE THE O-GLCNAC RESIDUES ON HISTONES, A FEW OF THE SITES ARE ON THE HISTONE TAIL WHERE THE SO-CALLED HISTONE CODE IS LOCATED. BUT SEVERAL OF THE SITES ARE IN FACT IN THE EXACT REGION WHERE DNA INTERACTS WITH THE HISTONE AND THIS IS PARTICULARLY INTERESTING AS I'LL SHOW YOU HOW BIG THIS MOLECULE IS. AND YOU MIGHT THINK IT'S JUST A STRUCTURAL MODIFICATION OF HISTONES BUT THAT'S NOT TRUE BECAUSE IF YOU LOOK AT THE CELL CYCLE, HE IS AN EXAMPLE WITH HISTONE H3. YOU CAN SEE DURING THE CELL CYCLE, SYNCHRONOUS EARLY AND LATE AND IN G1, OF THE GLC NATION GOES UP AGAIN. AND THEN IF YOU EXPOSE CELLS TO 45 DEGREES AND LET THEM RECOVER AND LOOK AT WHAT IS GOING ON WITH O-GLCNACATION OF THE HISTONES, IT GOES BACK UP AGAIN. SO IT'S CLEARLY CYCLING. IF YOU OVER EXPRESS THESE ENZYME THAT ADDS O-GLCNAC, ABOUT TWO FOLD IN CELLS, IT CAUSES THE HISTONE MODIFICATIONS, FOR EXAMPLE HISTONE H3 LYSINE 9 ACETYLATION GOES UP. HISTONE H3 SEREIN 10 PHOSPHORYLATIONS ARE KEY REGULATORY SITE GOES DOWN. METHYLATION GOES DOWN AND THIS METHYLATION AT THIS SITE GOES UP. AND THEN RECENTLY, A VERY EXCITING PAPER JUST APPEARED IN SCENES MAGAZINE SHOWING THAT O-GLCNAC NATION OF A2B AT SEREIN 112 REGULATES OUR FACILITATES ITS MONOUBIQUITINATION WHICH IS AN ACTIVATION EVENT FOR HISTONES AND THESE PEOPLE WHO IN THIS PAPER SHOWED THAT THE O-GLCNAC IS THE BINDING SITE FOR THE H2B UBIQUITIN LIGASE THAT PUTS UBIQUITIN ON THE PROTEIN. THERE IS CROSS TALK BETWEEN ALL OF THESE DIFFERENT POSTTRANSLATIONAL MODIFICATIONS. VERY BRIEFLY SAYING SOMETHING ABOUT THE ENZYMES INVOLVED IN CYCLING. THERE ARE TWO INCREDIBLY INTERESTING PROTEINS I'M TOTALLY UNBIZED BY THAT. THERE IS THE TRANSFERASE, A TWO DOMAIN PROTEIN. THE CATALYTIC SUBUNIT EVOLVED FROM FLY COJEN PHOSPHOR LAYS AND IT HAS LONG PROTEIN DOCKING DOMAINS AND THIS IS VERY IMPORTANT FOR HOW THIS ENZYME WORKS. THE ENZYME THAT REMOVES O-GLCNAC, IT ALSO HAS TWO DOMAINS. ONE IS THE CATALYTIC PART THAT REMAINS THE SUGAR AND THE OTHER IS A HISTONE TRANSFERAISE DOMAIN THAT MOST OF US IN THE FIELD DECIDE PUBLICATIONS TO THE CONTRARY, THINK IS NOT ENZYMATICALLY ACTIVE BUT PLAYS A ROLE IN TARGETING THE ENZYME TO CHROMATIN. DURING THE LATE STAGES OF APOPTOSIS, THE EXECUTION OF CASPASE CLEVES IT'S MOLECULE IN HALF AND THE O-GLCNAC HALF REMAINS ACTIVE. THE O-GLCNAC TRANSFERASE IS ABOUT 80% NUCLEAR AND 20% CYTOPLASMIC DEPENDING ON THE CELL TYPE BUT MOSTLY NUCLEAR ENZYME WHEREAS THE ENZYME THAT REMOVES O-GLCNAC IS LOCATED OTHER PLACES INCLUDING THE MITOCHONDRIA JOHN HAS SHOWN. BUT IF YOU -- THE JAMES OLESKE -- O-GLCNAC IS CYTOPLASMIC AND LESS IN THE NUCLEUS. THE INTERESTING THIS IS THAT THE PORTION THAT IS IN THE NUCLEUS OF O-GLCNAC IS HIGHLY LOCATED IN THE NUCLEUS AND A GRADUATE STUDENT IN MY LAB HAS SHOWN THAT O-GLCNAC PLAYS A DIRECT ROLE IN THE RIBOSOME BIOGENESIS IN AT LEAST 20 RIBOSOME PROTEINS THAT ARE MODIFIED AND THIS IS AN ONGOING PROJECT. SO IT'S DEFINITELY INVOLVED IN RIBOSOME ASSEMBLY. NOW I'LL COME BACK TO THIS UDP O-GLCNAC. THIS IS THE DONOR AND THIS IS THE O-GLCNAC TRANSFERASE IS SENSITIVE FROM NANOMOLAR UP TO THE SOLUBILITY OF UDP-DLCNAC, AND THE REASON WHY IT'S SO CRITICAL IS IT'S DIRECTLY REGULATE THE BY NUCLEOTIDE, ENERGY, GLUCOSE, AMINO ACID AND FATTY ACID METABOLISM WHICH EFFECT THE LEVELS OF THIS SUGAR DONOR IN THE CELL. AND JUST TO GIVE YOU AN EXAMPLE OF THIS AS I LIKE TO SHOW THIS, PARTICULARLY TO PEOPLE WORKING ON SIGNALING AND TRANSCRIPTION, IS THAT MOST PEOPLE DOING THESE STUDIES USE TISSUE CULTURE CELLS IN THEIR STUDIES. THEY GROW THEIR TISSUE CULTURE CELLS IN DIABETIC CONDITIONS. IF YOU LOOK AT THE COMPOSITION OF TISSUE CULTURE MEDIA, IT'S TYPICALLY 20-30 MILL I MOLAR GLUCOSE. THE NORMAL LEVEL OF GLUCOSE IS 5 MILL I MOLAR. AND JUST TO GIVE YOU AN EXAMPLE OF THAT, THIS IS CELLS GROWN IN 5 MILL I MOLAR GLUCOSE. AND THE PROTEINS ARE NORMALIZED AND I'M NOT SHOWING YOU THOSE. THAT THE LEVEL OF GLUCOSE AND THIS EXPOSURE, NOW I CAN EXSNOWS UNTIL IT WAS BLACK WITH 1000 SPOTS BUT THIS WOULD BE THE MOUNTAIN TOPS. HOWEVER, IF YOU GROW THESE SAME CELLS FOR A SHORT PERIOD OF TIME IN 30 MILL I MOLAR GLUCOSE, YOU CAN SEE THE EXTENT OF O-GLCNACATION HAS GONE UP DRAMATICALLY. IF YOU WAS STUDYING TRANSCRIPTION AND GROWING MY CELLS IN DIABETIC CONDITIONS, I MIGHT WORRY. WE DON'T KNOW ENOUGH ABOUT WHAT THIS IS DOING BUT IS AFFECTING MANY SIGNALING PATHWAYS AND TRANSCRIPTION. SO ORIGINALLY THE O-GLCNAC TRANSFERASE WAS CLONED IN A BACTERIAL ANALOGUE AND THIS IS SAY STRUCTURE -- THIS SLIDE WAS PROVIDED TO ME BY DONOVANAL TON AT DUNDEE. AND IT'S A BEAUTIFUL STRUCTURE. YOU CAN SEE THIS WHEN DON FIRST SHOWED THIS, HE HAD IT POSITIONED OVER A WINE BOTTLE AND OPENING UP LIKE IT WAS A CORK SCREW. THIS IS A CATALYTIC DOMAIN. THESE ARE THE TPR REPEATS HIGHLY CONSERVED AND THE IMPORTANCE OF THESE, THESE ARE WHERE ALL THE PROTEINS BIND THAT TARGET THIS ENZYME TO VARIOUS SUBSTRATES. VERY RECENTLY SUZANNE WALKER LABORATORY AT HARVARD DONE THE STRUCTURE OF THE HUMAN ENZYME AND YOU'LL NOTE IT'S VERY SIMILAR EXCEPT IN THEIR MODEL IT IS MORE LIKE A HINGE WHERE THE TARGETING PROTEINS AND SUBSTRATES BIND IN THIS HINGE OPENS UP TO ALLOW ACCESS TO THE ACTIVE SITE. AND SO WE ARE BEGINNING TO UNDERSTAND HOW THIS ENZYME WORKS. NOW I ALWAYS GET ASKED AT SEMINARS, THERE ARE 500 KINASES AND ONLY ONE OGT AND IF THEY ARE MODIFYING SIMILAR NUMBER OF PROTEINS, HOW IS THAT POSSIBLE? IT TURNS OUT THERE ARE MULTIPLE MANY O-GLCNAC TRANSFERRATIONS IT'S JUST THAT THEY EXIST AS TRANSIENT POLO ENZYMES AND SO SOME OF THE TARGETING PROTEINS WE HAVE BEGUN TO STUDY USING EAST 2 HYBRID ANALYSIS AS A START, THESE ARE PROTEINS THAT INTERACT WITH THE TPR DOMAIN AND OTHER REGIONS IN THE ENZYME AND TARGET IT TO SPECIFIC SUBSTRATES. SO THERE ARE ONES THAT HAVE BEEN SHOWN TO BE INVOLVED IF TRANSCRIPTION OR DEVELOPMENT, MOTOR PROTEINS, AND FOR EXAMPLE, ONE OF THE ONES THATILANCE WORKED ON WAS PROTEIN PHOSPHATASES ARE ASSOCIATE THE WITH OGT AND WHAT HIS STUDY SHOWED WAS IN MANY INSTANCES, THE SAME ENZYME THAT REMOVES THE PHOSPHATE AS THE O-GLCNAC WITHOUT LETTING GO UP SUBSTRATE. ONE EXAMPLE OF THIS IS WAS WORK BY MIKE HAUSLE WHEN HERE'S A STUDENT IN COLLABORATION WITH JOE ROGERS LOOKING AT REGULATION OF GLUCONY GENESIS. IT WAS KNOWN FOR MANY YEARS SINCE THE 50s, THAT PEOPLE WHO HAD DIABETES AND HAD HIGH GLUCOSE IN THEIR BLOOD, YOU WOULD THINK THEIR LIVER WOULD STOP MAKING GLUCOSE BUT THAT'S NOT WHAT HAPPENS. WHEN THE GLUCOSE GETS HIGH, GLUCNY GENESIS IS ELEVATED AND IN TERMS OF NORMAL REGULATION, THE INSULIN BIND TO THE INSULIN RECEPTOR, TRIGGERS THE CASCADE THAT ACTIVATES THE AKT KINASE AND IN NORMAL CELLS, THIS AND PHOSPHORALATES THREE KEY SITES CAUSING IT TO LEAVE THE NUCLEUS AND SHUTS OFF TRANSCRIPTION OF THE RATE LIMITED STEPS OFNY GENESIS. SO DURING THE NORMAL STATE, FOX O IS BOUND TO A CO-ACTIVATOR CALLED PGC1 ALPHA DISCOVERED BY BRUCE, AND IT'S THIS COMPLEX THAT THEN ACTIVATES GLUCNY GENESIS. I'M NOT GOING TO SHOW YOU DATA BUT TO GIVE YOU THE MODEL THAT RESULTED FROM THIS. PGC1 ALPHA IS ONE OF THE THESE TARGETING PROTEINS AND WORK FROM ANOTHER LAB HAS ALSO SHOWN THAT CRTC.2 IS A TARGETING PROTEIN AND THE WAY THIS REGULATION AND GLUCNY GENESIS WORKS IS UNDER HIGH GLUCOSE CONDITIONS, UDP POOLS GO UP AND BY MECHANISMS THAT WE DON'T FULLY UNDERSTAND YET, OGTCRTC2 CAUSING IT TO GO INTO THE NUCLEUS OR ACTIVATE IT, WHICH IT THEN ASSOCIATES WITH CRIB, TRANSCRIPTIONALLY ACTIVATES PG21 ALPHA ASSOCIATES WITH OGT AND CAUSE ITSELF TO BECOME SPECIFIC FOR FOX O AND THE GLOCK KNACK INDEPENDENT OF WHETHER IT HAS THOSE 3AKT PHOSPHORYLATION SITES OR NOT, GOES INTO THE NUCLEUS AND ACTIVATES G6PC AT THE RATE LIMITING STEPS OF NUKE NEOGENESIS. WHY WOULD THE LIVER DO THIS? IT IS RESPONDING TO OXIDATIVE STRESS DUE TO THE HIGH GLUCOSE LEVELS AND MITOCHONDRIAL DYSFUNCTION AND TRYING TO SURVIVE. THIS IS A SHORT-TERM STRESS RESPONSE BECAUSE AT THE SAME TIME THESE ARE BEING ELEVATED, WHICH ARE AFFECTING OTHER CELL TYPES IN THE BODY, MAGNES SOD AND CAT LAYS ARE ALSO REGULATED BY THIS SYSTEM AND THESE ARE THE ENZYMES THAT DETOXIFY OXYGEN RADICALS. OKAY. SO THE NEXT THINGS I WANT TO TALK ABOUT IS THIS CROSS TALK BETWEEN GLOCK KNACKALATION AND PHOSPHORYLATION AND HOW EXTENSIVE IS. SO WHAT DO I MEAN BY THAT? THERE ARE PROTEINS WHERE MANY PROTEINS WHERE THE PHOSPHATE RESIDUE AND THE GLOCK KNACK RESIDUES RECIPROCALLY OCCUPY THE SAME GROUPS SUCH AS THE C MYC ONCOGENE. OTHER PROTEINS WHERE THEY HAVE RECIPROCAL OCCUPANCY BUT AT DIFFERENT SITES. IN OTHER WORDS, IF THE GLOCK KNACK IS IN PROXIMITY OF A PHOSPHORYLATION SITE IT CAN'T BE PHOSPHORYLATED. THE INSULIN RECEPTOR SUBSTRATE IS A SCAFFOLD FOR EVERY KIND OF MODIFICATION YOU CAN THINK OF THAT IS PHOTS PHOSPHORYLATED AND GLOCK KNACK LATED SIMULTANEOUSLY AND THESE SPECIES SEEM TO MODULATE INSULIN SIGNALING IN WAYS WE DON'T UNDERSTAND AND I'LL TALK ABOUT KINASE 4. IT'S ANOTHER EXAMPLE. SO WHAT IS THE OUTCOME OF JUST -- THERE IS OVER 440 POST POSTTRANSLATIONAL MODIFICATIONS KNOWN. NO EXAMPLES I'M AWARE OF A PROTEIN THAT'S NOT POST TRANSLATION MODIFIED AND MONTH LEVELULAR DIVERSITY CREATED BY POSTTRANSLATIONAL MODIFICATION IS ILLUSTRATED BY THIS IDEA THAT IF YOU HAVE 10 MODIFICATION SITES ON A PROTEIN AND THEY WERE ONLY PHOSPHORYLATED OR DEPHOSOPHOROLATED, THE MOLECULAR DIVERSITY WOULD BE THIS YELLOW LINE DOWN HERE WHEREAS IF YOU JUST ADDED ANOTHER ONE THAT WAS ABOUT THE SAME ABUNDANCE LIKE O-GLCNAC, THE MOLECULAR DIVERSITY GETS AFTER THE NOM CALL IN A MURRAY. IF YOU START TO TALK ABOUT UBIQUITINATION AND ACETYLATION AND EVERY OTHER, MOLECULAR DIVERSITY BECOMES ENORMOUS IN A HURRY. SO I'LL GIVE YOU AN EXAMPLE OF THE C MECHANIC ONCOGENE. WE WERE STUDYING THIS C MECHANIC ONCOGENE AND THE MAJOR SITE TURNED OUT TO BE 38058. WHY IS THAT IMPORTANT? THAT'S THE MAJOR PHOSPHORYLATION SITE FOR GSK3 KINASE BETA. AND IF YOU LOOK AT THE MUTATIONS IN HUMAN LYMPHOMAS THAT OCCUR THAT CAUSE CANCER, CAUSE THIS PROTEIN TO BECOME AN ONCOJEAN, THIS IS 38058. THIS IS THE TRANSCRIPTION FACTOR AND THE GROWTH FACTOR SIGNALING CASCADE VIA IRK KINASE AND GSK3 BETA AND IRK KINASE, PHOSPHORALATES SEREIN 62 AND THEN AND ONLY THEN DOES THIS BECOME A PRIMING SITE FOR GSK3 BETA TO PHOSPHORYLATE 58. WHAT WE FOUND USING SITE SPECIFIC ANTIBODIES AND OTHER APPROACHES IN A NONGROWING CELL, THERE IS A GLK KNACK AT 58 AND IF YOU STIMULATE THE CELL TO GROW WITH GROWTH FACTORS OR SERUM AS FAST AS YOU CAN MEASURE IT, THE GLOCK KNACK IS NO LONGER THERE. IT HAS A ROLE IN THIS PROTEIN WITH THE TUMOR SUPPRESSOR. SO WE WANTED TO ASK A SIMPLE QUESTION ABOUT THIS RELATIONSHIP OF PHOSPHORYLATION AND WE DECIDED TO TAKE ADVANTAGE OF RECENT DEVELOPMENTS IN MASS SPECK TECHNOLOGY AND WE ASKED IN THE ABSENCE OF ANY OTHER STIMULI, IF WE JUST TOOK TISSUE CULTURE CELLS AND INDICATE NIH CELLS, ANDaQ=fz EXPOSEDçZu%THEM SHORT-TERM TO AN5oBbç INHIBITOR OF THEÚVSQm5)#l THAT+ R"÷ REMOVES O-GLCNAC AND THISJO8GLOBALLY ELEVATES O-GLCNAC ABOUT 2-3 FOLD ON HUNDREDS IF NOT THOUSANDS OF PROTEINS AND THEN ASKED WHAT DOES THAT DO TO SITE SPECIFIC PHOSPHORYLATION, SO THIS LOOKS COMPLICATED BUT IT'S NOT. IT'S A WORK FLOW FOR DOING THIS EXPERIMENT. SO THE ASSUMPTIONS WE MADE WAS IF A PHOSPHATE SITE WAS NOT AFFECTED BY HIGH LEVELS OF ACID, THIS IS A PHOSPHATASE INHIBITOR, THEN IT'S PROBABLY NOT PSYCHING. AND THEN WE USE O-GLCNAC INHIBITORS TO ELEVATE O-GLCNAC, AND THEN WE PURIFY THE PHOSPHOPROTEINS USING THE MOBILIZED CHROMATOGRAPHY AND THEN WE PURIFIED AFTER PROTEASE DIGESTION, PHOSPHOPEPTIDES USING TITANIUM DIOXIDE AND THESE WERE TAGGED WITH THE METHODS THAT ALLOW YOU TO DO MULTIPLEX QUANTITATION IN A MASS SPECTROMETER, AND THEN YOU CAN CALCULATE THE SITE OCCUPANCY BUT THE EXTENT OF THE PROTEIN, ABUNDANCE AND GET AN OCCUPANCY RELATIVE OCCUPANCY RATIO OF INDIVIDUAL SITES. AND THE WAY THIS DATA LOOKS IS, THIS IS A SITE THAT IS NOT PSYCHE LEAN SO FOR EXAMPLE, IN THE PRESENCE OF ACID, IT DOESN'T CHANGE. THIS HAPPENED T BE ON IRS2. HERE IS A SITE THAT IS CYCLING THAT THE ACID MADE IT GO UP AND INHIBITORS OF O-GLCNAC WHICH ELEVATED O-GLCNAC MADE IT GO DOWN. AND SO IN ANALYZING THIS, WE ANALYZED ABOUT 700 PHOSPHORYLATIONS IDENTIFIED AND QUANTIFIED AND THIS IS WORK BY AN OUTSTANDING GRADUATE STUDENT. WE FOUND IN NONSTIMULATED CELLS, NOT SURPRISINGLY, ABOUT 48% OF THE SITES WERE NOT CYCLING. BUT AT THE TIME, WHAT WAS REALLY STRIKING WAS WE WERE EXPECTING NOT THESE HUGE CHANGES. ALL OF THE SITES VIRTUALLY THAT WERE CYCLING WERE AFFECTED BY O-GLCNAC. 280 WENT DOWN AND 148 WENT UP. AND THESE THAT WENT UP ARE SURPRISING BECAUSE AT THE TIME WE WERE THINKING THESE WERE SORT EVER RECIPROCAL MODIFICATIONS BUT OBVIOUSLY IT'S MORE COMPLICATED THAN THAT. WELL, WHY IS IT MORE COMPLICATED THAN THAT? IT TURNS OUT THAT MANY KINASES, THIS IS A SCREEN THAT WAS DONE BY VIEWEDNER DIAZ WHEN HE WAS A POSTDOC IN THE LAB, MANY ARE MODIFIEDED BY O-GLCNAC AND SO FAR THE ONES THAT HAVE BEEN STUDIED ARE REGULATED BY THEM. THE FIRST ONE IS KINASE 4. A VERY IMPORTANT KINASE IN NERVOUS SYSTEM AND IN BETA CELLS. IT'S REGULATED BY CALCIUM CALL MOD LIN, CAMP KINASE, PHOSPHORALATES A CRITICAL RESIDUE AT 3200 TO ACTIVATE IT. WHEN IT'S ACTIVATED IT PHOSPHORALATES A BUTCH OF TRANSCRIPTION FANG ORGANIZATIONS, PARTICULARLY CRIB AND TURNS THEM ON. AND WE MAP THE MAJOR SITES OF O-GLCNAC ON THIS KINASE AND THE IMPORTANT ONE I WANT TO TALK ABOUT IS SEREIN 189. SO IF YOU TAKE NEURONS AND YOU DEPOLARIZE THEM USING THIS KINASE BECOMES ACTIVATED. YOU CAN SEE THE PHOSPHORYLATION GOES UP AND CONCOMINANT WITH THIS INCREASE, THERE IS A DECREASE IN GLCNACALATION OF SEREIN 189. AND IN ADDITION TO THAT, THE PHOSPHORYLATED ACTIVATED FORM OF THIS ENZYME BINDS O-GLCNAC SPECIFICALLY AND IF YOU MUTATE THE 200 SITES, PHOSPHORYLATION SITES TO AN ALANIN, THE LEVEL OF O-GLCNAC AT THE 189 SITE GOES UP. AND IF YOU MUTATE THE 189 SITE TO AN ALANIN TO PREVENT IT FROM BEING O-GLCNACIATED, THE ENZYME BECOMES CONSTITUTIVELY ACTIVE AND VERY ACTIVE ON THE CRIB TRANSCRIPTION FACTOR. AND THE MUTATED KINASE THAT IS MISSING THE GLCNAC AT 189 HAS A MUCH HIGHER AFFINITY FOR ATP. AND WHY IS THAT? WELL, IF YOU LOOK AT THE MODEL OF THE STRUCTURE OF THE CAM KINASE 4 AND ASK, WHERE IS THE GLCNAC RESIDUE AT SEREIN 189, IT TURNS OUT IT'S RIGHT IN THE MIDDLE OF THE ATP BINDING POCKET. IN OTHER WORDS, IF THIS KINASE HAS A GLCNAC PRESENT AT 189, IT'S A DEAD ENZYME AND IT CAN'T EVEN BIND ATP. AND JUST TO GIVE YOU A FEEL FOR THE DIFFERENT, THERE IS A SPACE FILLING MODEL OF A PHOSPHATE SITTING ON CAMP KINASE 4 AND THEN GLOCK SNACK ABOUT FIVE TIMES THE RADIUS OF A PHOSPHATE EVEN THOUGH IT DOESN'T HAVE A CHARGE. SO THIS LED TO THIS MODEL BECAUSE BACK IN 2007 SHOWED THAT CAMP KINASE 4 ITSELF PHOSPHORALATES OGT AND THE ACTIVATES IT. SO IT TURNS OUT THAT CAMP KINASE 4 SITTING IN A MEMBRANE WITH GLCNACOS IT IS A DEAD ENZYME AND BEFORE IT CAN GET ACTIVATED, THIS GLCNAC RESIDUE HAS TO BE REMOVED AND THEN IT GETS PHOSPHORYLATED AND ACTIVATED. TWO STEP MECHANISM. WHEN IT IS ACTIVATED, IT GOES BACK AND PHOSPHORALATES OGT AND RESETS THE CYCLE. AND THIS IS SORT OF AN EMERGING THEME THAT THIS IS LIKE A SAFETY VALVE OR A TWO STEP MECHANISM FOR TURNING ON AND TURNING OFTEN ZYME AND THE ADVANTAGES OF THIS MAY BE TO THE CELL IS IT MAKES IT MORE SENSITIVE TO NUTRIENT STATUS IN ADDITION TO ACTIVITY OF KINASES. ANOTHER EXAMPLE I'M GOING TO TALK ABOUT IS RECENT UNPUBLISHED WORKED ON PROTEIN KINASE KINASE 2. YOU CAN SEE THE SITES ARE LOCATED ON THE ALPHA AND ALPHA PRIME SUBUNITS AND NOT ON THE BAIT A AND THEN MAKE THE PEPTIDES AND BACK THEN WHEN WE DID THIS SITE MAPPING MANY YEARS AGO, WE WERE USING MANUAL EDMOND DEGRADATION TO MAP SITES, WHICH MEANT THE STUDENT THAT DID THIS HAD TO SPEND LIKE 15 HOURS IN THE HOOD DOING THIS. SO IT'S CRUEL AND UNUSUAL PUNISHMENT. BUT ANY WAY, THIS GLCNAC SITE GOT MAPPED TO SEREIN 347. AND SO PHIL COAL'S LAB, ONE OF THE WORLD'S EXPERTS AT DOING CHEMICAL LIGATION OF PROTEINS, DID THIS WITH A MADE TO RECOMBINANT 1-10341, AND THEN VARIOUS MODIFIED FORMS OF THE REASON THAT CONTAINED THE MODIFICATION SITES. SO THEY WERE ABLE TO MAKE THE PHOSPHOFORM AND THE GLCNAC FORM AND OTHER FORMS OF THIS ENZYME. AND WHAT WAS REALLY STRIKING ABOUT THIS FIRST OF ALL TO SHOW YOU THE PRESENCE OF A GLCNAC AT 347 PREVENTS PHOSPHORYLATION AT 344. THIS IS A VERY COMMON THEME. BUT WHAT WAS REALLY STRIKING AND I THINK IS GOING TO HAVE LONG TERM, ALMOST PROFOUND IMPLICATIONS TO OUR UNDERSTANDING OF SIGNALING, IS THAT THE CASING KINASE TWO WITH THE GLCNAC ON IT HAS A DIFFERENT SUBSTRATE SPECIFICITY THAN THE ONE WITH THE PHOSPHATE ON IT. SO THIS IS AN EXAMPLE OF THESE. THE WAY THIS WAS DONE WAS WITH A LARGE PROTEIN ARRAY WITH A DIFFERENT ISOMAKER SPECIES OF THE ENZYMES AND ASKED WHAT ARE THE SUBSTRATES WITH THESE SPECIES AND THAT SPECIES, SO YOU CAN SEE THIS IS A REALLY GOOD SUBSTRATE FOR THE O-GLCNAC ICE FORM BUT NOT FOR THE PHOSPHO. BUT THIS ENZYME IS A GOOD SUBSTRATE FOR THE PHOSPHOBUT NOT FOR THE O-GLCNAC. AND THIS ARRAY REVEALED A NUMBER OF PROTEINS THAT HAD THESE PROPERTIES, WHETHER IT WAS JUST THE ALPHA SUB UNIT ALONE OR THE ALPHA BETA TOGETHER, WHEREAS THE GLCNAC FORM PHOSPHOFORM, HAD ALL DIFFERENT SUBSTRATES. AND I THINK THAT THIS IS COMING OUT IN NATURE CHEMICAL BIOLOGY. AND I THINK IT'S GOING TO BE VERY IMPORTANT IN OUR VIEW OF REGULATION OF THE SPECIFICITY OF THESE ENZYMES BY POSTTRANSLATIONAL MODIFICATIONS. SO THE BASIC IDEA IS THAT ENZYME SPECIFICITY IS IN PART CONTROLLED BY MODIFICATIONS. SO WHEN IT HAS A GLCNAC AT 347, IT HITS A BUNCH OF SUBSTRATES THAT ARE DIFFERENT THAN WHEN IT HITS WHEN IT IS UNMODIFIED OR WHEN IT'S MODIFIED BY PHOSPHATE RESIDUES. OKAY, THE VERY LAST THING I WANT TO TALK ABOUT IS GIVE YOU AN EXAMPLE OF HOW THE CROSS TALK BETWEEN THESE DIFFERENT MODIFICATIONS IS IMPORTANT IN A CELLULAR PROCESS. AND THE EXAMPLE I'M GOING TO TALK ABOUT IS CYTOKENICEIS. SO WE KNEW A WHILE BACK THAT IF YOU OVER EXPRESS O-GLCNAC TRANSFERASE, ABOUT TWO FOLD, CELLS BECOME POLYPLOID. THIS IS A VERY COMMON FEATURE OF CANCER CELLS, BY THE WAY. SO, NUCLEAR DIVISION IS PROCEEDING NORMALLY BUT CYTOKINESIS OR CELL DIVISION IS NOT. SO YOU GET MULTINUCLEATED CELLS. AND CHAD, A POSTDOC IN THE LAB NOW AT KANSAS UNIVERSITY MEDICAL CENTER ON THE FACULTY, DID THIS VERY NICE WORK WHERE HE SHOWED THAT DURING THE CELL CYCLE, THE O-GLCNAC TRANSFERASE BECOMES HIGHLY ENRICHED AT THE MID BODY WHEN THE CELLS ARE GETTING READY TO PINCH OFF. AND THIS JUST SHOWS ANOTHER ILLUSTRATION OF HOW ENRICHED THIS ENZYME GETS AT THE TUBUAL. IF YOU OVER EXPRESS THE O-GLCNAC TRANSFERRATIONAISE IN -- TRANSFERASE IN CELLS, YOU GET A HUGE INCREASE IN O-GLCNACATION AND A DECREASE IN PHOSPHORYLATION AS MEASURED BY A PROLINE-DIRECTED PHOSPHATE SPECIFIC ANTIBODY OF THE IN ADDITION TO THIS, THIS SORT OF ILLUSTRATES HOW THE O-GLCNAC TRANSFERASE WORKS IN THAT IF YOU LOOK AT ASYNCHRONOUS CELLS WHICH ARE NOT AT THIS STAGE OF THE CELL CYCLE AND YOU ASK OR DO AN IMMUNOPRECIPITATION WITH AN IMPORTANT KINASE THAT IS INVOLVED IN CELL DIVISION, THE ONLY THING YOU BRING DOWN IS THE KINASE. BUT IF YOU LOOK AT THE SPECIFIC STAGE OF THE CELL CYCLE WHERE THEY ARE GETTING READY TO UNDERGO THIS CELL DIVISION EVENT AND YOU DO THE SAME EXPERIMENT, YOU BRING DOWN THE COMPLEX OF ENZYMES THAT CONTAIN O-GLCNAC, AGT, POLO KINASE, AURORA KINASE, PROTEIN PHOSPHATASES. SAME THING IF YOU DO OGT, YOU BRING DOWN AN ASYNCHRONOUS CELLS PREDOMINANTLY OGT WHEREAS AT THIS STAGE WHEN THIS COMPLEX FORMS, YOU BRING DOWN A COMPLEX AND WE ARE BEGINNING TO UNDERSTAND THE SUBSTRATES OF THIS COMPLEX THAT ARE MODIFIED, BOTH BY GLCNACALATION AND PHOSPHORYLATION AT THIS CLEAVAGE EVENT. BUT WE WANTED TO KNOW MORE DETAIL ABOUT WHAT IS GOING ON. HOW COME ELEVATING OGT TWO FOLD IN A CELL CAUSES CYTOKENIECEIS NOT TO WORK? SO WE AGAIN DECIDED -- IT TURNS OUT I'LL MENTION THAT CLASSICAL MASS SPECTROMETRY METHODS DO NOT WORK FOR O-GLCNAC. I HAD MASS SPECK LAB, THE BEST IN THE WORLD, UNABLE TO DETECT MODIFICATION. SO YOU HAVE TO USE OTHER METHODS AND I CAN TALK ABOUT WHY THAT IS BUT WE DEVELOPED THIS RATHER COMPLICATED METHOD THAT INVOLVES TAGGING THE DEMOCRACY KNACK RESIDUE ENZYMATICALLY AND THIS MADE USE OF A MUTANT GAL TRANSFERASE THAT WAS PROVIDED AND THERE IS A KIT I THINK THAT THE COMPANY SELLS WHERE YOU CAN ATTACH TO THE GLCNAC RESIDUE AND THEN YOU CAN USE A UV ACHIEVABLE BY TIN TAG TO TAG IT, DO A AFFINITY CHROMATOGRAPHY AND YOU GET A PEPTIDE WITH THIS UNIQUE STRUCTURE ON IT. THE BEAUTY OF THIS STRUCTURE IS YOU CAN THEN DO CLASSICAL COLLISION-INDUCED FRAGMENTATION AND OVER THE PEPTIDES THAT HAVE THIS AND THEN YOU CAN GO AND FIND OUT BICEP RATING THE PHOSPHOAND THE GLYCOTOGETHER, YOU CAN QUANTIFY OCCUPANCY OF BOTH THE SITES AND SO IN THIS STUDY, WE QUANTIFIED OCCUPANCY AT 450 PAIRS OF GLCNAC SITES PRESENT AT THE MID BODY. AND THIS WAS PUBLISHED IN SCIENCE SIGNALING. THE TAKE HOME MESSAGE OF THIS WAS -- AND IT TOOK LIKE A YEAR TO ANALYZE ALL THE DATA THAT CAME FROM THIS IN TERMS OF THIS SIGNALING PATHWAYS, WHICH IS NORMAL FOR THIS TYPE OF STUDY. BUT WHAT CAME OUT OF THIS WAS STRIKING. YOU KNOW THAT PSYCHE LEAN DEPENDENT KINASE IS THE MASTER REGULATOR OF CELL DIVISION. SO WHAT HAPPENS WHEN YOU ELEVATE O-GLCNAC TWO FOLD? ONE OF THE THINGS IS IT DECREASES THE TRANSCRIPTION OF KINASE ONE AND DECRESES MESSENGER RNA BUT DIDN'T ALTER THE PHOSPHORYLATION OF THIS PROTEIN. RESULTING EFFECT OF THIS WAS TO DECREASE THE PHOSPHORYLATION OF THIS NYT1 PROTEIN AND ALSO TO DECREASE THE AMOUNT OF CDC25. THE EFFECT OF THESE TWO WERE TO UPREGULATE TWO INHIBITORY PHOSPHATE KEY REGULATORY INHIBITORY PHOSPHATE EVENTS ON CDK1. THIS RESULTS IN DOWN REGULATION OF ALL THE PHOSPHORYLATION TARGETS OF CDK1 AND PROBABLY MAJORLY EFFECTS CYTOKENIECEIS. YOU CAN DO THE SAME THING FOR THE 60s OF AURORA KINASE AND POLO KINASES AND COME TO SIMILAR CONCLUSIONS. SO BOTH TRANSCRIPTIONAL EVENTS AND PHOSPHORYLATION EVENTS ARE INVOLVED IN REGULATING EACH PROCESS. SO IN CONCLUSION, WHAT I HAVE DESCRIBED IS THAT O-GLCNAC IS A MAJOR REGULATORY POSTTRANSLATIONAL MODIFICATION AND ALL MULTICELLULAR EUKARYOTES, PLANS PLANTS, ANIMALS, VIRUSES AND BACTERIA IS REQUIRED FOR LIFE IN MAMMALS AND PLANTS. PLANTS HAVE TWO O-GLCNAC TRANSFERASES. THE CROSS TALK AND INTERPLAY BETWEEN O-GLCNACATION AND PHOSPHORYLATION IS EXTENSIVE AND INVOLVED IN MANY CELLULAR PROCESSES. TURNING OUT TO BE VERY IMPORTANT IN TRANSCRIPTION AND I THINK SOME DATA THE TRANSCRIPTION COMMUNITY LE WAKE UP TO THIS FACT. IT'S PART OF THE HISTONE CODE WHERE MOST SITES ARE IN CONTACT WITH THE DNA. IT SERVES AS A METABOLIC SENSOR THAT REGULATES SIGNALING AND TRANSCRIPTION IN RESPONSE TO NUTRIENTS AND STRESS. AND WE THINK THAT ONE OF THE MAJOR AREAS WHERE THIS IS GOING TO IMPACT MEDICINE IS THAT IT REALLY EXPLAINS WHY HIGH GLUCOSE CAN BE SO TOXIC AS IN LONG-TERM EFFECTS OF DIABETES AND THAT IS ONE OF THE THRUST OF OUR LAB IS TO GET AT WHAT THE MECHANISM OF GLUCOSE TOXICITY AND MORE RECENTLY, IT BECOMES STRIKING THAT O-GLCNACATION IS DRAMATICALLY ELEVATED IN EVERY CANCER SO FAR THAT I KNOW OF THAT HAS BEEN LOOKED AT. THEY HAVE NO IDEA WHAT THAT MEANS NECESSARILY. BUT I THINK IT'S GOING TO PLAY A MAJOR ROLE IN MOLECULAR PROCESSES AS LEADING TO CANCER. AND SO THIS IS A NOW OLD PICTURE EVEN FRANK IS STILL IN IT, OF THE LAB. I NEED TO TAKE A NEW PICTURE. RECOLLABORATEED WITH PERRY'S LAB. WE HAVE BEEN WORKING WITH DON HUNT'S LAB FOR A WHILE WORKING ON DEVELOPING MASS SPECK METHODS TO STUDY O-GLCNAC AND WE HAVE A VERY NICE PROJECT GOING ON ON DIABETIC CARDIOMYOPATHY WITH ANNE MURPHY AND OTHERS HERE. OUTSTANDING PEOPLE. LOOKING AT DIABETIC CARD MYOPATHY. I DIDN'T HAVE TIME TO TALK ABOUT THAT TODAY. I THINK THAT'S IT. THANK YOU. [APPLAUSE] >> SO, IS THERE ANY WAY WE COULD MEASURE THIS STATUS OF YOUR O-GLCNAC MEASURE OF THE PROTEINS? BECAUSE I FOUND OUT THAT I SAW SOME INTERESTING DIVISION. ONE THING HA HAPPENED IS -- IS THAT A TRUE MEASURE OF THE FLU COSALATION OF THE PROTEIN -- [INAUDIBLE] >> SO I DIDN'T TALK ABOUT THIS AND WE HAD A STUDY GOING ON OVER WHERE WE ARE LOOKING AT O-GLCNAC AS A DIAGNOSTIC TOOL, PARTICULARLY FROM HUMAN CELLS. THE DATA LOOKS VERY PROMISING. AND THE BEAUTY OF IT IS YOU CAN DETECT THE ELEVATION IN BOTH O-GLCNAC AND SPECIFIC SITES ON PROTEIN AND THE ELEVATION OF O-GLCNAC ACE FAIRLY STRIKING IN HUMAN RED CELLS WELL BEFORE HEMOGLOBIN A1C BECOMES POSITIVE. WHEN IT BECOMES POSITIVE, YOU ALREADY GOT DIABETES AND IT'S PRETTY LATE IN THE PROCESS, WHEREAS, WHAT WOULD BE REALLY NICE IS IF YOU COULD DETECT IT SIX MONTHS TO A YEAR BEFORE YOU BECOME A1C POSITIVE AND THEN MAYBE YOU COULD CHANGE PEOPLE'S LIFESTYLE, GET THEM TO EXERCISE, QUIT EASING CHEESE BURGERS. WHATEVER. >> SO DIFFICULT TO CONTROL -- [INDISCERNIBLE] DOES IT HAVE AFFECT ON THIS PROCESS ON FLY COSALATION AND ALL THE OTHER -- >> I'M NOT SURE I UNDERSTOOD THE QUESTION. THE O-GLCNAC LEVELS ARE SENSITIVE NOT ONLY TOW GLUCOSE BUT TO AMINO ACIDS, LIPIDS, FATTY ACIDS. I MEAN ALL OF THESE THINGS CAUSE IT TO GO UP IN DIFFERENT MECHANISMS PROBABLY, BUT IT IS ELEVATED IN ALL OF THEM AND WE KNOW FOR A FACT IN DIABETIC INDIVIDUALS IT IS ELEVATED. ALSO ELEVATED IN PEOPLE WHO EAT GLUCOSAMINE. >> MY QUESTION IS IN RELATION TO THE SPLENDA, A SUGAR SUBSTITUTE. IF YOU LOOK AT THE STRUCTURE OF SPLENDA -- >> IT'S NEVER BEEN LOOKED AT BUT I WOULDN'T PREDICT SPLENDA WOULD ELEVATE IT. BUT IT'S NEVER BEEN LOOKED AT THAT I KNOW OF. SO BASED ON A NUMBER OF STUDIES BY OUR LAB AND JOHN'S LAB AND OTHERS THAT SUGGESTS THAT ONE OF THE THINGS THAT IS BECOMING CLEAR, AND I THINK THAT PHOSPHORYLATION PEOPLE ARE COMING TO GRIPS TO THIS AS WELL, IS THAT IT'S NOT NECESSARILY THE STOKEYOMITRY THAT IS IMPORTANT FOR THE BIOLOGY, IT'S THE CYCLING RATE. AND WE KNOW THAT IF YOU KNOCK DOWN OGT, THE O-GLCNAC ACE GOES DOWN IN PARALLEL EVEN THOUGH THEY ARE ON DIFFERENT CHROMOSOMES, WHICH IS KIND OF BIZARRE. SO THE CELL WANTS TO KEEP THE RATIO THE SAME. WE KNOW THAT THE ENZYME IS ACTIVATED BY TYROSINE PHOSPHORYLATION, O-GLCNAC TRANSFER ACE. WE KNOW THAT IT LOOKS LIKE IT IS SPECIFICITY IS REGULATED BY TARGETING BUT MORE WORK HAS TO BE DONE ON THAT. AND THEN A LOT OF TIMES, PARTICULARLY IN TRANSCRIPTION COMPLEXES, OGT AND OGA ARE FOUND IN THE SAME COMPLEX. SO, WE WANT -- THERE HAS TO BE A SWITCH OTHERWISE YOU