Bio 285 Exam #2 Review (Laura)- Allostery – a molecule that binds an enzyme somewhere other than the active site and controls the enzyme’s functiono To regulate activityo Causes enzyme to change shape & activity- **Mechanism of enzyme function stuff & mechanism of protein regulation stuff – should know for exam (basically memorize slides)- **Memorize pathways for exam- Q: What makes it an active site?o Enzyme is a molecule (usually a protein) that catalyzes a chemical reaction; active site defined as where substrate binds & where chemical reaction occurso Binding to somewhere other than the active site = changes enzyme shape = affects ability to bind substrates = change ability to catalyze chemical reaction (have to be in active site to catalyze chemical reaction)- Q: intrinsic kinase activity vs. extrinsic kinase activity?o Intrinsic = kinase activity of thing you’re talking about; its own kinase activityo Extrinsic = kinase activity of another molecule?- Autophosphorylation increases intrinsic kinase activity- Wildtype Abl kinase may dimerize & autophosphorylate, don’t run around the cell as dimerso Bcr gives them capability to tetramerize (or polymerize) brings two relations in close to each otherand that cross-phosphorylation activates that kinase activity- **Know parts of Abl (skip Bcr-Abl stuff), how does Abl control itself (autoinhibitory function, how regulated)- **Ras/GEF/GAP stuff – should know- Ras mutations cause cancer/Ran mutations do noto Ras and Ran structurally related, but who Ran and Ras bind to are differento Ran GEF/GAP different from Ras GEF/GAPo Essentially molecular switcheso Ras-GDP bound state = molecular switch = things to be regulated in cell- Q: compare/contrast Ras & G proteins?o Less related- Bad’s function is to inhibit Bcl2o For survival of cells, want Bad to be offo But sometimes cell survival is not right thing – sometimes cell should be activating programmed cell death pathways Not having programmed cell death/not regulating it correctly can be not goodo Bad’s function is to bind Bcl2 – then undergo cell deatho Bcl2’s job is to inhibit programmed cell death when activeo Bcl2 binds to something on mitochondria when it’s doing its job Actively inhibits cell deatho Bad tries to pull Bcl2 off mitochondria & sequester it Then becomes inactiveo Bad+Bcl2 = programmed cell death happeningo Bcl2 alone = programmed cell death not happeningo Active AKT = programmed cell death not happening b/c AKT phosphorylating Bad which can’t bind Bcl2o When AKT active, Bad is inactive When protein phosphorylated, protein is inactive in this caseo AKT must be phosphorylated twice (at two different sites) (active state when phosphorylated)- Sequential phosphorylationo Kinase active in cell cycle, kinase active later in cell cycleo First kinase must be phosphorylated for second kinase to be phosphorylatedo Might be happening to Ablo First site opens up possibility for second site- Q: does AKT need to be bound to PIP3?o Phosphorylated by two different kinases – “that’s good enough”o Does one of protein kinases need to be bound to PIP3 too? Yes – one anchored at PIP3 and one soluble- Q: if protein kinase 1 isn’t working, could protein kinase 2 phosphorylate AKT?o PK1 is dependent upon PK2o 1 needs to phosphorylate first, then active site opens for 2o Sequential- Survival signaling, dimerization, pip2->pip3, etc.- Q: how do proteins bind to PIP3?o PH- PTEN – o Takes phosphate off PIP3 and gives back to PIP2o Tumor suppressoro LOF in PTEN make pathway too active – survival pathway so making them too active leads to cancer- Q: overall picture in inhibiting apoptosis, survival signal transduction and cancer – how do they relate?o Cells with cancer, often find LOF in proteins that lead to programmed cell deatho Overall survival pathways are functioning too well/programmed cell death pathways aren’t working well enougho Pathways that lead to programmed cell death broken in some way- Q: RTK pathway leads to the inhibition of apoptosis, so if too active that leads to cancer?o Contributing factoro Lots of ways to break pathwayso Having overdivision pathwayo Having broken DNA damage response pathwayso PIP3 major regulator of response of bad things happening in cell – break that, can’t respond normally to cell damageo Which aspects of this pathway would lead to cancers? Anything that causes survival to happen when it shouldn’t happen- Q: question 12 on practice q’s – LOF Ras GAP? What would happen?o Ras would be bound to GTP all the timeo Any part of pathway that wouldn’t transduce signal? Problem with this? Couldn’t turn it off – potentially oncogenico Ras still binding to RTK = no- Go over JAX/STAT pathway:o Signal that causes receptor dimerizationo Receptor itself in JAX/STAT has no enzymatic activity – just brings this togethero JAK1 and JAK2 have enzymatic activity – noncovalently bound to the receptoro When JAK1 and JAK2 brought into close proximity to each other b/c signal binds so dimerizes – JAX are nonreceptor tyrosine kinases Nonreceptor tyrosine kinases are soluble tyrosine kinases who can float around cello JAK1 and JAK2 will phosphorylate each other & activate each other when close/dimerization brings them closeo Then activate downstream proteinso Once JAX are active they phosphorylate each other and then phosphorylate the receptoro That creates binding sites for the STATs Means STATs probably have a SH2 domaino STAT comes and binds, gets phosphorylated by GAPs, causes them to change shape, then dissociate from where on receptor and then STAT1 and STAT2 come together (dimerize) and they go into the nucleus and activate transcription STATs are transcription factors- **Bcr-Abl not necessary to know for exam - **Background on CML pathology not necessary to be memorized- Q: why does nonfunctional SH2 domain lead to activation of Abl?o Could argue SH2 either wayo SH2 could play important roles in downstream signaling for Abl, but also could play important autoinhibitory roleo Get rid of SH2, potentially have overactive kinase- **Go back and reread Abl review on Perusall- Abl – autoinhibitiono “CAP” – where myristate is bound SH2, SH3, linker – forms intramolecular interactions inhibited stateo Survival (cytoplasmic), cell mobility (cytoplasmic), DNA damage response (nuclear) DNA binding domain  DNA damage response Actin binding domain o Proliferation –