module 6 Flashcards
explain: dictyostelium cycle
- starts from unicellular amoeba -> slug -> fruiting body
- eukaryote
- not enough resources -> amoeba work together (aggregate) and form slug
⤷ happens in response to signaling molecule (cAMP from starved cells)
⤷ slug can move to resources (heat, light, food) - slug cells eventually differentiate into prestalk and prespore cells of fruiting body
⤷ ant. end = stalk, post. end = spore
explain: aggregation as a response to cAMP signal (components, purpose of activation)
- sig. = cAMP
- receptor = GPCR
- activation = cells reorganizing their intracellular actin cytoskeleton network to move towards source of signal
⤷ chemotactic resp.
explain: cell mvt. towards cAMP signal
- dynamic filopodia extend out to allow mvt.
- actin reorganization (allows mvt.):
⤷ nucleation
⤷ polymerization
⤷ depolymerization
explain: cell that can’t resp. to cAMP signal
- mutation in gene for clathrin heavy chain
- cell unable to form vesicle req. for transport to cell mem.
⤷ causes no mvt. to sig. - no prot. transport -> GPCR not moved to surface of cell -> cAMP not detected -> no mvt.
explain: human neutrophil cell (mvt.)
- neutrophils = WBC
⤷ have recep. on surface that bind to sig.
⤷ binding -> activation of internal changes that facilitate mvt. - function = neutrophil can capture and engulf bacterium in endocytosis
- neutrophils resp. to sig. from bac. that have invaded
⤷ bac. unintentionally prod. sig. bc have prot. w/ tripeptide (fMLP)
⤷ neutrophil (GPCR) can recog. fMLP - signal = fMLP
- receptor = fMLP receptor, GPCR
define: signaling
- transmission of information from one cell to another that induces a change in behaviour/response
- signal only useful if there’s a resp. to the sig.
explain: principles of signal transduction
- signalling cell prod. + releases sig. molecules
- target cell has recep. that specifically binds to that sig.
- binding of sig. activates recep.
⤷ initiates cascade of chemical events in target cell - cascade of events interpret and transduce sig. into what we refer as signal transduction pathway (STP)
- culminates in change in target behaviour
- resp. ex.:
⤷ changes in transcription
⤷ cell mvt. or growth
⤷ cell differentiation
⤷ changes in metabolism corresponding to enz. activation/inactivation - sig. needs to be removed to terminate target cell resp.
**only target cells w/ appropriate recep. able to resp.
explain: specificity of signal-receptor interactions
- complementary shapes allow 2 mol. to come closer together
- non-covalent interactions give specificity and high affinity
- one single AA change can reduce or eliminate sig. binding -> disrupt signaling
- rule: recep. will only bind to 1 natural ligand or closely-related molecules
name + explain: 2 levels of specificity of sig. response
- specificity of ligand for binding to recep.
- specificity of intracellular resp. that is mediated by STP
- many cells may be exposed to sig. but only ones w/ matching recep. can resp.
- diff. cells may receive same sig. but resp. differently through activation of diff. proteins
- but some cells may resp. to same sig.
name: example of fast cellular resp. and example of slow cellular resp.
FAST = changes in enz. activation
- sig. binds to mem. associated recep.
- cytosolic enz. activated
- cell quickly resp. to sig. by just changing activity of prot. already present in cell
SLOW = changes in gene transcription
- changes in protein lvls w/in cell = slower
- recep. in cytosol + gets activated by sig.
- recep. gets transported to nucleus
⤷ acts as transcriptional activator to make mRNA
- mRNA get translated to increase pro. lvls
- slow bc dep. on transcription, translation, prot. folding, prot. mod.
explain: secreted signals (2)
ENDOCRINE
- sig. released into circulatory sys.
- only cells w/ correct recep. resp.
- diff. tissues can resp. at the same time
- sig. cell and target cell usually far away from one another
- ex. secreting hormone
PARACRINE
- sig. released into extracellular space -> diffuse into neighbouring cells
- sig. cell and target cell = near one another
- ex. growth factors, neurotransmitters
explain: proximal signaling
- requires direct contact between target + sig. cells
- neighbouring cells can also comms by sharing cytosolic messengers
⤷ ex. in plants and animals
⤷ plants: plasmodesmata = junction between 2 cells (ex. transport sig. from roots to leaves)
⤷ animals: gap junctions (ex. allow cell to diffuse small molecules from one cell to another)
explain: autocrine signaling
- cell comms w/ itself
⤷ sig. cell = target cell - produces the secreted sig. = carries recep. for the sig.
- ex. growth factors to induce/stop cell division
name: types of cell-surface receptors (3 for this class in mod. 6)
- g-prot. coupled recep. (GPCR)
⤷ activates to prod. cAMP to reg. cell metabolism - cytokine recep.
⤷ JAK/STAT = control prod. of RBC - recep. tyrosine kinase (RTK)
⤷ linked to phosphorylation cascade through small G-prot. (Ras) to reg. gene exp.
explain: Epo signal
- Epo = erythropoietin
- response = cells proliferate
- used for ethrocytes (RBC)
- Epo exp. = regulated by oxygen binding transcription factor in kidneys
- cells carry Epo receptor
- Epo = cytokine, EpoR = cytokine recep.
- recep. = linked to JAK-STAT sig. transduction pathway
- resultant resp.:
⤷ inhibition of cell death
⤷ changes in gene exp. pattern
⤷ differentiation - no Epo = erythroid progenitors undergo apoptosis
explain: components of the Epo pathway
- signal = Epo prot.
- recep. = erythropoietin recep.
- intracellular sig. transduction pathway = JAK-STAT
⤷ JAK kinases
⤷ STAT transcription factors - resp. = transcription of STAT targets + inhibition of apoptosis
**normally if Epo available, sig. reacts w/ recep. -> dimerization
explain: 3 domains of Epo recep. (+autophosphorylation)
- cytosolic domain
- transmembrane alpha-helix domain
- extracellular domain
- each recep. associated w/ JAK kinase
- unphosphorylated JAK = unactive, very weak kinase activity
⤷ but Epo binding -> dimerization -> the 2 JAK kinases get moved closer together - move so close they are enough to phosphorylate a nearby JAK kinase (autophosphorylate)
explain: JAK kinase (as a tyrosine kinase)
- specifically a tyrosine kinase
- only tyrosine gets phosphorylated
question: how does JAK kinase phosphorylate tyrosine?
- Epo recep. get activated
- phosphorylated docking sites get phosphorylated -> become available for prot.-prot. interactions
⤷ can bind w/ STAT transcription factors - binding makes STAT prot. go from inactive to active
- STAT has domain = SH2
- SH2 recognizes phosphorylated tyrosine
- STAT accumulates on Epo docking sites (bc tyrosine)
- STAT gets phosphorylated by JAK -> gets dimerized
- now dimerized STAT can be transported to nucleus to activate target genes
question: how does STAT recognize phosphorylated tyrosine?
- allows prot. to bind to specific target substrates
- links prot. in a pathway
- SH2 recognizes specific AA prot. seq. and will bind w/ high affinity if tyrosine is phosphorylated
⤷ low affinity if unphosphorylated - do not bind to corresponding unphosphorylated peptide
⤷ allows prot.-prot. binding to be reversible
question + explain: what are the target genes of STAT transcription factors? (1 ex.)
- Bcl-XL gene -> Bcl-XL prot.
⤷ inhibitor of apoptosis
⤷ allows erythroid progenitor cells to persist and eventually differentiate - bone marrow = primary source of erythrogenesis but also from liver
⤷ more visible in fetal liver
explain: result of mouse fetal liver + Epo recep.
- STAT5 activation regulates genes that differentiate Epo. cells into RBC
⤷ gene ex.: Bcl-XL (inhibitor of apotosis)
WILDTYPE MOUSE
- bright red
- bc liver creating RBC
MOUSE
- no Epo -> no RBC being made
- no red
- homozygous for loss of func. allele of Epo gene
question: how to turn off Epo formation signaling path? (3)
-
reversing phosphorylation (short term inac.)
- phophatase prot. dephosphorylates modified AA
- ex. SHP1
- SHP1 has 2 SH2 domains that allow it to dock where STAT docks and dephosphorylates JAK
- inactivates JAK (short term)
- allows fast reactivation of JAK -
SOCS prot. (long term inac.)
- SOCS = suppressor of cytokine signaling
- SOCS can bind to phosphorylated docking sites via SH2 domain
- SOCS exp. when high O2 lvls
- block access of STAT to docking sites on Epo. recep.
- SOCS is also an E3 ubiquitin ligase
⤷ targets JAK
- removing JAK -> turns off pathway
⤷ reactivation = slow bc needs exp. of new JAK proteins -
recep. recycling + sig. release (long term inac.)
- sig. turn off when recep. gets internalized through endocytosis and ligand dissociates
- recep. can be recycled back to surface of cell when needed
- if Epo lvls go back down, recep. won’t be reactivated
question: pros and cons of RBC formation (continuous)?
- disabling Epo formation = bad
- continuous Epo formation - bad
- overproduction of RBC -> elevated haematocrit
- elevated haematocrit -> increased viscosity -> blockages of vessels
- can be good for athletes (Epo doping)
⤷ more RBC -> increased capacity to carry O2 + increased endurance
name: sig. of receptor tyrosine kinase and Ras
- NGF (nerve growth factor)
⤷ induces neural cell differentiation - PDGF (platelet derived growth hormone)
- EGF (epidermal growth factor)
- insulin
- in all cases, hormone interacts with a transmembrane receptor tyrosine kinase
⤷ will activate intrinsic kinase activity of that receptor
explain: RTK (functions, description)
- receptor tyrosine kinases
- associated w/ Ras G-prot. activation
- pathways:
⤷ cell differentiation
⤷ cell survival
⤷ apoptosis
⤷ cell division
⤷ proliferation
⤷ changes in cell metabolism
explain: components of the RTK pathway
**RTK = ligand gated (ligand = sig.)
- ligand = growth hormone
- recep. = RTK
- have extracellular sig. binding domain, single-pass transmem. domain, intrinsic kinase activity
- ligand binding leads to dimerization of receptor + autophos.
- RTK sig. = longer than cytokine + involve activating Ras (g-prot.)
explain: regulation of Ras in RTK pathway
- requires prot. that link it to the activated RTK
⤷ adaptor prot. = GRB2
⤷ Ras effectors = GEF and GAP - activation leads to kinase cascade -> activation of MAP kinase
- MAP kinase modulates cell beha. by phosphorylating transcription factors + changing gene exp. patterns
question: how does RTK get activated?
- ligand binds
- leads to recep. dimerization
- RTK recep. on its own = weak
⤷ dimerization -> can phosphorylate (or autophos.) lip of tyrosine kinase - phosphorylating -> increases intrinsic kinase activity of recep.
⤷ allows more prot. to get phos. - tyrosine on docking sites of RTK become targets of kinase
- phosphorylated docking sites become potential binding sites for prot.-prot. interaction domains
explain: adaptor proteins role at RTK docking sites
- carry 2+ prot. interaction domains that allow prot. to act as linkers between other prot.
- indirectly links prot. in the pathway to the RTK recep.
- adaptor prot. = scaffold prot.
explain: example of adaptor prot. (for RTK)
- GRB2
- has 3 prot.-prot. interaction domains
⤷ 1 SH2, 2 SH3 domains - SH2 recog. tyrosine
- SH3 bind to the next prot. in the pathway
- SH3 always bind to proline rich domains but SH2 binding dep. on reversible phosphorylation of docking sites
explain: activation of Ras (from RTK pathway)
- Ras = G-prot.
- RTK activation -> Ras activation
- regulated by GTP binding
⤷ bound to GTP = active
⤷ bound to GDP = inactive - G-prot. has intrinsic GTPase activity
⤷ always active but can be modulated
ACTIVE
- arms interact w/ terminal phosphate on GTP in binding site
- GPT fits specifically in binding pocket
- pull the arms/switches together into “ON” conformation
⤷ interacts w/ glycine and threonine on each switch
INACTIVE
- needs GTPase to turn “off” prot.
- GTPase hydrolyzes GTP -> GDP
- GDP in binding pocket but no terminal phosphate so switches aren’t held inwards
- arms open into “off” position
- GDP = low affinity for binding pocket -> GDP leaves
- stays off until GTP comes in binding pocket
explain: prot. that regulate Ras inac./activation (3)
-
GEF (guanine nucleotide exchange factor)
⤷ promotes dissociation GDP
⤷ allows GTP to bind
⤷ accelerates activation of Ras -
GAP (GTPase activating prot.)
⤷ enhances intrinsic GTPase activity (100-fold)
⤷ inhibits Ras activation -
GDI (guanine nucleotide dissociation inhibitor)
⤷ increases affinity of binding pocket for GDP
⤷ GDP stays longer -> Ras “off” for longer
⤷ inhibits Ras activation
recap: Ras-GDP/GTP cycle (question: what would happen w/out GAP?)
- Ras = OFF
- GDI promotes GDP binding
- GEF promotes GDP dissociation
- GTP comes into pocket
- Ras = ON
- interacts w/ target prot
- GAP promotes GTPase activity
- Ras = OFF
**Ras stays ON for a fixed amount of time depending on presence of GAP
- no GAP = increased ON time
⤷ more signaling
⤷ more of target prot. is activated
name + explain: GEF for Ras
- Ras interacts w/ GEF called SOS
- SOS = prot. relocated to mem. through indirect assoc. to activated RTK recep.
- SH3 domains of GRB2 hold SOS close to mem.
- brings SOS close to Ras
⤷ promotes release of GDP and binding of GTP -> activating Ras
name: 3 conformation states for Ras
- inactive Ras-GDO
- SOS binding (displaces GDP)
- active Ras-GTP
name + explain: GAP for Ras
- Ras interacts w/ GAP called NF1
- enhances GTPase and accelerates hydrolysis of GTP
- inactivates Ras + shortens length of time Ras is active
- evidence: mut. causing loss of NF1 -> elimination of GAP -> increased Ras active time
question + explain: is Ras downstream of RTK in the pathway?
- 4 conditions tested w/ EGF
- adding EGF -> - sig. binds to EGF-RTK recep.
- typically induces cell div.
-
RTK activates Ras (RTK upstream)
- YES cell proliferation
- control -
Ras activates RTK (RTK downstream)
- remove Ras by adding antibody before adding EGF
- NO cell division
- despite activation of RTK by EGF, no downstream step -> no signaling - RTK and Ras = parallel, indep. paths and either lead to cell division
-
RTK and Ras = parallel + both needed for cell division
- replaced Ras w/ always active ver. = Ras-D
⤷ lacks GTPase
- no EGF added + no RTK activated
- YES cell division
- bypassed effect of inactive RTK by having an always active downstream
- assoc. w/ over proliferation (tumourigenesis)
- result: Ras = downstream from RTP
⤷ dep. on RTK recep. activation
question: how does disruption in RTK pathway lead to cancer?
- mutations causing always active Ras -> cancers
- ex. single glycine elimination in Ras
⤷ blocks binding of GTPase accelerating prot. - ex. Her2
⤷ RTK linked w/ hereditary forms of breast cancer
⤷ wildtype Her2 = recep. for EGF sig.
⤷ mutant Her2 = does not resp. to sig. + always activated bc always dimerized
⤷ causes uncontrollable cell division - ex. NF1
⤷ when absent, leads to uncontrollable cell division
explain: Raf activation
- caused by Ras activation
- Raf has phosphorylated AA bound by 14-3-3 adaptor prot.
⤷ holds Raf in inhibited conformation - Ras binds + releases Raf from prot.
define + explain: Raf + MAP kinase
- serine/threonine kinase prot. at the top of a kinase cascade in the RTK path
⤷ phosphorylates serine and threonine - Raf = MAP kinase kinase kinase (MAPKKK)
- activation -> phosphorylates target prot. (MEK)
- MEK phosphorylates MAP kinase at tyrosine and threonine -> activating the prot.
- MAP kinase = serine/threonine kinase
⤷ dimerizes when activated and moves to nucleus
explain: purpose of MAP kinase (target)
- target = P90 RSK kinase
- P90 RSK kinase gets phosphorylated -> translocated to nucleus
- MAP kinase also translocated to nucleus
- in nucleus: they each phosphorylate a target transcription factor
⤷ TCF (ternary complex factor) by MAP
⤷ SRF (serum response factor) by P90 - transcription factors bind to DNA seq. (serum resp. element SRE)
- SRE = enhancer sequence upstream of a collection of genes
- binding promotes assembly of RNA poly. + transcription of target gene
- ex. C-fos has the upstream SRE gene
⤷ C-fos = codes for transcription factor that enhances rate of transcription of genes that reg. cell cycle
explain: struc. of GPCR (examples of GPCRs)
- common struc. = 7 transmem. alpha helix domains that loop through mem. to form final functional receptor
- creates 4 extracellular segments (E1 - E4)
⤷ fold to form signal-binding domain - creates 4 cytoplasmic segments (C1 - C4)
⤷ fold to form internal domain that interacts w/ trimeric G prot.) - ex.:
⤷ recep. that initiate stress resp.
⤷ light activated rhodopsins in eye
⤷ odourant recep. in nose
⤷ hormone + neurotrans. recep.
⤷ plant growth hormone recep.
⤷ glucose sensing in yeast
explain: components of GPCR pathway (adrenergic stress resp.)
- sig. = catecholamines
⤷ epinephrine (adrenaline), norepinephrine (noradrenaline) and dopamine - recep. = GPCR
- release from adrenal medulla of adrenal glans = part of fight/flight resp.
- involves activation of recep. assoc. trimeric G-prot.
⤷ also involves activation of effector prot. = adenylyl cyclase - adenylyl cyclase modulates cytosolic conc. of cAMP
- effects of increased cAMP:
⤷ changes release of stores E for fight/flight - fast resp req. activation of enz
- slow resp. activates transcription
explain: subclasses of adrenergic recep.
- 2 subclasses
- alpha-2-recep.
- beta adrenergic recep.
- epi. can bind to both
⤷ but gives diff. resp. - beta = stimulator, alpha = inhibitory
explain: beta adrenergic recep. (location, result)
- stimulatory
- in liver + adipose -> stim. glycolysis and lipolysis for fuel mobilization
- in heart musc. -> increase contraction -> increased blood supply
- in smooth musc. in intestine -> increase musc. relaxation so all E can be focused on fueling locomotory musc. for fight/flight
explain: alpha 2 adrenergic recep. (location, result)
- inhibitory
- in cells of blood vessels of skin, kidney
- in smooth musc. cells of intestine-
- overall resp.: cause arteries to constrict + reduce supply of blood to periphery
explain: catecholamines
- water soluble sig. in bloodstream
- prod. from adrenal glands
- secretes hormones (epi.) and steroids (aldosterone and cortisol)
- binds to both alpha and beta GPCR
⤷ induces diff. resp. dep. on which recep.
-
question: how to activate GPCR + connection to effector (adenylyl cyclase)
- GPCR inactive = not associated w/ trimeric G-prot.
- binding -> conformational change in intracellular domain of GPCR
⤷ allows it to act w/ high affinity to trimeric G prot. - causes conform. change -> GDP dissociates from G-prot.
⤷ allows binding of GTP to pocket - GTP binds to G-prot. -> active
- trimeric G-prot. dissociates releasing G-alpha subunit
- subunit can move laterally to interact w/ effector enz.
⤷ enz. only active if G-prot. = associated - length of activation for enz. = dep. on GTPase activity of G-prot.
- GTP hydrolyze -> GDP
- G-prot. = inactive
- G-alpha subunit = released from effector + inactivates effector enz.
explain: activation for effector enz. (adenylyl cyclase) + impact of beta adrenergic recep.
- has 3 subunits
⤷ alpha, beta, gamma - activation -> alpha subunit dissociates from trimeric complex + binds to adenylyl cyclase
- adenylyl cyclase role = increase intracellular conc. of cAMP
- hydrolysis causes GTP -> GDP
⤷ inactivates G alpha and dissociates it from adenylyl cyclase - absence of adenylyl cyclase -> a lot of cytosolic enz.
⤷ decrease cAMP conc.
question: how does adenylyl cyclase make cAMP?
- converts ATP into cyclic AMP while releasing diphosphate
- active adenylyl cyclase = high cAMP conc.
explain: degradation of cAMP
- constant phosphodiesterase counteracts actions of adenylyl cyclase
- phosphodiesterase catalyzes breakdown of cAMP into non=cyclic form of 5’AMP
- if active adenylyl cyclase makes cAMP and phosphodiesterase breaks it down, cAMP still high
- if adenylyl cyclase inhibited, cAMP conc. low
explain: function of cAMP + define: PKA
- important role in secondary messenger in cells
- responds to GPCR pathways
- determines activation or inactivation of next step in sig. pathway
⤷ so it modulates activity of target prot. - ex. protein kinase A (PKA)
⤷ PKA = serine/threonine kinase that phosphorylates targets
explain: activation of PKA
- inactive PKA = tetrameric
⤷ 2 regulatory subunites
⤷ 2 catalytic subunits - reg. subunits have binding sites that bind cAMP
- low cAMP = no cAMP in binding sites -> inactivation
- if cAMP conc. increases and cAMP binds -> conformational change in pseudo-substrate domain of regulatory subunit releases the catalytic subunit
- PKA = active
question: what is the difference between active and inactive PKA (struc.)?
ACTIVE
- cAMP bound
- pseudo-substrate retracts
- allows for activation of PKA enz./catalytic subunit
INACTIVE
- cAMP released
- pseudo-substrate domain extended
- blocks substrate binding domain of PKA
question: what are the targets of PKA? what is the resp. to PKA?
- resp. to epi. sig. = increase in E supply to tissues in the body
- to release glucose to cells, body needs ATP
⤷ so PKA targets glycogen as a source of glucose - glycogen = can be broken down into glucose by glucose phosphorylase
- stress resp. = inhibit glycogen synthase and promote glycogen phosphorylase
⤷ make less glycogen, break it down into glucose
question: what happens for short term resp. in fight/flight in terms of PKA and energy (in musc, in liver)?
MUSC.
- glycogen breaks down into glucose-6-phosphate
- glycolysis produces pyruvate and NADH for ATP production
- more ATP powers skeletal musc. for fight/flight
LIVER
- glycogen breaks down into glucose-6-phosphate
- PKA phosphorylates phosphorylase kinase which activates glycogen phosphorylase
- liver cells also inhibit prod. of more glycogen
⤷ bc PKA inactivates glycogen synthase
- releases free glucose into blood stream for fast transport to body
^fast + short term resp. to epi. bc just mod. enz. already present in cell
question: what happens for long term resp. in fight/flight in terms of PKA and energy (w/ CREB)?
- PKA starts inactivated
- catalytic PKA subunit translocated to nucleus
- transcription factors
⤷ CREB binds to cAMP resp. element CRE - CREB bound to CRE enables assembly to initiate transcription
- slower resp. but targets genes req. for prod. of glucose
⤷ ex. genes for phosphorylase kinase and glycogen phosphorylase
explain: amplification of the signals in signaling pathways
- can be amp. between more and more numbers of prot. in a pathway
- ex. stress resp. = between steps of adding of epi. - prod. of glucose = 10^8 fold amplification
- amp. often seen at steps involving enz. activation (ex. adenylyl cyclase)
