BIOLOGY 3000 Exam 3

Microtubule-associated proteins (MAPs) regulate MT structure & dynamics

+TIP protein EB1 stabilizes MTs by stimulating rescue Fig 18-14

cAMP binding to R subunits of PKA is cooperative:

-Binding of 1st cAMP to CNB-B increases affinity of 2nd cAMP to bind CNB-A -cAMP is an allosteric activator of PKA: Changes activity by binding to regulatory, not catalytic site

How do protein kinases distinguish between S/T & Y?

-IRK: Deep cleft to accommodate; Serine too "short" to contact y phosphate -CDK2: Shallow Cleft to accommodate Ser/Thr; Tyr too large to fit into cleft

Calcium-Calmodulin Complex

-Increases cAMP Phosphodiesterase activity from 50% to 100%

Comparison of binding & physiological response curves for a "generic" ligand-receptor complex Fig 15-8

-Kd = [ligand] required to bind 50% of receptors = [R] [L] / [RL] -In general, [L] necessary to elicit 1/2 maximal physiological response is significantly less than Kd i.e. requires only a small fraction of bound receptors. There is not a linear correspondence between # bound receptors & physiological response. -Illustrates that signal amplification occurs during signal transduction pathway.

Redundancy

-PKA & PKC recognize & phosphorylate the same Thr on GS to inhibit its activity -PKA & BARK recognize & phosphorylate the same S/Ts on B-Adrenergic receptor to desensitize it

The fundamental problem of cell signaling: How does a cell "transduce" the binding of an extracellular ligand to elicit both short-term and long-term intracellular physiological responses?

-Short-term, immediate effects due to altering activities of pre-existing proteins (e.g., changes in glucose metabolism by GPCR signaling) -Long-term effects due to altering gene expression & protein synthesis (e.g., changes in cellular proliferation by RTK signaling)

Terminating the Signal II

-There is always an excess of PP relative to IP so PP is never completely inhibited -This small pool of active PP rapidly dephosphorylates IP when PKA activity drops & rapidly generates additional PP to dephosphorylate other PKA targets (GPK, GP & GS)

Different trimeric G protein complexes alter the activities of effector enzymes

1) Adenylyl Cyclase - cAMP (increased) - B-Adrenergic Receptor and Glucagon Receptor 2) Phospholipase C - IP3 and Dag (increased) - A-Adrenergic Receptor and B-Adrenergic Receptor 3) Phospholipase C - IP3 and Dag (increased) - Acetylcholine receptors in endothelial cells

Progression of MT Assembly Fig 18-3

-Beta-tubulins exposed -GTP on beta-tubulin hydrolyzed to GDP after polymerization newly added (+) end has GTP cap -13 protofilaments make up one singlet (majority exist as)

PKC

-Ca2+ binds directly to PKC causing it to migrate to the plasma membrane, where it can interact with DAG -PKC phosphorylates & inhibits glycogen synthase (GS) -GS only relevant PKC target -PKC inhibition of GS is redundant with PKA, not additive!

Calcium as Second Messenger

-Ca2+ stimulates GPK activity 50% & increases glycogen degradation -Ca2+ also stimulates cAMP phosphodiesterase from 50% activity to 100% & reduces [cAMP] -Ca2+ does not bind directly to either protein

Caffeine & Theophylline (Tea)

-Caffeine & Theophylline are competitive inhibitors of cAMP Phosphodiesterase (PDA1) Net Result: -PKA & GPK remain hyper-active (& so do you - Hence the buzz) But glycogen stores are depleted; brain & muscle are glucose-depleted -Result? - Hit the coffee or soda again cycle repeats

Cholera toxin ADP-ribosylates Gas & inhibits its GTPase activity

-Causes persistent activation of Adenylyl Cyclase & mimics binding of GMPPNP or GTP-y-S to Gas -ADP-ribose forms an ester bond between the COO- of a Glutamic acid on Gas & the 3'OH of Ribose

The Kinesin Strut Fig 18-21

-Each kinesin step covers 16 nm -Each kinesin walks along the same protofilament on the same MT -Kinesin transports vesicles 3µm/second

Why do most S/T kinases prefer S over T?

-F in cleft confers preference for Ser. (F present in most S/T kinases) -V in cleft confers preference for Thr

Cofilin

-Fragments ADP-F-actin increases # of (-) ends & enhances depolymerization Fig 17-11

GPK

-GPK = (abyd)4 -y (gamma) = Catalytic Subunit -ab (alpha & beta) phosphorylated by PKA -PKA stimulates GPK to 50% max. activity -(delta) = Ca2+•Calmodulin; also stimulates GPK to 50% max activity -100% GPK activity requires both PKA phosphorylation and Ca2+•Calmodulin

Kd

= [ligand] required to bind 50% of receptors As Kd goes up, affinity goes down

"FRET" Fluorescence Resonance Energy Transfer

-X & Y are any 2 interactive proteins each fused to a different FP -Emission spectra of "donor" overlaps with absorption spectra of "acceptor" -Excite X-CFP with ~430 nm: If X & Y are not associated, no Y-YFP observed at ~530 nm & see only X-CFP at ~475 nm -Excite X-CFP with ~430 nm: If X & Y are associated, observe Y-YFP at ~530 nm (X-CFP emission is "quenched" by Y-YFP) -FRET most sensitive & widely-used experimental approach to assay real-time changes in protein-protein interactions in living cells

Rates of GTP hydrolysis control dynamic instability

-alpha-tubulin on the "newest" dimer is a GAP that stimulates GTP hydrolysis on the adjacent "older" -tubulin -Therefore, only an actively elongating MT has "new" GTP•Tubulin at its (+) end -If growth stops, then slow GTP hydrolysis ultimately converts (+) end to GDP cap & catastrophe results -In summary: A growing MT tends to keep growing while a static MT will tend to shrink

Relaxation of Muscle Pathway Fig 15-38

1) Acetylcholine 2) Acetylcholine GPCR 3) Gq 4) Phospholipase C 5) IP3 6) Calcium/Calmodulin 7) NO Synthase 8) "NO" a biological "neutrino" 9) Guanylyl Cyclase - NO binds directly to Guanylyl Cyclase in smooth muscle cells 10) cGMP 11) Protein Kinase G 12) Relaxation of Muscle Cell *cGMP phosphodiesterase (PDE5) converts cGMP to 5' GMP blocks muscle relaxation & promotes vasoconstriction

How did this modification evolve & how can phosphorylation be used to either activate or inactivate certain proteins?

1) Constitutively active protein when non-phosphorylated 2) Constitutively active protein requiring an R-E interaction 3) E to S substitution inactivates protein activity 4) Protein activity now negatively regulated by phosphorylation (e.g., glycogen synthase) 5) Phosphorylation now positively regulates protein activity

Terminating the Signal

1) Dissociate [HR] Complex 2) Convert Gas•GTP to Gas•GDP & inactivate Adenylyl Cyclase 3) Activate cAMP Phosphodiesterase - Converts cAMP to 5' AMP (1 function of Ca2+ is to fully activate cAMP Phosphodiesterase)

MT polarity necessitates the use of directional motor proteins Fig 18-28

1. (-) end motors (e.g., dyneins) carry cargoes only towards (-) end 2. (+) end motors (e.g., kinesins) carry cargoes only towards (+) end

3 classes of protein kinases based on amino acids phosphorylated on target proteins

1. Serine/Threonine (S/T) Kinases (~400) 2. Tyrosine (Y) Kinases (~90) 3. "Dual Specificity" Kinases can phosphorylate S/T or Y (~40)

Determining MF organization: Actin cross-linking proteins form bundles or networks

1. Tight bundles 2. Loose Bundles 3. "Spoke-like" connections to plasma membrane 4. Large networks 5. Linear connections to plasma membrane Figure 17-20

What happens at the end of the road?

Kinesin motors on MTs hand off cargoes to myosin motors on MFs

In general, motors directionally transport cargoes (e.g., vesicles or organelles) on stationary MTs Fig 18-20

Kinesin-1 and Kinesin-5 However, in the mitotic spindle, the "cargo" can also be another MT

Microtubule-associated proteins (MAPs) regulate MT structure & dynamics

Kinesin-13 hydrolyzes ATP to "curve" protofilaments into GDP-conformation & promote catastrophe by dissociating terminal tubulin dimers

Switch II domain of Gas•GTP activates adenylyl cyclase by bringing its catalytic domains together

Figs. 15-23, 26

The Hormone-Receptor Complex functions as a GEF to activate Galpha-s

Figure 15-16

GTP Activates Ga By Enabling Its "Switch" Domains to Associate

Figure 15-5

Formin

Formin nucleates long MFs in response to extracellular signals that activate Rho•GTP

Actin

Most abundant protein in eukaryotic cells 10% by weight of total protein in muscle cells 1-5% of total protein by weight in non-muscle cells = 5 X 108 molecules/cell Ancient: Arose from bacterial ancestral protein, MreB Highly conserved

CapZ

Prevents (+) assembly when [G-actin] ≥ C+c

Tropomodulin

Prevents (-) disassembly when [G-actin] < C-c

Receptor desensitization/adaptation occurs in response to constant exposure to hormone

Prevents persistent elevation of cAMP levels & constitutive activation of PKA

Acetylcholine

Promotes vascular smooth muscle relaxation & lowers blood pressure

Which family of proteins dynamically alters microfilament (MF) dynamics to direct cell migration in response to extracellular signals?

Rho G proteins (Slide #3 Part 1 Cytoskeleton Slides, Fig. 17-41)

How can you prevent a kinase substrate from ever being phosphorylated?

S/T changed to A Y changed to F (Eliminates phosphorylation site)

How can you alter a kinase substrate so that it functions as though it is always phosphorylated?

S/T changed to E Y changed to E (Mimics phosphorylation)

Taxol

Taxol inhibits GTP hydrolysis on Beta-tubulin ("Tubulin" refers to the alpha-beta-tubulin dimer)

All MTs Originate From a Pair of Centrioles Within the Centrosome

The (-) ends of all MTs terminate within the centrosome with a ring of y-tubulin associated with other proteins termed the y-tubulin ring complex (y-TuRC) (Fig 18-7)

MFs have polarity

The 2 ends are structurally & functionally distinct Myosin "decorates" MFs & "points" towards the (-) end ATP cleft "exposed" at (-) end & "hidden" at (+) end -F-Actin is a "right-handed" double-helix

All MTs originate from a single source

The Microtubule Organizing Center (MTOC) aka the Centrosome (Centrosome contains 2 centrioles)

Dynamic Instability

The key to understand MT function Explains simultaneous growth & shrinkage of MTs in a given cell

Kinase-substrate complexes are relatively long-lived.

This means they can be biochemically isolated and you can identify the substrates for a given kinase by coimmunoprecipitating complexes using an antibody specific for the kinase.

Thymosin-Beta4

Thymosin-Beta4 prevents incorporation of ATP-G-actin at either (+) or (-) ends Fig 17-11

Signal-induced changes at the leading edge of migrating cells : Branched MFs

e.g., Fibroblast Growth Factor (FGF) Platelet-Derived Growth Factor (PDGF)

MT polymerization is [tubulin]-dependent

As (-) ends are "embedded" in MTOC, virtually all MT growth & shrinkage in cells occurs at (+) end [tubulin]cell = 10-20 µM

Getting IP3, DAG & Ca2+ Involved

B-adrenergic receptor activates trimeric Gao or Gaq complexes in liver cells that activate phospholipase C (PLC)

MT polymerization & depolymerization are temperature-dependent

Cells incubated at 4°C will lose their entire MT cytoskeleton Return them to 37°C & the MTs grow back!

PKA Regulates Both Glycogen Synthesis & Degradation Fig 15-24

- Glycogen Synthase - Directly and Indirectly Inhibited by PKA (PKA directly & indirectly inhibits glycogen synthase (GS) ) - Glycogen Phosphorylase - Indirectly Activated by PKA (PKA indirectly activates glycogen phosphorylase (GP) via a kinase cascade)

Simple FRET

- Proteins not interacting: Excite CFP; Visualize CFP - Don't see YFP - Proteins interacting: Excite CFP See YFP

Dynamics of Actin polymerization & depolymerization: ATP & ADP caps

-ATP is hydrolyzed after G-actin polymerizes into F-actin, but Pi does not immediately dissociate -"New" (+) end has ATP cap & low C+c -"Old" (-) end has ADP cap & high C-c

Signaling by Plasma Membrane-Attached Proteins

-Applicable to embryogenesis & tissue formation -Signaling & responding cells in direct physical contact

Paracrine Signaling

-Applicable to growth factors, nerve impulse transmission & embryogenesis -Signaling & responding cells in close proximity

Signal Amplification

-10^6 - 10^7 Amplification from [Epinephrine] to [Glucose] in blood -50% increase in blood [glucose] ~ 5 mM levels

Main Features of Galpha-s Activation & Initial Signal Amplification Figure 15-14

-Gas•GTP dissociates from Gbg -Gbg also dissociates from HR -Gas•GTP binds AC -HR complex acts as GEF by binding to GbyGa & causing GDP dissociation -Gas binds GTP causing it to dissociate from Gby -1 HR activates 100 Gas == 1 Gas activates 1 AC ==1 AC generates 100 cAMP -Therefore, 1HR = 10,000 cAMP

Cholera toxin ADP-ribosylates Gas & inhibits its GTPase activity Part II

-Hyper-activated PKA phosphorylates & constitutively "opens" CFTR resulting in efflux of Cl- -Na+ & H2O move from bloodstream through interstitial space separating adjacent cells -Result: Severe diarrhea causing dehydration and ion imbalance

MF polymerization is [G-Actin]-dependent

-If [G-Actin] is < Cc (Critical Concentration) no polymerization occurs -If [G-Actin] is > Cc net polymerization occurs At steady state, [G-Actin] = Cc & no net growth occurs

Cells can regulate preferential assembly/disassembly at (+) or (-) ends independently of [G-actin]

-If [G-actin] > 0.6 µM, (+) end grows ~12X> than (-) end -If [G-actin] > 0.12 µM & ≤ 0.6 µM, only (+) end grows & (-) end shrinks -No net growth = Treadmilling Fig. 17-10

Microtubule-associated proteins (MAPs) regulate MT structure & dynamics

-MAP2 & Tau modify the "caliber" of an axon that contains a fixed # of MTs -MAP2 & Tau bind to the surface of MTs & stabilize them -MAP2 has a long "spacer" arm & increases spacing between MTs -Tau has shorter "spacer" so MTs are more closely packed

Autocrine Signaling

-Most applicable to cell proliferation &tumorigenesis -Positive feedback growth response for individual cells

Endocrine Signaling

-Most common signaling in multicellular organisms -Site of synthesis & release distinct from target site of action

The B-Adrenergic Receptor Mediates the "Flight or Fight" Response Fig 15-37

-Muscle & liver are the 2 primary target tissues & 2 simultaneous metabolic responses are elicited: 1) Inhibit glycogen synthesis 2) Convert glycogen to glucose -Goal: Increase blood & intramuscular [glucose] as energy source for muscle contraction

Dynamic instability of MTs is determined by the presence of GTP or GDP caps (Fig 18-11)

-Newly added beta-tubulin on growing MT has GTP [tubulin] > Cc & slow GTP hydrolysis favors continued growth & (+) end has GTP "cap" GTP cap maintains linear protofilaments -GTP hydrolyzed to GDP after polymerization "internal" beta-tubulin has GDP -GDP protofilaments curve outward

Dynamics of actin polymerization & depolymerization: "Nuclei" limit polymerization

-Nuclei can be generated in cells by formins & Arp2/3 -Nuclei can be generated in cells by severing existing F-actin filaments -rate-limiting step for polymerization

Profilin

-Profilin accelerates exchange of ADP for ATP but... -Profilin-ATP-Actin can only bind to (+) end -Profilin prevents (-) growth >C-c Fig 17-11

PDE5 attractive drug target to treat angina &/or hypertension - Identify cGMP analogues that inhibit PDE5

-Viagra potent inhibitor of PDE5 -Viagra ineffective treatment for angina or hypertension -Viagra effective treatment for Erectile Dysfunction by promoting vasodilation

Actin-related proteins (Arp2/3) & WASp

Actin-related proteins (Arp2/3) & WASp nucleate branched MFs in response to extracellular signals that activate Cdc42•GTP

Hormonal Stimulation in Liver Cells

Activates cAMP, Ca2+, DAG & IP3 pathways in liver cells

Hormonal Stimulation in Muscle Cells

Activates only cAMP pathway in muscle cells -PLC not activated; no elevation in Ca2+, DAG & IP3

Phosphorylated IP

Activation of a PP Inhibitor ensures target proteins remain phosphorylated for duration PKA is active

Homologous desensitization

BARK is activated by PKA & only phosphorylates the B-Adrenergic receptor

Antagonist Fig 15-9

Bind receptor & block hormone action Ex: Therapeutic application = b-blockers -Treat cardiac arrhythmia & tachycardia

Agonist Fig 15-9

Bind receptor & mimic hormone action Ex: Therapeutic application = anti-asthmatics

How does Calcium modulate its protein targets?

Ca2+ binding to Calmodulin is cooperative & allosteric

Neuronal Stimulation in Muscle Cells

Directly increases intracellular [Ca2+]; activates only Ca2+ pathway

Dominant active Rac, Cdc42, & Rho

Dominant active Rac, Cdc42, & Rho cannot hydrolyze GTP & perturb MF morphology

Dominant negative Rac, Cdc42, & Rho

Dominant negative Rac, Cdc42, & Rho cannot exchange GDP for GTP & prevent cell migration

Extracellular signals activate the Rho family of small G proteins

Dynamically alter MF organization & cellular architecture

Multiple, noncovalent interactions determine ligand-receptor binding affinities

Ex: Epinephrine - Hydrophobic Interactions w/ F201 F306 W303

A given cell can simultaneously receive "conflicting" signals Some GPCRs activate Gai that inhibits AC & reduces [cAMP]

Ex: Epinephrine is stimulatory and Adenosine is inhibitory, but both are going after the same adenylyl cyclase - one to turn it on and one to turn it off

GPCRs are coupled to heterotrimeric G protein complexes (e.g., Gs, Go, Gq Families) Go/Gq

Go/Gq == Phospholipase C === IP3 + DAG (DAG & IP3 are both derived by cleavage of plasma membrane phosphatidyl inositol) (Primary biological function of Ca2+ is to mediate intracellular signaling)

GPCRs are coupled to heterotrimeric G protein complexes (e.g., Gs, Go, Gq Families) Gs

Gs == Adenylyl Cyclase === cAMP goes up

Why coffee, Red Bull or tea give you a buzz & then a crash: Extracellular Effects

In addition to inhibiting cAMP phosphodiesterase; Caffeine competes with Adenosine for binding to its receptor which prevents down regulation of AC

Signal-induced changes at the leading edge of migrating cells : Linear MFs

Lipoprotein A pro-inflammatory cytokine stimulates macrophage

Dynamic instability of MTs can be observed in vitro & in vivo

MT growth & shrinkage occur simultaneously Fig 18-9

cAMP sends the signal to the cytosol by activating cAMP-dependent protein kinase (aka PKA)

Note: By this point, the initial signal triggered by 1 hormone•receptor complex has been amplified to 10,000 cAMPs! -PKA is activated by different hormones & triggers diverse responses in different tissues

Target protein activity is regulated by changing [phospho-protein]:[non-phosphorylated protein]

Note: Phosphorylation activates some proteins & inactivates others! Fig 15-3

Heterologous desensitization

PKA phosphorylates all GPCRs that activate AC -Allows for Beta-Arrestin to bind to B-Adrenergic receptor thus allowing it to be endocytosed and degraded in the lysosome

Spectrin

Spectrin cables connect MFs to the erythrocyte plasma membrane to provide strength & flexibility -Band 3 is the HCO3-/Cl- antiporter -Mutations in Spectrin, Band 4.1, or Ankyrin generate anemias due to deformed erythrocytes (Elliptocytosis and Spherocytosis)

Microtubule-associated proteins (MAPs) regulate MT structure & dynamics

Stathmin enhances GTP hydrolysis & binds to curved GDP containing protofilaments to promote catastrophe Stathmin-tubulin complexes cannot be reincorporated into other MTs

Cytoskeleton Functions

Strength/Support/Flexibility, Intracellular Transport, Morphology, Motility, Cell Division Key: The Cytoskeleton is Dynamic, Not Static!

Substrate specificity

Substrate specificity due largely to differences in charge (blue = K, R; red = D, E) & hydrophobicity of surface residues within the ATP binding cleft

Actin

Under physiological conditions, monomeric G-Actin binds Mg2+ & ATP Polymerization (G-Actin to F-actin) does not require ATP hydrolysis But... ATP hydrolysis influences polymerization kinetics

FRET demonstrates that receptors activate coupled G proteins by rapidly dissociating Gby from Ga

You could also show that FRET between CFP•Ga & YFP•AC Increases as FRET between CFP•Ga & YFP•G Decreases

Protein phosphorylation is THE major mechanism to regulate protein function

~20-30% of proteins present in a cell are regulated by altering their phosphorylation states!