MCDB 3145 Unit 4

Purposes of signaling

Coordinate activities with other cells, response to specific stimuli; affects virtually every aspect of structure/function; can stimulate, inhibit, or change resulting in cell differentiation

Ligands

Bind to extracellular receptors; begin signaling process via GPCRs, RTKs, immune B & T cell receptors

Autocrine signaling

Cells stimulate themselves

Paracrine signaling

Cells stimulate others in proximity

Endocrine signaling

Cells stimulate others at a distance (via blood)

Direct contact signaling

Cells stimulate others via contact

Effectors

Inner surface, activated by GPCR, generate second messengers

Second messengers

Activate/inactivate specific proteins

Signal transduction pathways

Consist of kinases/phosphatases, change protein structure and function

Kinase self-inhibited inactive state

Converted to active (or back to inactive) by ligand binding or phosphorylation of other kinases

Kinase scope on substrate

Numbers vary per kinase/phosphatase, some have numerous proteins while others act on only a single residue of a single protein

Epinephrine

GPCR→blood glucose increase→extra energy

Heterotrimeric G protein

Lingand binds and causes receptor conformation change, leads to GDP→GTP exhange and effector activationA

Adenylyl cyclase

Converts AMP to cAMP

General pathway series, step 1

Ligand binding activates receptor, conformation change increases G-alpha affinity

General pathway series, step 2

G-alpha exchanges GDP for GTP

General pathway series, step 3

G-alpha leaves G-beta/gamma (active) and activates effector

General pathway series, step 4

Effector generates second messenger (ex cAMP)

General pathway series, step 5

GTP hydrolysis via G-alpha (or sometimes helper proteins, depending on receptor)

General pathway series, step 6

Desensitization blocks additional G-protein activation

GRK

Receptor kinase, blocks G-alpha ability to interact with receptor loops

General pathway series, step 7

Arrestin arrests receptor signaling

GPCR purpose

Allows signal amplification (1 ligand→hundreds of G-proteins→hundreds of thousands of effectors), controlled by timer

Glucose mobilization

cAMP activation inhibits glycogen synthase and activates glycogen phosphorylase, glycogen breakdown stimulated to increase blood sugarC

CREB

Transcription factor targets glucose production genes, transforms non-carbohydrates substrates into glucose

Muscarine ACh receptor

Smooth muscle GPCR, activates phospholipase C→IP3→Ca2+ increase→muscle contractionP

Phospholipase C

Activates multiple second messengers, splits phospholipid head group PIP2 off glycerol to convert to 2nd messenger, binds via PH domain

PIP2

Converted to PIP3 (phosphate) and DAG (lipid)

IP3

Binds smooth ER Ca2+ channel receptor and stimulates Ca2+ release

DAG

Diffuses through membrane to protein kinase CA

rrestin

Binds phosphorylated GPCR and AP2→clathrin endocytosis

Growth factor signaling

Mostly RTKs (tissue specific) and rely on growth factors

EGFR

Dimerizes upon binding epidermal growth factor

Transphosphorylation

RTK on one receptor cytoplasm-side phosphorylates the other receptor

Bivalent ligand

Binds 2 receptors simultaneously (ligand-mediated dimerization)

Phosphorylation generates ___

Binding sites for cellular signaling protein

SH2 & PTB domain

Scaffold/adaptor proteins help make a signaling conplex/platform

Grb2

One SH2 domain and 2 SH3 domains that bind to next protein

SH3

Bridge SOS GEF, exchanges Ras GDP for GTP

Ras

Oncogene, inherently weak small GTPase, acts as a binary switch (off when GDP bound), active form binds downstream kinases and helps activate

Ras-GDP

Must be lipid-modified to localize to plasma membrane and be activated by GEF (highly regulated)

MAP3K pathway

Promotes growth

P13K/AKT pathway

Cell survival

Interphase

Everything outside of mytosis

G0

“Quiet” stage, cells can reenter cycle at some point

Amphibian oocytes

Used as model; respond to outside-surface human progesterone→maturation

Progesterone

Extracellular administration to oocyte signals MPF intracellular factor production that directs maturation in G2 egg

Maturing-egg cytoplasm

Triggers maturation in naive egg

MPF

Most active in mitosis, controlled proteolysis degradation in regulation of major cellular activity; contains Cdk1 (catalytic subunit) and Cyclin B (structural subunit); cyclin levels are dynamic, Cdk1 levels are static

Fission yeast

Clearly visible division stage phenotypes

Budding yeast

Very clearly buds and elongates, but no clear G2 phenotype

Yeast culture

Mutate yeast→plate at permissive temperature→split into permissive/restrictive temperatures

Restrictive-growing yeast

Temperature-resistant

Cdc mutant yeasts

Continued growth at low (but not high) temperatures; arrest with a single cell morphology; grow permissive then shift to lock a phenotype

Rescue experiment (Cdc cloning)

Cdc28-st grown at 25C, transformed with plasmid, yeast that grow at restrictive temp (35C) are rescued cells and can have plasmid isolated, allows sequencing showing Cdc2 kinase

Cdc2 kinase activity peak

G2/M as shown by antibodies

Cdc2-ts

Can be rescued with Cdk1 gene

Fission yeast checkpoitns

G1/S and G2/M, same Cdc2 activated but by different cyclins

Allosteric regulation of Cdk1

Cyclin increases and binds Cdk1 (catalytic domain)

Cdk1

Phosphorylates chromatin, regulates proteins (especially condensin); disassembles interphase cytoskeleton and assembles mitotic spindle

Cdk1/Lamin

Phosphorylates/inactivates lamin, breaks down nuclear envelope at prophase

CAK

Kinase, phosphorylates Thr161 (stim) and activates Cdc

Wee1

Phosphorylates Tyr15 (inhibitory) and overrides CAK Thr161-P

Cdc25

Dephosphorylates Tyr15-P to help Cdk become fully active at right time (late G2) and allow cell division

Wee1 mutation

Smaller cell size, enters mitosis too early

Cyclin B threshold levels

Rapid MPF activation/inactivation (enter/exit mitosis)

Mitosis exit

Requires turning off Cdk1→G1; Cyclin regulated by transcription/degradation (rapid degradation by ubiquitin-proteasome upon cell cycle completion)

Anaphase-promoting complex

Contains E3, ubiquitin enzymes E1 and E2 mark Cyclin B

Cyclin B degradation

Turns off MPF, mitosis exit, anaphase switch at end of M; blocking means cell can’t leave mitosis

G2/M checkpoint first responders

Sense DNA damage and initiate repair

G2/M checkpoint second responders

Signal to cell that damage has occurred and repair is underway

G2/M checkpoint effectors

Contribute to outcome; repair damage, cycle arrest, apoptosis, etc

CKIs

Bind to active Cdks or Cdk-cyclin and inhibit; last resort to inhibit fully active complex (emergency brake); key checkpoint protein

G1

Cells responsive to growth factors that stimulate growth and division

EGF addition

Early response genes activate 30 minutes later, delayed response 120 minutes, decrease after activation

Fos & Myc

Early response transcription factors, \~1 hour

ELK

Transcription factor, phosphorylation-activated by MAPK

SRF

Associates with ELK to activate SRE genes (response element)

Fos

Leucine zipper containing SRE, forms heterodimer with jun to form AP1 complex

AP1

Activates proliferation and growth genes including cyclin D1

ELK/SRF/jun

Quiescent cell nucleus, normally inactive until phosphorylated by MAPK pathway

Cell cycle entry

Done via Cyclin Ds, Cyclin E, Cdk2 and Cdk4

Prophase

Chromosome condensation, cytoskeletal disassembly, golgi/ER fragment, envelope dispersion, centromere chromatin connection

Prometaphase

Chromatin MTs attach to kinetochores; envelope dissolution, spindle assembly, central positioning

Aurora/polo/Cdk1/cyclin positive feedback loop

Keeps kinases active during mitosis

Cohesin complex

Holds sister chromatids together by wrapping around DNA strand

Condensin

Forms ring around supercoiled DNA

Centrosome duplication

End of G1, finished by S, controlled by Cdk2, acquires PCDM + gTuRC

Centrosome separation

Late G2 via myosin 2 and cytoplasmic dyenin I

\+end kinesin 5 (& 12)

Push centromeres apart, spindle bipolarity

Chromosome alignment errors

Too many centrosomes, no bipolar spindle, no or multiple metaphase plates

Kinesin 5 inhibitor

Monopolar spindle and can’t divide

Aster formation

Building the mitotic spindle, followed by centrosome separation and movement

Polo-like kinase

Phosphorylates PCM, stimulates nucleation

Free MT end

“Search” for centrosomes via dynamic instability

Kinetochores

Assemble at each centromere and attach chromatid to MT bundles (metaphase plate formation)

Kinetochore fibers

Captured and stabilized MTs

CENP-E

\+end motor

Kinesin 13-MCAK depolymerase

Depolymerizes MT

Ndc80

Tethers kinetochore to + end