Exam 3 NEED TO KNOW CSF BIOL 3040

CDK

cyclin dependent kinase - gets enzymatically activated when binds to cyclin

Cyclin

activates CDK

M-cyclin

Regulatory protein that binds to mitotic Cdk to form M-Cdk, the protein complex that triggers the M phase of the cell cycle.

the cyclin that acts in G2 to trigger entry into M phase

S cyclin

Regulatory protein that helps to launch the S phase of the cell cycle.

CDK helps initiate DNA synthesis

G1/S Cdk

Protein complex whose activity triggers entry into S phase of the cell cycle; consists of a G1/S cyclin plus a cyclin-dependent protein kinase (Cdk).

S-Cdk

Protein complex whose activity initiates DNA replication; consists of an S cyclin plus a cyclin-dependent protein kinase (Cdk).

CAK

CDK- activating kinase phosphorylates an activating site in Cdks

Wee1 kinase

phosphorylates inhibitory sites in Cdks; primarily involved in suppressing Cdk1 activity before mitosis

Cdc25 phosphatase

activates CDKs

removes inhibitory phosphates from Cdk1 so cyclin B can bind to activate mitosis

opposes Wee1

PP2A-B55

PP2A-B56

PP2A

Heterotrimer phosphatase: catalytic + 2 regulatory subunits. Regulated by phosphorylation, methylation. Substrate serine-threonine

Protein phosphatase 2A

regulatory subunit is SPECIFICITY DEPENDENT

PP1

protein phosphatase 1

Greatwall Kinase

phosphorylates Ensa

phosphorylates

Ensa

inactivates PP2A-B55

phosphorylates

Protein Phosphate CDK Regulatory Pathway

M-Cdk

Protein complex that triggers the M phase of the cell cycle; consists of an M cyclin plus a mitotic cyclin-dependent protein kinase (Cdk).

p27

binds to cyclin (cdk) to block entrance of cell to the S phase of cell cycle

protein that binds to cyclin and cdk blocking entry into S phase

inhibitor

p21

Cdk inhibitor protein

cell cycle regulatory protein that inhibits the cell cycle; its levels are controlled by p53

APC/C

the ubiquitin ligase that promotes the destruction of a specific set of proteins, thereby promoting the separation of sister chromatids and the completion of M-phase

anaphase-promoting complex/cyclosome

Cdc20

activates APC

triggers initial activation of APC/C at metaphase to anaphase transition - stimulated by M-CDK

Cdh1

Activating subunit which binds and activates APC/C in late mitosis through early G1

APC/C-activating subunit that maintains APC/C activity after anaphase and throughout G1; inhibited by Cdk activity

KNOW THIS GRAPH!

GEF

guanine nucleotide exchange factor - ON = GTP

GAP

GTPase activating protein - OFF = GDP

GPCRs

-G Protein Coupled Receptors: membrane proteins involved in signal transduction

-binding of an intracellular G protein turns on an active site to affect an intracellular pathway

3 kinds: Gs, Gi, Gq

activated RTK - receptor tyrosine kinase

upon binding to RTKS, monomers can form dimers and phosphorylated each other

PLC-beta pathway

X= IP3

Y= Ca2+

Z= Dag

EGF binding

different than receptor tyrosine kinase

GPCR binding

Three G-proteins (alpha, beta, and gamma) associate with the GPCR but only the G-alpha binds to the GTP after receptor activation.

binds on the cytosolic side

adenylyl cyclase

an enzyme that converts ATP to cAMP in response to an extracellular signal

activates PKA

cAMP

cyclic adenosine monophosphate

binds to PKA and activates it - binds to the regulatory subunit of PKA and that releases the active catalytic subunits

activates protein kinase A

Molecular interaction domains

PTB domain

phosphotyrosine binding domain

SH2 domain

A protein domain that binds phosphorylated tyrosine residues.

SH3 domain

binds proline rich sequences

Pleckstrin homology (PH) domain

binds to inositol phospholipids

phospholipase C

G-protein linked receptor signals phospholipase C

splits PIP into IP3 and DAG:

1. IP3 increases intracellular Ca2+

2. DAG activates kinases (PKC) to activate enzymes

IP3

One of the products of PIP2 cleavage in the Ca++/phosphoinositide signal pathway.

Moves to cytoplasm to trigger Ca++ release from intracellular stores leading to Ca++/calmodulin cascade.

DAG

diacylglycerol

Another of the products of PIP2 cleavage

activates protein kinase C - PKC

guanylyl cyclase

enzyme that catalyzes transformation of GTP to cyclic GMP

allows for muscle relaxtion

nitric oxide binds to guanylyl cyclase to make cGMP

GPCR desensitization

Receptor desensitization prevents sustained activation that could be harmful to cells

gets phosphorylated and then arrestin binds and ARRESTS

arrestin

proteins that bind phosphorylated GPRCs and deliver them to clathrin coated pits for endocytosis

enzyme that participates in desensitization of GPCRs by phosphorylating them after they have been activated by ligand binding

binds to GPCR so G proteins cannot interact w GPCR

RTK activation of Ras

RTK is activated through autophosphorylation.

Adaptor proteins bind to the RTK which requires SH2 domains, which then binds SOS (Ras Guanosine Exchanger factor GEF), which promotes the exchange of GDP to GTP. Once GTP is bound, Ras is activated.

1. signal molecule binds and activates RTK - autophosphorylation

2. An adaptor protein (Grb2) docks on a particular phosphotyrosine on the phosphorylated RTK

3. the adaptor recruits a Ras guanine nucletide exchange factor (Ras-GEF) (Sos) that stimulates Ras to exchange its bound GDP for GTP (Ras is activated when in GTP form)

4. Activated Ras can now stimulate downstream signalling pathways→ex: cell division pathways

What can Ras signaling do?

activate MAP kinase

MAP kinase kinase kinase

MAPKKK

Raf

Phosphorylates and activates MAP-kinase-kinase.

- ATP → ADP

MAP kinase kinase

MAPKK

Mek

Phosphorylates and activates MAP-kinase.

- ATP → ADP

MAP kinase

MAPK

Erk

Mitogen-activated protein kinase. Protein kinase that performs a crucial step in relaying signals from cell-surface receptors to the nucleus.

It is the final kinase in a three-kinase sequence called the MAP kinase cascade.

scaffold proteins

organize groups of intracellular signaling molecules into signaling complexes

improve the efficiency of a signaling cascade by holding all the participating enzymes in close proximity

PI-3-kinase-Akt signaling pathway

this promotes cell survival and it is activated by a RTK.

phosphorylates an inositol phospholipid that is embedded in the cytosolic side of the plasma membrane. this attracts intracellular signaling proteins that have a special domain that recognizes them.

Akt is a protein kinase that is released from the plasma membrane and phosphorylates various downstream proteins on specific serines and threonines.

this promotes cell survival

PDK1

a kinase that phosphorylates another kinase Akt to become active

Akt

A serine/threonine kinase that is activated by PIP3 and plays a key role in signaling cell survival.

Bad protein

promotes apoptosis

inactivates apoptosis inhibitory protein

mTORC2

a protein complex that regulates cellular metabolism as well as the cytoskeleton

What can active Akt do?

can phosphorylated bad, allowing for an active apoptosis inhibitory protein, blocking apoptosis so NO CELL DEATH HOORAY

Bcl2

inhibits apoptosis

PI 3-kinase

phosphorylates PIP2 to PIP3

Enzyme that phosphorylates inositol phospholipids in the plasma membrane, which generates docking sites for intracellular signaling proteins that promote cell growth and survival.

PI-3 Kinase-Akt-mTORC1 signaling pathway

TSC

Rheb GAP - OFF

Rheb

Ras homolog that activates mTORc1

A monomeric Ras-related GTPase that in its active form (Rheb-GTP) activates mTOR, which promotes cell growth.

mTOR

mammalian target of rapamycin

mTORC1

Mechanistic target of rapamycin complex 1, kinase

Increases translation therefore, increases protein synthesis

Raptor protein

part of the MTORC1 COMPLEX

Rag

binds to GTP and raptor and mTOR making complex mTORC1!

Ragulator

Rag GEF - ON!

Gator1

Rag GAP - OFF!

Gator2

inhibits Gator1 allowing mTORC1 complex to be made and activated

Wnt/beta-catenin signaling pathway

without Wnt, beta catenin becomes unstable, and transcription DOES NOT occur

WITH Wnt signaling, beta catenin is stable YAY, and transcription DOES OCCUR!

with APC mutations beta catenin is always stable and results in UNCONTROLLED GROWTH AND POLYPS

Wnt

Local signaling protein; activates receptors of the Frizzled family

regulates cell proliferation

soluble factor

resting cells: NO WNT; B catenin complexed with APC and gets destroyed

WNT stimulation deactivates destruction complex, increases beta catenin intracellular, which goes to nucleus and binds TCF, activates cell cycle genes and causes proliferation

Frizzled

receptor in the Wnt pathway

Groucho

protein that binds to LEF1/TCF and does NOT allow for transcription

LEF1/TCF

gets activated by beta catenin binding when its stable to bind

groucho gets kicked off

BrdU

A chemical that identifies newly-divided cells S PHASE

Flow cytometry

looking at number of cells expressing markers

ex: separate T cell populations using CD4/CD8 markers

method:

1. cells are stained with antibodies linked to fluorescent tag

2. cells passed through beam of light one at a time

3. light scattering and light emission is measured

4. fluorescence-activated cell sorting (FACS)- sort cells based on expression of markers

LOOK AT GRAPH

geminin

inhibits cdt1

Cdt1

Controls the timing of DNA replication

opposes geminin

Cell cycle control system

A cyclically operating set of proteins that triggers and coordinates events in the eukaryotic cell cycle.

APC/C

anaphase-promoting complex/cyclosome

DNA Damage p53 and p21 response pathway

DNA damage

activates ATM/ATR

Chk1/Chk2 activation

that phosphorylates p53 releasing Mdm from p53

^^^Mdm degrades p53

then there is stable p53

this binds to p21 gene yay

that allows for transcription

then translation p21 cdk inhibitor

inhibits Cdk complex

• DNA damage can temporarily halt progression through G1 phase

• p53- Checkpoint protein and a tumor suppressor

Activation of Checkpoint Kinase 1 (Chk1) by DNA damage stalls the cell cycle in S or G2 Phase by inhibiting the Cdc25 phosphatase

Chk1 inhibits Cdc phosphatase

^^^gets activated by ATR / ATM kinases

Protein Degradation by APC/C Complex

PTEN

PTEN = Tumor Suppressor and inhibits hyper activation of PI3K

NO PTEN→ hyperactivation of PI3K→ cell proliferationand growth

EMT

epithelial to mesenchymal transition

epithelial to mesenchymal transition

Acquisition by epithelial cells of the phenotypes of mesenchymal cells such as fibroblasts.

process by which epithelial cells lose their cell polarity and cell-cell adhesion, and gain migratory and invasive properties to become mesenchymal stem cells

CANCER

Mad2

(Mitotic Arrest Deficient) -component of Mitotic Checkpoint Complex (MCC) - inhibits CDC20

signal that chromosomes are not completely attached to microtubule spindles by blocking Cdc20 action

One of several proteins recruited to unattached kinetochores, it binds and inhibits Cdc20-APC/C and prevents cells from passing through the spindle assembly checkpoint until all chromatids are properly attached to spindle poles

Spindle Assembly Checkpoint (SAC)

Operates at the metaphase/anaphase transition to check for misaligned chromosomes (the third critical checkpoint).

Mad2 activated when misaligned and checkpoint is ON and inhibits anaphase

when all chromosomes are aligned hooray Mad2 is not active and securin is degraded and separin cleaves cohesin scc ring and anaphase occurs

anaphase A

movement of chromosomes toward the poles

anaphase B

spindle poles move apart

BOLES POLES

kinesin 5 and dyenin = astral

How is contractile ring assembled?

aurora b and central spindilin come first

Rho GEF = Ect2

Rho GAP

active RhoA binds to formin = actin

active RhoA binds to ROCK (kinases) = myosin

CONTRACTILE RING!

phragmoplast

In dividing plant cells, a structure formed by overlapping microtubules that guide vesicles containing cell wall components to the middle of the cell.

no astral microtubules since then have no centromeres