BIOL 4004 Exam 4

Cdk

-Cyclin-dependent kinases
-Cdks phosphorylate substrates which control major cell cycle events
-activity of Cdks oscillate throughout the cell cycle

Cyclins

-Bind Cdks and act as their major regulator
-Cdks are only active when a Cyclin-Cdk complex is formed
-Cyclic changes in cyclin levels control Cdk activity
-Cyclins also direct Cdks to their target proteins

G1/S-cyclins

Help trigger cell division commitmentS

S- cyclins

trigger chromosome duplication and early mitotic events

M-cyclins

trigger entry into early mitosis

Structural basis of Cdk activation: inactivation state

activation site is blocked by the T-loop

Structural basis of Cdk activation: Partial activation

cyclin binding causes the T-loop to move out of the active site

Structural basis of Cdk activation: Full activation

Cdk-activating kinase (CAK) phosphorylated a Tyrosine residue near active site

CAK

• Cdk-activating kinase

• activates a Cdk by phosphorylation

Wee1 kinase

• phosphorylates active site of Cdk

• inhibitory phosphorylation turns off Cyclin-Cdk activity

Cdc25 phosphatase

• removes inhibitory phosphate at active site of Cdk

• activates Cyclin-Cdk

CKI

• Cdk inhibitor protein

• binding of CKI interferes with the active site and/or ATP binding site of Cdk

• examples: p21, p27

M-Cdk

• cyclin B(M-cyclin) and Cdk1 form M-Cdk complex

• trigger entry to mitosis

• activity is rapid and irreversible to progress through stages

APC/C

• anaphase promoting complex/cyclosome

• APC is ubiquitin ligase that transfers poly-ubiquitin to target proteins, promoting their degradation proteosomes

• APC targets: Cycling B and securin

Cdc20

• activating subunit of APC

• binds in mid-mitosis

Cdh1

• activating subunit of APC

• binds in late mitosis

Condensin

• forms ring like structure which encircles DNA loops within each sister chromatid

• M-Cdk phosphorylates condensin subunits to stimulate condensin activity

Kinesin-5 motors

• attach to non-kinetochore microtubules and push MTs (Lengthening spingle)

• plus end directed

Kinesin-14 motors

• attach to non-kinetochore microtubules and pull MTs (shortening spindles)

• minus end directed

Dynein motors

• attach cell cortex to astral MTs and pull centrosomes

• minus end directed

Chromokinesins

• Kinesin 10 and 4

• associated with chromosome arm and push the chromosome away from the centrosome

Ndc80 complex

• mediates MT attachment to kinetochore

• one end of Ndc80 complex is anchored in the kinetochore, the other end interacts with sides of kinetochore MTs

• lateral binding

Aurora- B kinase

• tension sensing mechanism

• incorrect attachment: it is able to phosphorylate MT attachment, reducing affinity

• correct attachment: unable to reach MT attachment site, increases affinity of MT binding

Cohesin

• rings hold sister chromatids together until anaphasde

Mad2

• part of spindle assembly checkpoint

• unattached kinetochores catalyze conformational change

• change binds and inhibits Cdc20-APC complex

RhoA

• small GTPase

• triggers assembly and contraction of contractile ring

• RhoA-GTP activates formins that nucleate the assembly of actin filaments

• activates Rock that inhibits myosin phosphatase. keeping light chains phosphorylated, stimulating myosin II filament formation

G1 checkpoint

senses cell size, physiological state of the cell, and environmental conditions

Myc

• gene regulatory protein

• triggers increased G1-Cdk activity

E2F

• transcription factor required for the transcription of S phase genes

• controls the initiation of S phase

• Rb binds to E2F and inhibits it

Rb

• retinoblastoma protein

• binds and inhibits E2F

• phosphorylation inactivates Rb, leads to active E2F

G1-Cdk

• cyclin dependent kinase that phosphorylates Rb to activate E2F

• Promotes G1/S phase transition by activating E2F

G2 Checkpoint

• ensures that DNA is replicated properly

• entry into mitosis is blocked by incomplete DNA replication since unreplicated DNA inhibits Cdc25 phosphatase and prevents activation of M-Cdk

Hydroxyurea

inhibits DNA synthesis

Caffeine

blocks checkpoint mechanism (in S phase of cell cycle)

Metaphase checkpoint/Spindle assembly checkpoint

• ensures that all chromosomes are properly attached to the spindle

• sister-chromatid separation does not occur until all chromosomes are properly attached

DNA Damage Checkpoint (G1)

• DNA damage triggers the activation of protein kinases (ATM, ATR, Chk1, and Chk2)

• a major target of these kinases is p53

p53

• usually bound by Mdm2, resulting in degradation of p53 in proteasomes

• phosphorylation of p53 blocks Mdm2 from binding and allows it to accumulate

• p53 moves to the nucleus and binds p21 which arrests cells or trigger apoptosis

Mdm2

binds p53 and tags for degradation

p21

• Cdk inhibitor (CKI)

• arrests cells in G1 after binding and inactivating G1/S-Cdk and S-Cdk

DNA damage checkpoint (G2)

• DNA damage triggers Chk1 and Chk2 activations

• they phosphorylates and inhibit Cdc25, blocking progression into mitosis since M-Cdk is inactive

mTORC1

• activated by signaling

• stimulates protein synthesis, lipid synthesis, and reduces protein turnover

2 heritable properties of cancer

1) reproduce in defiance of normal restraints on growth and division

2) invade and colonize territories normally reserved for other cells

Key properties of cancer cells

1) disregard signals that regulate cell proliferation

2) avoid apoptosis

3) escape replicative senescence and avoid differentiation

4) genetically unstable

5) invade surrounding tissues

6) survive and proliferate in foreign sites

carcinomas

arise from epithelial cells

adenocarcinomas

arise from glandular tissue

sarcomas

arise from connective tissue and muscle cells

Leukemias

derived from WBC and precursors

Lymphomas

derived from lymphocytes

Chronic Myelogenous Leukemia (CML)

• leukemia white blood cells have Philadelphia chromosome created by a translocation between chromosome 9 and 22

• site of breakage is identical is all cases of cancer

Warburg Effect

• tumor cells have abnormally increased glucose uptake

• reduced oxidative phosphorylation

• higher conversion of lactate and small molecule building blocks for cell growth

Metastasis step 1

• break through basal lamina

• involves epithelial-mesenchymal transition

• -loosen adhesions by downregulation of expression of cadherins and integrins

• degrades ECM

Metastasis step 2

invade capillary

Metastasis step 3

travel through bloodstream, circulating tumor cells

Metastasis step 4

adhere to vessel wall

Metastasis step 5

exit from vessel

Metastasis step 6

From metastasis at distant site (different tissue)

oncogenes

• normally promote cell proliferation

• gain-of-function mutations lead to cancer by overactivity

• can be identified through their dominant transforming effects

tumor suppressor genes

• normally suppress cell proliferation

• loss-of-function mutations lead to cancer

• can be identified by genetic and molecular approaches

Burkitt’s Lymphoma

• caused by chromosome 8 and 14 rearrangement

• abnormal activation of Myc gene under the control of B lymphocyte regulatory sequences leads to lymphoma

Ras

• ras oncogene has point mutations which make it hyperactive

Hereditary retinoblastoma

• both eyes effect

• descend from parents to children

non-hereditary retinoblastoma

• one eye affected

• extremely rare

recessive effect

• cells with one functional copy of tumor suppressor are normal

Cytochrome p-450 oxidases

• enzyme in liver

• normally convert toxins into harmless chemicals

• certain chemicals can be converted to highly mutagenic products

aflatoxin

• derived from mole that grows on grains and peanuts when stored under humid conditions in the tropics

• cause of liver cancer rates in tropics

Problems of conventional cancer therapy

1) side effects

2 heterogeneity of tumor cell population

3) acquired mutations

Brca1/Brca2

• involved in pathway to repair single strand DNA breaks

PARP

• another pathway to repair single stranded break

• drug that blocks PARP can selectively kill Brca-deficient genes

Imatinib (Gleevec)

• binds and blocks the activity of Bcr-Abl and halts CML

• Gleevec binds to the ATP binidng site selective to Bcr-Abl

Drugs ending in “nib”

• small molecule inhibitors

• can enter cells and inhibit intracellular reactions

Drugs ending in “mab”

monoclonal antibodies

target RTK

not incorporated into cells, functional blocking

Her2

• EGFR related RTK

• overexpressed in 25% of breast cancers

Tramuzumab (herceptin)

• anti-Her2 antibody

• blocks Her2

• effective to cure breast cancer

CTLA4

• inhibitory receptor regulating T cell activation

• cancer cells downregulate T cell response by activating these inhibitory receptors

PD1

• inhibitory receptor regulating T cell activation

• cancer cells downregulate T cell response by activating these receptors

Ipilimumab

can overcome immunosuppressive environment by preventing cancer cells from inhibiting T cell response

allows immune attack on cancer cells