Cancer 5: Signaling + mechanisms in growth and division

Question 1

a) What is the significance of c-Myc?
b) How is it upregulated?
c) What happens when c-myc = mutated?

Answer 1

a) - c-Myc is an oncogene
- acts as transcription factor - stimulates expression of cell cycle genes
- -> growth factor increases [Myc]

b) How it is upregulated:
cell receives signal –> stimulates an intracellular sginalling cascade –> causes upregulation of transcription factors e.g c-Myc
• c-Myc stimulates the expression of cell cycle genes, and has an important role in G1

c) Mutated Ras and c-Myc act as oncogenes, leading to uncontrolled cell division and tumour proliferation

Question 2

What are 3 key components of signaling pathways?

Answer 2

1. Regulation of enzyme activity by protein phosphorylation (kinases)
2. Adapter proteins
3. Regulation by GTP-binding proteins

Question 3

Describe the process of growth factor stimulation of signaling pathways?

Answer 3

This can be divided into two phases:
• growth factor binding to the cell surface
receptor protein tyrosine kinase (RPTK)

o RPTK = phosphorylated –> activates kinase cascade
- which provides additional docking sites + activates Ras (small G protein).

o Ras binding stimulates –> early-response gene expression from MAPK/ERK cascade, eg c-Myc expression
–> activation of the cell-cycle control system

E.g. anti-HER2 Ab Herceptin used in HER2-positive metastatic breast cancer

Question 4

EGFR/HER2, mutationally activated or overexpression can cause: ____ ____

Answer 4

breast cancer

Question 5

What is RAS?

and what does it do to kinase cascades?

Answer 5

RAS = oncogene

activates kinase cascades

Question 6

How does peptide growth factors take part in signaling?

Answer 6

• Phosphorylated receptor tyrosine kinase recruits adaptor + signalling proteins
• Intracellular signalling proteins bind to phosphorylated tyrosines –> allows adaptor proteins to bind to them
• E.g. anti-HER2 Ab Herceptin used in HER2-positive metastatic breast cancer
• -> blocks binding of ligand

Question 7

Following RPTK phosphorylation, describe the process of Ras complex activation

Answer 7

Following RPTK phosphorylation, additional docking sites are formed. adapter proteins such as Grb2 (growth-factor receptor binding protein 2) binds (not Ras)

• Binding of Grb2 recruits inactive Ras protein to the plasma membrane.
–> The inactive Ras protein = associated with a GDP molecule

• Ras-activating proteins –> then activates Ras. (using exchange factor Sos - which exchanges a phosphate from a GTP (from ras-activating protein) –> for GDP (from Ras)
• The Ras complex with GTP is now active

Question 8

How does receptor protein TKs signal to RAS?

Answer 8

• Sos binds Grb2 (adapter) via SH3 domains –> Sos exchanges GDP for GTP –> activates Ras
• Ras must bind plasma membrane to become activated

Question 9

How is RAS activated oncogenically?

what re the 2 mutations involved in this process?

Answer 9

Activated by mutations increasing amount of active GTP-loaded Ras

2 mutations involved:
o V12Ras = glycine –> valine at position 12 –> prevents GAP binding

o L61Ras = glutamine –> leucine at position 61 –> prevents GTP hydrolysis

Question 10

How does active ras activate protein kinase cascade?

Answer 10

• Active Ras binds + activates kinase –> causes kinase cascade

2 key ones:
o Specifically - Extracellular signal-regulated kinase (ERK) cascade

o Generically - Mitogen-activated protein kinase (MAPK) cascade

Question 11

What is a ERK Cascade?

what effect does it have on the cell cycle ?

Answer 11

When active: Ras activates protein kinase cascade
• Generically, this is referred to as a
mitogen-activated protein cascade
(MAPK).
• More specifically, Ras activates an
Extracellular signal-regulated kinase cascade (ERK)

ERK –> stimulate changes in cell proteins + gene expression (eg c-Myc) –> to promote cell division

Question 12

How do kinases stimulate changes in cell proteins/ gene expression to promote division?

Answer 12

Myc + Ras = can be oncogenes if mutation/ expressed in high amount

o Phosphorylate proteins (non-kinases) –> promote division

o Phosphorylate gene regulatory proteins –> change gene expression e.g. c-Myc that stimulates cell proliferation

Question 13

What are cyclin dependent kinases (Cdks) ?

what are they regulated by?

Answer 13

Cdks = controls cell cycle

activity of Cdks = regulated by:
o interaction with cyclins
o Phosphorylation

Question 14

What is the significance of Cyclins?

Answer 14

• proteins which activate Cdks.
• transiently expressed at specific points in the cell cycle
• form cyclin-Cdk complexes –> different complexes triggers different events in the cell cycle

Question 15

What do activated Cdks do:?

Answer 15

• They phosphorylate proteins (on Serine or Threonine) –> to drive cell cycle progression
  e. g CDK1 (w/ mitotic-cyclin) –> phosphorylates substrates at mitosis e.g. nuclear lamins (form nuclear envelope); when phosphorylated –> envelope broken down
  e. g CDK2 (w/ cyclin E) –> start kinase (tumour suppressor)
  e. g Cdk1 + Mitotic Cyclin B–> forms MPF: –> stimulates mitosis

Question 16

Note: Cdk1 + Mitotic Cyclin B–> forms MPF: –> stimulates mitosis

Answer 16

• Mitotic Cyclin (B) forms a complex with Cdk1, acting as the mitosis promoting factor (MPF)

Question 17

How is Cdks regulated via phosphorylation?

Answer 17

inactive MPF = formed by CDK1 + cyclin B –> activated by phosphorylation by:
o CDK-activating kinase (CAK)
o Inhibitory kinase (Wee1)

a) These 2 kinases phosphorylate separate residues = one activated + one inhibited
b) Then phosphatase removes inhibited phosphate –> to form a fully active MPF

requires BOTH:
- activating phosphorylation + removal of inactivating phosphorylation

Eg 1. MPF targets nuclear lamins; the phosphorylation causes breakdown of nuclear envelope (key step in prophase)

• Eg 2. Cdk4/6-Cyclin D targets retinoblastoma protein (pRB); phosphorylation inactivates the protein.
–> which releases E2F transcription factor, driving gene transcription of Cyclin E and allowing progression of the cell cycle from G1 > S phase

Question 18

Different cyclins and different Cdks are required at different stages of the cell cycle
• G1 involves Cyclin ___ and Cdk___
• S phase involves cyclin ____ and Cdk____

Answer 18

Different cyclins and different Cdks are required at different stages of the cell cycle
• G1 involves Cyclin E and Cdk2
• S phase involves cyclin A and Cdk2

Question 19

What is the function of Anaphase Promoting Complex C

what is it activated by?

Answer 19

Anaphase Promoting Complex C:

Ub ligase that catalyses Ubn + degradation of cycB.

o Activated by Cdc20 (binds phosphorylated APC/C, phosn. done by MPF)

o So, Cdk1/cycB promotes own degradation.

o Active APC/C-Cdc20 released from attached kinetochores

Question 20

Cyclin D binds/activates CDK4/6 to stimulate synthesis of cyclin E:

Answer 20

C-myc –> activates cyclin D

LOOK at diagram

Question 21

Regulation of gene expression by Rb:

Answer 21

• important tumour suppressor
• at G0, RB = activated
  holds onto transcription factor E2F (inactive)
• in proliferating cell
• cdk4/6 - cyclin D –> phosphorylate Rb protein
• inactivates rb protein
• release e2f
• bind to dna
• start transcribing next cyclin requires for the cycle
  Cyclin E

in a similar method,
then Cyclin E acts on cdk2 –> release E2F –> forms cyclin A –> release TF –> forms cyclin B which acts on cdk1–> cause mitosis etc.

Question 22

describe the regulation of Rb in cell cycle

Answer 22

• C-Myc stimulates cyclin D production
• CDK4/6- D phosphorylates pRb –> release some E2F–>cyclin E production etc.
• When pRb phosphorylated further –> further inhibition –> larger E2F release

Question 23

Cdks are also regulated by CDK inhibitors (CKI)

How do CKI work?

Answer 23

CKIs

• -> inhibits Cdks
• -> prevent progression through the cell cycle (which is important in maintaining integrity of the cell cycle)

Question 24

What are the 2 CKI FAMILIES?

Answer 24

• G1 phase CKIs (INK4) = inhibit CDK4/6 by displacing cyclin D, prevent entry into G1
• S phase CKIs (CIP/KIP) = inhibit all CDKs by binding to CDK/cyclin complex, prevent entry into S-phase (hold in G1)

–> regulated by degradation

Question 25

Grb 2 = contains SH3 + SH2 + SH3
SH = src homology regions

SH2 = recognizes phosphorylated tyrosine
- SH3 = proline rich regions –> docking site for other proteins

Answer 25

recruits ras

ras binds to memb –> becomes activated –> then transmits signals downstream

Question 26

What are characteristics of Cyclin?

Answer 26

• transiently expressed at spcf point in the cell cycle
• regulated at level of expression
• synthesized –> then degraded
• susceptible to degradation –> which causes cyclical activation

Question 27

NOTE: Signal from fully attached kinetochores
causes cyclin B to be degraded:

• Cdk1 inactivated
• key substrates dephosphorylated
• mitosis progresses

Answer 27

-

Question 28

P27KIP1 = tumour suppressor –> reduced expression = poorer prognosis in malignancy

Answer 28

-

Question 29

How is Ras deactivated?

Answer 29

GTP-binding (G) proteins

• G- proteins act as a molecular switch
• turns Ras off
• by GTP hydrolysis (of the GTP molecule associated with the active Ras) using GTPase activating proteins (GAP proteins)

Question 30

NOTE: ONCOGENES

• EGFR/HER2, mutationally activated or overexpressed in many breast cancers (Herceptin antibody for the treatment of HER2-positive metastatic breast cancer)
• Ras, mutationally activated in many cancers (inhibitors of membrane attachment)
• Cyclin D1, overexpressed in 50% of breast cancers
• B-Raf, mutationally activated in melanomas (kinase inhibitors in trials)
• c-Myc, overexpressed in many tumours

Answer 30

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