Signalling and Mechanisms in Growth and Division

Question 1

What is c-Myc?

Answer 1

oncogene - overexpressed in many tumours

c-Myc is a transcription factor, so if it is being highly expressed, there is stimulation to express cell cycle genes. The levels of c-Myc then go down, when cells enter the S phase

Question 2

Describe the levels of myc in the cell cycle?

Answer 2

G0 - very low
S - rapid rise triggered by cell divison -> plateau
G1 - plateau

Question 3

What are the key components of the signalling pathways?

Answer 3

• Regulation of enzyme activity by protein phosphorylation (kinases)
• Adapter proteins
• Regulation by GTP-binding proteins

Question 4

How do GFs stimulate the signaling pathway?

Answer 4

Mitogenic GF (e.g Hepatocyte GF released after liver damage) -> tyrosine kinase type receptor -> GTP- binding protein (Ras) -> kinase cascade -> immediate early genes (e.g c-myc) - control expression of other genes

Question 5

How can signalling by peptide growth factors be manipulated in cancer treatment?

Answer 5

in HER2 positive breast cancer - herceptin (antibody) inhibits HER2 receptor tyrosine kinase

blocks early stage of growth stimulation

Question 6

What is Grb2?

Answer 6

important adaptor molecule that’s recruited in breast cancer

When the dimeric GF binds to two receptor tyrosine kinase molecules, it brings them closer together -> tyrosine kinase domain is able to cross-phosphorylate the partner receptor (you get multiple cross-phosphorylation of several tyrosine residues) -> Phosphorylated domains on the tyrosine kinase receptors act as docking sites for adaptor proteins (which are recruited to the activated tyrosine kinase receptors) -> contribute to downstream signalling

Question 7

What are adaptor proteins?

Answer 7

proteins that are able to recognise specific phorsphorylation motifs and interact to different proteins

Protein-protein interactions: bring proteins together

• modular and contain domains - different domains that are mixed and matched to give the protein different properties, and are important in molecular recognition

Question 8

What types of protein-protein interations does Grb2 have?

Answer 8

SH2: binds to the phosphorylated tyrosines of the receptor

SH3 (2 copies): bind to the proline-rich regions of other protein

Question 9

What is Ras?

Answer 9

• GTP binding protein
• either on (GTP bound) or off (GDP bound):
  > Under the influence of appropriate signals, the GTP can replace the GDP to make Ras active
• NOT phosphorylation – it is merely the exchange of GDP for GTP (catalysed by Sos)

GTP binding protein can hydrolyse GTP to GDP to turn itself off

Question 10

What is the cycle of GTP binding proteins controlled by?

Answer 10

• Exchange factors (e.g. Sos) that turn it ON

- GTPase activating proteins (GAPs) that turn it OFF (clip the phosphate off

Question 11

How is Ras activated?

Answer 11

Grb is bound to the RPTK via its SH2 domain and it binds to a protein called Sos (exchange factor for Ras) through its SH3 domains

When the RPTK becomes activated, you get phosphorylation of the receptor -> Grb2 (with Sos attached) binds to these phosphorylated tyrosine domains

Sos is then close enough to the membrane to activate Ras -> allows the exchange of GDP for GTP in Ras to form a GTP bound form of Ras
-> conformatinal change -> active state

Question 12

What are common oncogeninc mutations of Ras?

Answer 12

V12Ras - glycine (simple hydrogen side chain) residue in position 12 changed to VALINE (hydrophobic) due to mutation -> prevents GAPs from binding to Ras -> can’t turnt off very easily

L61Ras - Glutamine (amide side chain) in position 61 is converted to LEUCINE (hydrophobic). This is a single base change in the genome -> inhibits the intrinsic GTPase activity -> constantly being in the GTP bound (on) state -> gives growth stimulatory signals

Question 13

What is the ERK cascade?

Answer 13

Extracellular signal-regulated kinase (ERK) cascade (specific) - also known as Mitogen-activated protein kinase (MAPK) cascade (general family is called MAPK)

ERK - kinase cascade that is involved in the growth stimulatory signalling

Question 14

What are cyclin- dependent kinases?

Answer 14

family of kinases

• serine-threonine kinases
• CDKs are in the cell throughout the cell cycle but not activated until they bind to an activating protein called cyclin
• ALSO controlled by phosphorylation –

Question 15

What are cyclins?

Answer 15

• activate CDKs
• transiently expressed during cell cycle
• degraded once they’ve activated CDKs

Question 16

What CDK complex controls the progression throughout mitosis?

Answer 16

M-phase promoting factor (combination of cyclin and CDK) - triggers mitotic machinery

Question 17

What do aactivated CDKs do?

Answer 17

phosphorylate proteins (on Serine or Threonine) to drive cell cycle progression

• CDK1 will bind to the mitotic cyclin (cyclin B) to phosphorylate substrates at mitosis e.g nuclear lamins ( cause breakdown of nuclear enverlope)
• CDK2, which binds to G1 cyclin (cyclin E). In this case, the active kinase will be phosphorylated at the start of synthesis e.g retinoblastoma protein ( tumour suppressor)

Question 18

What is mitosis promoting factor?

Answer 18

consists of:

• CDK1
• Mitotic cyclin (usually cyclin B)

interaction will induce cells to undergo mitosis

Question 19

How are CDKs regulated?

Answer 19

CDK1 binds to cyclin B -> complex is usually inactive on its own - has another level of regulation, which is PHOSPHORYLATION

There are 2 phosphorylation reactions to regulate CDK activity. CDK has to be activated at specific sites to become activated. This activation is performed by CDK activating kinase (CAK)

Question 20

How is the phosphorylation of CDK1 balanced?

Answer 20

CDK activating kinase (CAK) puts an activating phosphorylation onto Cdk1

Wee1 (inhibitory kinase) puts an inhibitory phosphorylation onto Cdk1

Question 21

What does Cdc25 do?

Answer 21

CDK1 is bound to cyclin BUT it needs the phosphate to be removed before it can act

Cdc25 (phosphatase) takes off the inhibitory phosphate that was put on by Wee1 -> an active MPF

Question 22

When does dephosphorylation of CDK1 happen?

Answer 22

end of interphase

Question 23

describe the positive feedback action of cdc25

Answer 23

Active MPF is able to phosphorylate Cdc25 to increase its activity - drives mitosis

Question 24

Which cyclins control each stage of mitosis?

Answer 24

• G1/S: CDK2 AND Cyclin E
• S: CDK2 AND Cyclin A
• M: CDK1 AND CyclinB
• same CDK is being used in G1/S phase and S phase but they are doing different jobs - when cyclin binds to CDK it actually changes its substrate specificity so that it can phosphorylate different substrates depending on which cyclins are bound to it, it also changes substrate accessibility

Question 25

What are the effects of GF stimulation of signalling pathways on the cell cycle?

Answer 25

promotes G0 to G1 transition

• bind to the receptor protein tyrosine kinase
• through Ras, this triggers a kinase cascade -> phosphorylation of transcription factors that turns on the expression of c-Myc
• Immediate early gene transcription factors are turned on (e.g. c-jun, c-Fos and c-Myc)

Question 26

What does cyclin D do?

Answer 26

binds to CDK4 and CDK6 –> controls the entrance to the cell cycle and stimulates synthesis of cyclin E

• regulates the G0 -> G1 transition

Question 27

What regulates cyclin D?

Answer 27

TF c-Myc

Question 28

How does retinoblastoma regulate gene expression?

Answer 28

G0 : unphosphorylated
- binds to and sequesters a family of TF called E2F -> held in cytoplasm -> everything turned off

CDK4/6-cyclin D kinase phosphorylates retinoblastoma protein -> loses afinity for E2F -> E2F TFs bind to promotors in nucleus of genes involved in cell cycle progression

• acts as brake in cell cycle - TSG

release of E2F stimulates the production of cyclin E -> forms complex with CDK2 -> further phosphorylation of retinoblatsoma -> increased E2F -> binding at lower afinity

**The cyclin A gene promoter is not activated until the E2F concentration is high enough. This cycle continues throughout the cell cycle

Cyclin A binds to CDK2 -> cyclin B and CDK1 complex formed -> mitosis

Question 29

What do E2F TFs do?

Answer 29

regulate proto-oncogenes (including Myc proteins) and control genes involved in S phase (e.g thymidine kinase)

Question 30

What are the two types of CDK kinase inhibitors?

Answer 30

INK4 Family

CIP/KIP Family

Question 31

When are the CDK kinase inhibitors active?

What do they do?

Answer 31

INK4 family is active in G1

• Inhibitors of the G1 phase
• They inhibit CDK4/6 by displacing cyclin D

CIP/KIP family is active in S phase

• Inhibitors of the S phase
• They inhibit ALL the CDK/cyclin complexes by binding to them