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Advanced Cell Signalling > Control of Immediate Early Gene Transcription > Flashcards

Control of Immediate Early Gene Transcription Flashcards

1
Q

What is the activation of a signalling pathway?

A

Transient

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2
Q

What are the effects of signalling pathway activation?

A

Transient or long term

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3
Q

What is the usual half life of most proteins?

A

Less than 24 hours

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4
Q

How can the output be varied by activating a signalling pathway?

A

Amplitude
Duration
Sub cellular location
Context

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5
Q

What are PC12 cells?

A

Cell line derived from rad adrenal medulla

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6
Q

What do PC12 cells response to?

A

NGF and EGF

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7
Q

What do NGF and EGF activate?

A

The classical ERK1/2 MAPK cascade

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8
Q

How do NGF and EGF act differently in PC12 cells?

A

NGF activates ERK1/2 for longer
EGFR is internalised faster
NGF promotes stronger nuclear activation of ERK1/2
NGF activates Rac1 at the membrane, EGF does not do this well
NGF causes neuronal differentiation, EGF induces proliferation

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9
Q

What causes the difference in NGF induced neuronal differentiation and EGF induced proliferation?

A

The different in ERK activation

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10
Q

How does NGF increase ERK1/2 nucleur activation?

A

ERK1/2 has to translocate from the cytoplasm to the nucleus and therefore the longer the activation, the more ERK can translocate

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11
Q

What is gene transcription controlled by?

A

Transcription factor recruitment / activation of gene promoter
Chromatin environment

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12
Q

How does transcription factor recruitment / activation of gene promoters effect gene transcription?

A

Promoters can be regulated by multiple transcription factors
Multiple pathways converge on one transcription factor

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13
Q

How does chromatin environment effect gene transcription?

A

Chromatin structure can be stably modified during cell development or after cell stimulation
- e.g. methylation allows you to have open and closed areas for chromatin (closed inhibits transcription)
This changes promoter exposure

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14
Q

How are immediate early genes induced?

A

quickly after stimulation

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15
Q

What do immediate early genes not need which secondary response genes do?

A

Synthesis of new proteins e.g. transcription factors

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16
Q

What is IL-10 and why?

A

An immediate early gene

It is transcribed quickly after activation and cyclohexamide does not effect its transcription

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17
Q

What cyclohexamide do?

A

Inhibits the production of new proteins

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18
Q

What is IL-6 and why?

A

A secondary response gene

It is produced slower than IL-10 and is inhibited by cyclohexamide addition

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19
Q

Give example of transcription factors which are immediate early genes

A

c-fos
c-jun
nur77

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20
Q

Give an example of an immediate early gene which modifies the cytoskeleton

A

Arc

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21
Q

Give examples of enzymes which are immediate early genes

A

iNOS

PTGS2/ cox2

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22
Q

Give examples of cytokines/ growth factors which are immediate early genes

A

IL-10
IL-1
HB-EGF

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23
Q

How does phosphorylation control transcription factors?

A

It promotes cytoplasmic location in some cases

Can also promote nucleur location

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24
Q

Give an example of how phosphorylation induces cytoplasmic location

A

Akt induces FOXO phosphorylation which causes 14-3-3 binding and cytoplasmic location
Inhibition of Akt induces FOXO movement into the nucleus for transcription

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25
Q

Give an example of how phosphorylation causes nuclear location

A

JAK causes phosphorylation of 2 STAT molecules

The STAT molecules dimerise and move into the nucleus

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26
Q

Describe CREBs function

A

Promotes immediate early gene transcription which causes:

  • development
  • regulation of metabolism
  • synaptic function
  • Innate immune function
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27
Q

What is CREB?

A

A transcription factor

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28
Q

What family is CREB a member of?

A

bZIP

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29
Q

What are bZIP domains?

A

DNA binding domains with alpha helical motifs containing a sequence specific DNA binding region and a leucine zipper which mediates dimerisation

30
Q

In DNA, what does CREB bind to?

A

cAMP response element (CRE)

31
Q

Why are not all CRE’s active?

A

They may be inaccessible - tightly bound to the nucleosome or in condensed chromatin
Binding of TFs to adjacent sites may block CREB binding to CRE
Motif may not be in the gene promoter or be able to contact the transcriptional machinery

32
Q

As well as the bZIP domain, what are the other CREB domains?

A

Q1
KID
Q2

33
Q

What is a Q1 domain?

A

Glutamine rich domain

34
Q

What is the KID domain?

A

Kinase rich domain

35
Q

What is CREM?

A

It only has a Q2 and bZIP domain and therefore still binds CRE but cannot drive transcription
May be a CREB inhibitor

36
Q

What is ATF1’s relation to CREB

A

They have similar structures and are regulated in a similar way and therefore may compensate for one another

37
Q

What is the PKA signalling pathway

A

GPCR -> GalphaS subunit -> Adenylate cyclase -> cAMP -> PKA

38
Q

What inhibits cAMP?

A

Phosphodiesterase

39
Q

What are CBP and p300?

A

Two related transcription co-activators

40
Q

Apart from the transcription factor binding domains, what other domais are in CBP and p300?

A

HAT
BR
PHD

41
Q

What is the function of the HAT domain?

A

Causes histone acetylation which is required for transcriptional regulation

42
Q

What is the function of PHD?

A

Chromatin binding

43
Q

What is the CREB binding domain?

A

KIX

44
Q

What domain of CREB binds to KIX?

A

KID domain

45
Q

What does the unphosphorylated form of CREB KID have?

A

A disordered domain

46
Q

What is the function of phosphorylating CREB on Ser133?

A

Stabilises the structure

47
Q

Once CBP or p300 are recruited to CREB, what do they do?

A

Acetylate histones to increase transcription

48
Q

Apart from PKA, what other kinases can phosphorylate CREB on S133?

A

CaMK

MSK1/2

49
Q

Despite MSK1/2 phosphorylating S133 on CREB, what does it not do?

A

Recruit CBP and p300

50
Q

What are CRTCs?

A

Alternative CREB co-activators

51
Q

What are the 3 isoforms of CRTCs?

A

1
2
3

52
Q

Does CRTCs have catalytic activity?

A

no

53
Q

How does CRTCs bind to CREB?

A

The N-terminal of CRTC binds to the bZIP domain of CREB

54
Q

Can CRTCs bind to CREB when phosphorylation on S133 has not occured?

A

yes

55
Q

What are the possible functions of CRTCs?

A

To stabilise the interaction between CREB and CBP or between CREB and components of the RNA polymerase complex

56
Q

In unstimulated cells, where are CRTCs localised to?

A

The cytoplasm

57
Q

How is CRTCs bound to 14-3-3 ?

A

By phosphorylation on 3 sites

58
Q

What does 14-3-3 binding to CRTCs do?

A

Masks a nuclear localisation sequence and therefore CRTCs stay in the cytoplasm

59
Q

What does dephosphorylation of CRTCs induce?

A

CRTCs translocate to the nucleus where it can bind to CRE

60
Q

What promotes CRTCs dephosphorylation?

A

cAMP-PKA stimulation

61
Q

What is SIK?

A

Salt inducible kinase

62
Q

What are the 3 isoforms of SIK?

A

1, 2 and 3

63
Q

When are SIK activated?

A

When they are phosphorylated by LKB1

64
Q

What is the function of SIKs?

A

They phosphorylate CRTCs on the sites required for 14-3-3 binding

65
Q

How is SIKs function inhibited?

A

By PKA phosphorylating it

66
Q

What is the evidence against MAPK being able to stimulate CREB dependent transcription?

A

MAPK activation does not drive the transcription of classical CREB dependent luciferase reporter genes
MAPK activation does not promote p300 and CBP recruitment
Mutations in p300 and CBP do not effect MAPK induced transcription
MAPK activation does not promote nucleur localisation of CRTCs
Many CREB dependent genes also contain serum response elements in their promoters which could explain the regulation by MAPKs
CREB may play a basal role in transcription - even if MAPK activation did induce CREB target genes in CREB KO this could be due to a basal and not a stimulated role for CREB

67
Q

What is the evidence for MAPK stimulating CREB dependent transcription?

A

Many IEs induced by MAPKs are CREB targets
There is some overlap between PKA-induced and MAPK-induced CREB target genes but there is also some differences
Knockdown of CREB can reduce gene induction downstream of MAPK
Deletion or inhibition of MSK1/2 reduced the induction of several IE genes downstream of stimuli which activate MAPK

68
Q

As well as CREB, what else can MSK1/2 phosphorylate?

A

Histone H3

Trim28

69
Q

Since histone H3 can be phosphorylated by MSK1/2, what does this mean in terms of CREB?

A

MSK1/2 can induce IE genes independently of CREB

70
Q

What happens if you KO Ser133 in CREB in mice?

A

They die shortly after birth

71
Q

What happens if you change the Ser to Ala in Ser133 in CREB in mice?

A

They are viable and fertile but are born at less than the expected Mendelian frequency

72
Q

What is required for the production of IL-10? How can you increase IL-10 expression

A

LPS

Increase it by adding PGE2 stimuli too

Advanced Cell Signalling (12 decks)

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