Phosphorylation Signalling Flashcards

1
Q

What cellular processes are facilitated by signalling pathways?

A

• Mitogenic signalling
o Membrane-nucleus signalling, eg hormones that affect transcription
• Sensing cellular stress
o Response to nutrients/ATP/AA/O2 levels
• Chromatin organisation and transcription control
• Cell Cycle control
• Sensing DNA Damage

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2
Q

What is the simplest kind of signalling pathway?

A

Two componenet signalling, where a single protein has a sensing domain that responds to stimulus and activates or inactivates a signalling domain that acts upon another protein with a reciever domain, causing the effector protein to output a response to the change in stimulus.

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3
Q

What kind of phosphorylation signalling do bacteria use that eukaryotes don’t?

A

Autophosphorylation of Histidine residues

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4
Q

What is the relative frequency of use of the different PTMs used in signalling?

A
Phosphorylation
Acetylation
Hydroxylation
Methylation
Ubiquitination
SUMOylation
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5
Q

How much more common is phosphorylation than acetylation?

A

A full order of magnitude

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6
Q

Why is phosphorylation commonly used in signalling?

A

Adding a phosphate group to a protein can have a large effect on it, which is useful for causing large conformational changes or altering catalytic/binding ability.

Phosphate groups introduce a large steric change to the protein as well as being a large concentration of negative charge which can affect all the surrounding protein structure.

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7
Q

How many human kinases are there?

A

450

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8
Q

Which two phosphorylable residues are very similar?

A

Serine and threonine are very similarly structured amino acids, differing only by an extra carbon in the serine side chain before the hydroxyl group.

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9
Q

Which phosphorylable residue is dissimilar to the others?

A

Tyrosine, having a very different shape. As such, tyrosines are very rarely phosphorylated by the same kinases as serine and threonine (which often are done by the same unspecific kinase) but there are exceptions to this, notably in MAPKKs such as MEK1 and 2 which phosphorylate serine and tyrosine on the same target.

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10
Q

Which residue is phosphorylated least?

A

Tyrosine phosphorylation is much rarer than the others, accounting for 1 in 2000 phosphorylation events. It does however play a key role in mitogenic signalling.

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11
Q

What are some examples of RTKs?

A

These include receptors for EGF2, ErbBs, IGF2 and insulin as well as HER2 receptors.

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12
Q

How do RTKs transmit the signal?

A

The RTK intracellular domains usually consist of two arms, each with kinase domains that phosphorylate each other (transphosphorylation) at multiple tyrosine residues.

These phosphate groups act as docking sites, allowing the binding and activation of various adaptor proteins that go on to activate signalling pathways.

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13
Q

Of the signalling pathways initiated by RTKs, why is STAT5 notable?

A

STAT5 is activated by the RTKs directly, and itself directly regulated the genes with no intermediate cascade.

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14
Q

What domains bind second messengers to the phosphorylated RTK arms?

A

SH2 or PTB.

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15
Q

What characterises PTB phosphotyrosine binding?

A

Not all members of the PTB domain family actually bind pY, their structure is more similar to that of the PH domains responsible for binding to phospholipids in the plasma membrane and this is in fact the role of many of them. The PTB domains that are pY-binding only bind pY in a specific sequence context: N-P-x-pY.

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16
Q

What are RTK receptor substrates?

A

Instead of only transphosphorylating the two arms, some RTKs such as the insulin and IGF-1 receptor recruit a substrate whose tyrosines are phosphorylated to recruit the adaptor proteins. Some cross-phosphorylation does still tend to occur.

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17
Q

What is the RTK receptor substrate for insulin and IGF-1 receptors?

A

IRS1/2 (insulin receptor substrate)

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18
Q

What is the RTK receptor substrate for the fibroblast growth factor receptors?

A

FRS1/2

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19
Q

What are HER3 RTKs?

A

Also known as ErbB3 receptors, these are a member of the EGFR family. They are often found to be overactive in breast cancer, hence antibody drugs have been used to disable them.

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20
Q

What is the mechanism of HER3 signal transduction?

A

Upon binding the ligand HER3 must form heterodimers with other RTKs, often HER2 or EGFR (AKA ErbB1 or HER1), to compensate for its own lack of kinase ability. Because HER3 cannot phosphorylate itself it must recruit other receptors to do it for it, but when it is activated it creates a very potent signal that can have strong downstream effects.

21
Q

What is the role of HER2?

A

HER2 is an active kinase receptor, but does not bind any known ligands so may only be present in order to function as a heterodimer with HER3.

22
Q

What different effects can phosphorylation have on a protein?

A

Creation of protein docking sites - RTK signalling

Disruption of Complexes – E2F/pRb/HDAC

Enzyme Activation – MAP Kinase Pathways

Enzyme Inactivation – c-SRC and CSK

De/Stabilisation of Proteins – Myc Level

Modulation of Subcellular Location -FOXO

23
Q

What is E2F involved in?

A

E2F is a transcription factor which causes progression from the G1 to S phase, at which cells make a commitment to divide. Its targets include DNA pol.

24
Q

What represses E2F?

A

It is repressed by a tumour suppressor called pRb which binds E2F with a long C-terminal tail domain. It also binds a HDAC, which provides a level of regulation we will not look at.

25
Q

What relives the inhibition of E2F?

A

In early-mid G1 phase, cyclin D-CDK4/6 complex double phosphorylates the c-terminal domain of pRb, destabilising it and causing it to release the HDAC and lowers its affinity for E2F.

Three further phosphorylations by the Cyclin D-CDK4/6 and Cyclin-E-CDK2 complexes causes a conformational change and total complex disruption leading to full activation of E2F.

26
Q

What are MAPK pathways? What stimulates them?

A

Mitogen activated protein kinases are a family of signalling cascades in which kinases phosphorylate the next kinase in the step in order to amplify the signal of a receptor. They are an example of enzyme activation by phosphorylation.

These are often stimulated by Ras, Rac, CDC42 and RHO.

The three main MAPK pathways are Erk, JNK and p38

27
Q

What are Erk and Ras involved in?

A

Erk is responsible for proliferation and differentiation, and is primarily activated by Ras. Thus it is no surprise that mutations in Ras are the most common mutations associated with cancer.

Ras, a small GTPase, is activated by membrane-associated events and activates Raf (MAPKKK), which phosphorylates MEK1/2 (MAPKK), which goes on to phosphorylate two very closely located threonine and tyrosine residues on ERK (MAPK). ERK can then go on to activate transcription factors in the nucleus.

28
Q

What are the parts of a MAPK signalling cascade?

A

There are three MAPKs involved in each pathway, which can be generalised as MAPKKK, MAPKK and MAPK. MAPKKK tends to phosphorylate serine residueson MAPKK, which phosphorylates MAPK on closely related threonine and tyrosine residues.

29
Q

What is the structure of c-SRC?

A

Human c-SRC is composed of four SH domains, in descending order from the N-terminus where SH4 links it into the lipid bilayer.

30
Q

What is the role of the c-SRC SH2 domain?

A

The SH2 domain inhibits the kinase activity of the SH1 domain when bound to it, which only occurs when the SH1 Tyr530 is phosphorylated by CSK.

31
Q

How is the SH1 domain of c-SRC activated?

A

The phospho-tyrosine must be removed by phosphatases before the SH1 kinase activity can be activated, and full activity also required another, this time activating phosphorylation to the SH1 domain at Tyr419.

32
Q

What are the phosphorylable sites on c-Myc and what effect do they have?

A

When c-Myc is phosphorylated at its Ser-62 this acts to stabilise it, preventing degradation. Thr-58 phosphorylation has the opposite effect, often leading to polyubiquitination and degradation.

33
Q

What kinases phosphorylate c-Myc?

A

Ser-62 is phosphorylated by ERK, and Thr-58 by GSK-3.

34
Q

How does cellular signalling regulate c-Myc?

A

Growth stimulatory signals that act through Ras increase the concentration of c-Myc by activating ERK through the Raf/MEK pathway and inhibiting GSK-3 via PI3K/Akt.

35
Q

What is FOXO?

A

FOXO is a transcription factor and so is only active when within the nucleus.

36
Q

What inhibits FOXO?

A

Phosphorylation by Akt and de-ubiquitination by USP7 cause nuclear export to the cytoplasm where it may be polyubiquitinated and degraded.

37
Q

What activates FOXO?

A

Phosphorylation at different sites by JNK or MST1 promotes nuclear import, stimulating the FOXO to enter the nucleus and activate its stress response.

38
Q

What are Non-phosphorylatable Analogues?

A

In order to study the effects of a protein not being phosphorylated when it would otherwise be the amino acids in question can be replaced with residues of similar shape and size that cannot be phosphorylated.

39
Q

What are the non-phosphorylatable analogues for the three phosphorylable residues?

A

Serine and threonine can both be replaced by alanine with little or no structural disturbance. Tyrosine is similarly replaced with phenylalanine.

40
Q

What are Phosphomimetics?

A

In order to study the effects of constant phosphorylation of those residues, Ser and Thr can be replaced with acidic amino acids that approximately mimic the larger shape and negative charge of a phosphate group.

41
Q

What phosphomimetics are used?

A

Serine is often replaced with aspartate and threonine with glutamate to reflect the different chain lengths of the smaller amino acids.

42
Q

How many phosphatases are there in humans?

A

Enzymes that remove phosphate groups are far less common than kinases, around 154 compared to 450.

43
Q

What are the classes of phosphatases?

A
•	Serine/Threonine phosphatases
      o	43 known examples
      o	PP1, PP2A, PP2C
•	Tyrosine Phosphatases
      o	56 known examples
               	22 Receptor Protein Tyrosine Phosphatases (PTPs)
               	34 Non-Receptor PTPs
•	Dual Specificity Phosphatases
      o	Can dephosphorylate serine, threonine or tyrosine
      o	41 known examples
      o	DUSP1, VHR
•	Lipid Phosphatases
      o	Remove phosphates from phospholipids
      o	14 known examples
      o	PTEN, SHIP1, SHIP2
               	PTEN also dephosphorylates proteins in vitro, both important for function
44
Q

How does the active site of phosphatases allow for environmental regulation?

A

All PTPs, dual specificity phosphatases and also PTEN contain a cysteine residue in the active site that is essential for catalysis. The cysteine is within a conserved sequence of HC(X5)R.

This cysteine can be oxidised or reduced, but is only active in its reduced sulphydryl form. This allows for phosphatases to react to the oxidative state of the cell. During a oxidative burst this turns off all the phosphatases.

45
Q

Why is phosphate signalling relevant to medical research?

A

Aberrant mitogenic signalling is a hallmark of cancer because it is responsible for, amongst many other things, controlling proliferation, growth, angiogenesis, genome instability, apoptosis, immune system evasion and avoiding growth suppressors.

46
Q

How can analysis of signalling pathways lead to improved cancer treatment?

A

Having identified so many mutations/problems in kinase signalling that can cause cancer has led to the development of a new generation of drugs that aim to undo the effects of the mutations.

This includes the ability to personalise therapies to the patient by identifying where in their signalling pathway the amplification is occurring, but because these pathways are so vital to so many processes these often do have severe side effects.

47
Q

What have CDK4/6 inhibitors recently shown promise in the treatment of?

A

Breast cancer treatment

48
Q

What does Sorafenib treat?

A

It is an inhibitor which activates autophagy and blocks downstream signalling. It can be used to treat melanomas in which there is a V600E mutation in Raf.