13 - Turning On/Off the Signals - Williamson

Question 1

describe the structure of most kinases and draw a diagram of this

Answer 1

• kinase contains 2 domains; one for binding ATP and the other holds the substrate (kinase domain), positioning it next to the activation loop and ATP (ie in the AS)

Question 2

when can the substrate bind to the kinase? draw a diagram of the S binding

Answer 2

when the activation loop has been P therefore more rigid structure (once loop has been P it can form H bonds with the protein -> rigidifying it) and can form a B sheet with the substrate

Question 3

why is it important that the ON state kinases all show a similar structure ?

Answer 3

similar ON state rigid structure allows recognition of these kinases by multiple different proteins etc. important for OFF state to have a v different structure because does not want to be accidentally recognised as being ON

Question 4

describe ALL of the mechanisms (giving examples and drawing diagrams) that allows the kinases to become active

Answer 4

• P of the activation loop. forming B sheets with substrate therefore locking in conformation
• presence of pseudo substrate loop. similar shape to the S and blocks the AS
• proteins can also only be active as dimers. dimerisation interface present to promote dimerisation so their AS can reach configuration. eg P of the Activation loop of ERK2 necessary for correct organisation of dimerisation interface. this can then bind its dimer and move into the nucleus

Question 5

describe how Cdk cell cycle regulatory proteins are regulated. draw a diagram

Answer 5

• the activation loop firstly needs to be P
• for the ATP to P the substrate correctly, needs to be held in correct conformation, with correct metal ions bound etc
• cyclins binding to the dimerisation interface of the CDR regulatory proteins positions the PSTAIRE helix with the crucial Glu residue
• this can then position the ATP correctly so can phosphorylate the substrate

Question 6

are most kinases activated in the same way? if so, what is the exception to the rule and why is this so?

Answer 6

yes most kinases activated in the same way

• apart from the activation of Raf by Ras
• because Raf is the start of the kinase cascade, then after activation no going back so need to ensure that this activation is done correctly. also many key signals/pathways feed into Ras so need to ensure correct activation

Question 7

Describe the structure and draw a diagram of the molecule that assists in Raf activation

Answer 7

14-3-3 molecule

• dimer
• 2 binding sites that recognise a phosphorylated Ser/Thr (activation of Raf requires Ser/Thr P not Tyr)
• sites are at certain distance apart to recognise phosphorylated residues
• 14-3-3 has rigid structure, known as molecular anvil in which proteins can be bent so their phosphorylated sites fit the 14-3-3 binding sites
• 14-3-3 binds to Raf but differently depending on which molecules phosphorylated

Question 8

how can the cell control the activity of 14-3-3?

Answer 8

P of Ser58 residue @ the dimer interface disrupts the dimer and stops binding to targets

Question 9

describe how Raf. is activated then inactivated again. draw a diagram of this cycle

Answer 9

• resting raf (inactive kinase domain) clamped shut by 14-3-3 which recognises the phosphorylated residues on Raf - S259 and S621. recognition of these phosphorylated sites only occurs if @ correct distance therefore for correct recognition Raf needs to be in close conformation
• Ras - GTP binds to Raf and causes it to open up and expose the S259 P site when 14-3-3 is displaced
• phosphatase PP2 binds and deP S259
• 14-3-3 detachment uncovers Y341. allows it to be P and Raf is activated
• 14-3-3 unbound arm can bind other kinases eg PKC leading to > P of Raf. kinase cascade begins
• negative feedback; rephosphorylation of S259, 14-3-3 can rebind again, clamps Raf shut and inactivates it
• phosphatases can remove the additional phosphorylated residues

Question 10

what is the evidence that kinases aren’t v specific?

Answer 10

• human genome encodes around 25000 genes are estimated 500,000 phosphorylation sites
• only 518 kinases therefore can’t be specific (eg over 1000 targets each - not taking into account specific S/T and Y kinases)
• only 1/3 of that are phosphatases therefore even LESS specific

Question 11

if kinases aren’t that specific then how can specificity (which is so important be achieved)?
broad terms

Answer 11

proximity of proteins using scaffolds and adaptors allow the correctly shaped S to fit into the active site of its R

Question 12

draw an example of a scaffold protein connected to a G protein and state why it is not a rigid strcture

Answer 12

not a rigid structure because the S needs to be able to move and fit into the kinase AS

Question 13

draw a scaffold protein that binds proteins involved in the kinase cascade. give the name of this scaffold

Answer 13

343 - 13

Question 14

what happens if we overexposes scaffold proteins?

Answer 14

eg one scaffold protein will bind Raf, another one MEK therefore will not get any interactions between these 2. need to be held on the SAME scaffold so that the proteins can find each other

Question 15

how do scaffolds and adaptor proteins differ?

Answer 15

adaptors eg SOS have clearly defined ends eg one end binds SH2, the other SH3 and scaffolds do not have this. however they have similar functions in both being involved in signalling and bringing proteins together to interact

Question 16

draw some diagrams and explain insulin signalling

Answer 16

• insulin dimeric R is is already attached when unbound to insulin however too far apart for crossP
• upon insulin binding, the 2 halves come into closer proximity , cross P and the P receptor and the insulin receptor now binds IRS1 scaffold protein
• scaffold IRS1 allows assembly of more proteins @ the R and increases both specificity and integration of the signals from other pathways
  IRS1;
• PTB; recognises pY on R
• PH anchored to membrane
• 3rd domain is P by the active receptor kinase
• once this 3rd domain has been P it can be recognised by many other adaptor proteins eg Grb2 which can then bind to Sos, more proteins

Question 17

describe and draw diagrams of the 3 main ways that a R can be turned OFF. giving examples

Answer 17

1) internalisation of L bound R. R can either then be degraded or recycled
2) phosphatases can be linked to their substrates via adaptors or scaffold -> deP and inactivation
eg JAK/STAT
3) crucial part of the system can be degraded using the Ub system eg JAK/STAT; activation, SOCS expression, SOCS SH2 -> Phosphorylated receptor -> SOCS BOX recruits E2 ligase. eg Smad turned off by smurf

Question 18

how are G proteins turned off?

Answer 18

• GAP

- arrestin binds to the GPCR, downregulats the R leading to internalisation -> recycling/digestion

Question 19

how does the cell turn off ion channels?

Answer 19

enzymes to degrade the S

and pumping the ions (v hard work)

Question 20

what is colocation and why is it so useful?

Answer 20

most steps of RTK are not enzyme reactions, they are binding events that function to get a component into different place eg next to membrane or nucleus

Question 21

give 2 examples of where colocation is useful?

Answer 21

PH domains;

• bind to membranes and inositol phosphates
• attach components to membranes In a defined orientation. therefore now a 2D search rather than 3D, allows components to find each other much more easily
• PH domains can also recognise specific lipids/parts of membranes eg lipid rafts
• they can also recognise PIP3 - product of PI3 kinase (enzyme activated from other signalling pathways)

Ras Isoforms;

• 3 isoforms of Ras that all have similar structure but act differently due to their location
• K Ras = farnesyl chain and a basic patch therefore attached to membrane
• N/H Ras = palmitoyl chain. therefore > likely to be in the cell membrane and Golgi membranes
• palmitoylation is reversible and controls where they are

Question 22

why is it that just inhibiting a protein/kinase cannot give the desired function?

Answer 22

• kinases are not specific - specificity may arise from binding elsewhere
• location is vital. proteins may have different functions in different places

Question 23

describe lipid rafts briefly

Answer 23

• regions of the membrane that are rich in cholesterol and sphingolipids therefore > rigid and thicker than other regions of the membrane
• can collate functionally related proteins together eg signalling proteins + cytoskeletal proteins (Mts, actin fibres)
• can link extracellular activity to intracellular activity because signals can rapidly pass down MTs to nucleus etc
• not permanent structures so can be assembled and taken apart
• assembly = natural result of lipid segregation (spontaneous). signalling function is probably natural result of evolution