L16 The use of antibodies in the study of cell signalling pt2

Question 1

How can proteome be studied in cell signalling?

Answer 1

Identification of PTMS (eg phosphorylation) using mAbs

Question 2

Which three amino acids are phosphorylated?

Answer 2

Serine, threonine and tyrosine.

Question 3

Give examples of kinases that phos. each amino acid

Answer 3

Serine - PKA

Threonine - PKC

Tyrosine kinase - EGF (self-phos.)

Question 4

What three approaches can be taken to study protein phosphorylation?

Answer 4

1. Is protein phosphorylated?
2. Which aa’s are phosphorylated?
3. Where is the site of phosphorylation?

Question 5

How can it be investigated if a protein of interest is phosphorylated?

Answer 5

Metabolically label cells with radioactive phosphphate group.

SDS PAGE isolated protein.

Weight of POI can be seen, known if weight is phos. or not.

Question 6

What disadv does radioactive phos. labeling have?

Answer 6

Doesn’t say which amino acids are labelled, or where the site is.

Question 7

How can the type of aa undergoing phos. on POI be determined?

What disadv. does still this have?

Answer 7

POI hydrolysed and can be analysed via TLC or HPLC to see which aa’s in the sample are phos.

Still doesn’t determine specific sites

Question 8

How can the site of phos. on POI be determined?

Answer 8

Many ways,

2D phosphopeptide mapping of tryptic digest.

HPLC of tryptic digest.

site directed mutagenesis

2D gel EF

Question 9

How does 2D phosphopeptide mapping of tryptic digest determine site of phos.?

Answer 9

Peptides show a ‘barcode’ of weight and charge on gel. Determined from a database if present.

POI spot can be excised from gel and analysed furtehr by MS

Question 10

After 2D PAGE, how are proteins visualised?

Answer 10

Using staining.

Coomassie blue, silver stain or syproRuby for example

Question 11

Which stains are used for studying PTM?

Answer 11

Pro-Q Diamond is a phosphorlyation stain.

Seen in blue

Question 12

How are antibodies used to study phos?

Answer 12

Anti-pTyr

Anti-pSer

Anti-pThr

Question 13

How at mAbs generated for pTyr?

Answer 13

Mouse immunised with phosphotyrosine coupled to KLH (adjuvant)

Question 14

How are anti-pTyr antibodies used in studying cell signalling/PTM?

Answer 14

1. Looking for gross changes in protein tyrosine phos across proteome.
2. Monitor specific proteins using isolated POI’s.
3. Used for immunoprecipitation of phosphorylated proteins only.

Question 15

How is BAD investigated using phos. specific Abs?

Answer 15

Bad is pro-apoptotic, but inhibited by phos. of Ser 112 and Ser136.

This is done by Akt at Ser136 and promotes cell survival.

Question 16

What are MAPKs?

Answer 16

Serine/threonone kinases. 3 main subfamilies

p42/44 - ERK1/2

JNKs

p38

Question 17

What are the activators of MAPKs and what role do they play?

Answer 17

TKR’s and GPCRs.

Involved in cell prolif, diff, apop, inflamm and response to environ. stress

Question 18

How are antibodies used to study ERK1/2 pathway?

Answer 18

1. Monitor expression and distrution of signalling proteins involved in disease or health for e.g.
2. Phospho-specific Abs to site, to monitor changes in protein phos.