L20 Antibodies in cell signalling pt 3

Question 1

What is protein phosphorylation?

Answer 1

Addition of phosphoate group to protein, from kinase.

Question 2

What role does phosphorylation play on BAD?

Answer 2

BAD is a pro-apoptotic Bcl2 family member protein.

Phosphorylated, it is inactivated.

Question 3

How is BAD pro-apoptotic?

Answer 3

Displaces Bax from Bcl-2/xL. Means Bcl-2 can act and cause apoptosis.

Unphos. it binds Bcl-2.

Question 4

What two sites is BAD phos?

Answer 4

Ser112 and Ser136.

Caused by IL-3 (example).

Question 5

How are phospho-specific antibodies generated?

Answer 5

Rabbits/mice immunised with synthetic 12-14 aa peptide containing phospho-amino acid (coupled to KLH).

Antibodies purified from it using protein A/G and affinity chromatography.

Question 6

What are MAPKs?

Answer 6

MAP kinases.

p42/44

JNKs

p38

Involved in cell proliferation, differentiation, apoptosis, inflammation and response to stress.
activated by tyrosine kinases.

Question 7

How does EGF receptor signal?

Answer 7

Exists as monomer, signal causes homodimerisation, that causes change in intracellular domain, and auto-phosphorylates tyrosine.

Grb2 attracted by P-tyr.

Grb2 brings SOS close to membrane.

When SOS is close to membrane, attracts Ras-GDP (Inactive) which exchanges exchange to GTP, active Ras.

Ras recruits Raf, which activates downstream MAPK, Mek and ERK. Phosphorylates downstream proteins

Question 8

What role does Shc have?

Answer 8

Shc (Adaptor protein) binds to P-tyrosine of activated EGFR. Attracts Grb2 and downstream signaling occurs.

Question 9

How can anti-Shc be used to study cell signaling?

Answer 9

Confirm whether Shc is involved (phosphorylated) in singaling or not, indicate targets etc.